Antigen-coated Plate Research Articles

Production of a monoclonal antibody against aflatoxin M1 and its application for detection of aflatoxin M1 in fortified milk

Aflatoxin M1 (AFM1) is a toxic metabolite of the fungal product aflatoxin found in milk. For food safety concern, maximum residual limits of AFM1 in milk and dairy products have been differently enforced in many countries. A suitable detection method is required to screen a large number of product samples for the AFM1 contamination. In this study, monoclonal antibodies (MAbs) against AFM1 were generated using a conventional somatic cell fusion technique. After screening, five MAbs (AFM1-1, AFM1-3, AFM1-9, AFM1-11, and AFM1-17) were obtained that showed cross-reactivity with aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) but with no other tested compounds. An indirect competitive enzyme-linked immunosorbent assay (ELISA) using a partially purified MAb and antigen-coated plates yielded the best sensitivity with the 50% inhibition concentration (IC50) and the limit of detection (LOD) values of 0.13 ng/mL and 0.04 ng/mL, respectively. This indirect competitive ELISA was used to quantify the amount of fortified AFM1 in raw milk. The precision and accuracy in terms of % coefficient of variation (CV) and % recovery of the detection was investigated for both intra- (n = 6) and inter- (n = 12) variation assays. The % CV was found in the range of 3.50–15.8% and 1.32–7.98%, respectively, while the % recovery was in the range of 92–104% and 100–103%, respectively. In addition, the indirect ELISA was also used to detect AFM1 fortified in processed milk samples. The % CV and % recovery values were in the ranges of 0.1–33.0% and 91–109%, respectively. Comparison analysis between the indirect ELISA and high performance liquid chromatography was also performed and showed a good correlation with the R2 of 0.992 for the concentration of 0.2–5.0 ng/mL. These results indicated that the developed MAb and ELISA could be used for detection of AFM1 in milk samples.

FIRST REPORT OF BEAN YELLOW MOSAIC VIRUS IN ALPINIA GALANGA IN INDIA

Alpinia galanga (family Zingiberaceae), commonly called greater galangal or blue ginger, is a rhizomatous herb distributed in various parts of India and throughout Southeast Asia. In a garden of the Indian Agricultural Research Institute (New Delhi), 90% of the plants exhibited spindle-shaped yellow or light green streaks in the interveinal tissues and midribs. Based on symptoms and leaf dip preparations that revealed flexuous filamentous particle of ca. 750×12 nm, infection by the potyvirus Banana bract mosaic virus (BBrMV) was suspected. Antigen- coated plate (ACP)-ELISA with a general potyvirus antiserum (DSMZ, Germany) reacted positively, whereas there was no reaction with an antiserum to BBrMV raised at ICAR-National Research Centre for Banana (Tiruchirappalli). Total RNA extracted from symptomatic and healthy leaf tissues using a RNeasy plant mini kit (Qiagen, USA) was tested by RT-PCR, using Chen et al. (2001) degenerate potyvirus-specific primers that amplify a ca.1.7 kb fragment from the 3' genome end (FP: 5' GGNAAYAAYAGYGGNCARCC 3; RP: 5' GTTTTCCCAGTCACGAC(T)15 3’). A product of 1.649 kb was amplified from symptomatic but not from healthy plants, which was cloned and sequenced (GenBank accession No. KM198742), revealing 87-94% and 91-94% identity at the nucleotide and amino acid level, respectively, with Bean yellow mosaic virus (BYMV) isolates from various crops. In ACP- ELISA using a polyclonal antiserum to BYMV (NBPGR, New Delhi), 10 infected leaf samples tested positive, whereas healthy sample were negative. Thus, the symptoms observed in A. galanga, which resemble those elicited by BBrMV in A. purpurata in Hawaii (Wang et al., 2010) are caused by BYMV. To the best of our knowledge this is the first report of the natural occurrence of BYMV in A. galanga in India.

Multi-color quantum dot-based fluorescence immunoassay array for simultaneous visual detection of multiple antibiotic residues in milk

Antibiotic residues, which are among the most common contaminants in animal-based food products such as milk, have become a significant public health concern. Here, we combine a multicolor quantum dot (QD)-based immunofluorescence assay and an array analysis method to achieve simultaneous, sensitive and visual detection of streptomycin (SM), tetracycline (TC), and penicillin G (PC-G) in milk. Antibodies (Abs) for SM, TC and PC-G were conjugated to QDs with different emission wavelengths (QD 520 nm, QD 565 nm and QD 610 nm) to serve as detection probes (QD-Ab). Then a direct competitive fluorescent immunoassay was performed in antigen-coated microtiter plate wells for simultaneous qualitative and quantitative detection of SM, TC, and PC-G residues, based on fluorescence of the QD-Ab probes. The linear ranges for SM, TC and PC-G were 0.01-25 ng/mL, 0.01-25 ng/mL and 0.01-10 ng/mL, respectively, with detection limit of 5 pg/mL for each of them. Based on fluorescence of the QD-Ab probes, residues of the three antibiotics were determined visually and simultaneously. Compared with a commercial enzyme-linked immunosorbent assay kit, our method could achieve simultaneous analysis of multiple target antibiotics in multiple samples in a single run (high-throughput analysis) and improved accuracy and sensitivity for analysis of residues of the three antibiotics in authentic milk samples. This new analytical tool can play an important role in ameliorating the negative impact of the residual antibiotics on human health and the ecosystem.

Occurrence of Cucumber mosaic virus Subgroups IA and IB Isolates in Tomatoes in Nigeria.

Tomato (Solanum lycopersicum L.) is a major economic crop consumed globally in fresh or processed forms. During a routine field survey of major tomato-producing areas of southwestern Nigeria in May/June 2013, tomato plants cv. Roma VF showing virus-like symptoms including stunting, chlorosis, and narrowing of leaf blades were observed in 10 farmers' fields with varying levels of incidence averaging ~27%. Moderate to high aphid infestations were also observed in affected fields, and fruit production was significantly impacted based on visual observations. Since symptoms observed on affected plants are similar to those described for Cucumber mosaic virus (CMV) infection in tomato (5), leaf tissue samples collected from a total of 92 tomato plants across 10 commercial farms were subjected to antigen coated plate (ACP)-ELISA essentially as described previously (2). In ACP-ELISA using a CMV polyclonal antibody, 24 of the 92 samples (26.1%) derived from 7 of the 10 survey locations spread across Oyo, Ogun, Ekiti, and Osun states of southwestern Nigeria tested positive for CMV. Based on the ACP-ELISA results, one randomly selected sample from each of the CMV-positive survey locations, seven samples in total, was subjected to total nucleic acid extraction (1) followed by one step-single tube RT-PCR using primers CMV1/CMV2 and conditions described previously (4) with appropriate virus-positive and -negative controls. A ~500 bp DNA band was amplified from these seven ACP-ELISA-positive samples, thus confirming the presence of CMV. To further confirm these results and to enable molecular typing of CMV isolates from southwest Nigeria, the amplified DNA fragments were precipitated with the addition of 70% ethanol and centrifugation and directly sequenced using the ABI 3130xL Genetic Analyzer (Applied Biosystems, California) at the Bioscience Center of the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. Following the removal of primer- and 3'UTR-specific sequences, the remaining 366-bp partial CP-specific sequences (GenBank Accession Nos. KM091952 to 58) and corresponding sequences of global CMV isolates obtained from GenBank were subjected to multiple alignments using the MEGA 6.0 software. This analysis showed that tomato-infecting CMV isolates from southwest Nigeria shared 91.6 to 99.4% and 94.9 to 99.1% nucleotide (nt) and amino acid (aa) identities among themselves and 91.6 to 98.0% and 94.1 to 98.3%, 89.4 to 94.1% and 93.2 to 98.3%, and 75.2 to 78.8% and 84.0 to 87.3% with corresponding nt and aa sequences of representatives of CMV isolates belonging to subgroups IA (D10538), IB (AB008777), and II (M21464), respectively. Maximum likelihood phylogenetic analysis revealed the clustering of four and three CMV isolates obtained in this study into subgroups IA and IB, respectively, with >70% bootstrap support. CMV has been detected in tomato seeds (3) and its very wide host range includes cultivated crops and weed species (5). It is therefore plausible that contaminated seed lots and alternative weed and crop host plants serve as sources of CMV inoculum to cultivated tomato in affected farms. Although CMV has been reported from tomato from several countries worldwide, to our knowledge, this is the first empirical evidence for the occurrence of CMV subgroups IA and IB in cultivated tomato in Nigeria.

First Report of Maize chlorotic mottle virus Infecting Maize in the Democratic Republic of the Congo.

Maize (Zea mays L.) is a major food and fodder crop cultivated on 1.54 million ha in the Democratic Republic of the Congo (DRC). In December 2013, unusually severe chlorotic mottle symptoms and pale green streaks were observed in local varieties (Mudishi 1 and 2, Bambou, Kasayi, H614, H613, and Mugamba) and exotic varieties (H520, H624, H403, HDK8031, and ZM607) in Beni, Lubero, and Rutshuru territories at 1,015 to 1,748 m elevation in North Kivu Province. Symptoms were prominent on newly emerging leaves that later developed marginal necrosis resembling the symptoms of maize lethal necrosis (MLN), caused by a dual infection of Maize chlorotic mottle virus (MCMV, genus Machlomovirus) and Sugarcane mosaic virus (SCMV, genus Potyvirus). Each of these viruses, but particularly MCMV, is also known to cause severe mosaic and mottling symptoms in maize (4). In January 2014, symptomatic and asymptomatic samples (n = 20) from disease-affected fields in Beni and Lubero provinces were collected for virus testing using Whatman FTA Classic Cards (1) and analyzed for MCMV (2681F: 5'-ATGAGAGCAGTTGGGGAATGCG and 3226R: 5'-CGAATCTACACACACACACTCCAGC) and SCMV (8679F: 5'-GCAATGTCGAAGAAAATGCG and 9595R: 5'-GTCTCTCACCAAGAGACTCGCAGC) by reverse transcription (RT)-PCR (4). Samples were also analyzed for Maize streak virus (MSV, genus Mastrevirus), an endemic virus in DRC, by PCR using MSV specific primers (MSV215-234: CCAAAKDTCAGCTCCTCCG and MSV1770-1792: TTGGVCCGMVGATGTASAG) (3). A DNA product of expected size (~520 bp) resulted only for MCMV in all the symptomatic plant samples. None of the samples tested positive for SCMV or MSV. RT-PCR analyses were performed to ascertain the absence of potyviruses using the degenerate potyvirus primers (CIFor: 5'GGIVVIGTIGGIWSIGGIAARTCIAC and CIRev: 5'ACICCRTTYTCDATDATRTTIGTIGC3') (2) were also negative. Occurrence of MCMV in symptomatic samples was further confirmed by antigen-coated plate (ACP)-ELISA using anti-MCMV rabbit polyclonal antibodies produced at the Virology Unit, IITA, Ibadan, Nigeria. The RT-PCR product of MCMV was purified and sequenced in both directions (GenBank Accession No. KJ699379). Pairwise comparison of 518 bp nucleotide sequence corresponding to p32 and p37 open reading frames of MCMV by BLASTn search revealed 99.8% nucleotide sequence identity with an MCMV isolate from Kenya (JX286709), 98 to 99% identity with the isolates from China (JQ982468 and KF010583), and 96% identity with the isolates from the United States (X14736 and EU358605). MCMV is a newly emerging virus in Africa, first detected during a severe MLND outbreak in 2011 in Kenya (4). This disease has since become a serious threat to maize production in East Africa. MCMV has been reported in maize from Kenya, Rwanda, Tanzania, and Uganda. To our knowledge, this is the first report of MCMV occurrence in DRC. This finding confirms the further geographic expansion of MCMV and illustrates the need for further studies to identify vectors and also create awareness about the disease and to strengthen surveillance to prevent its further spread in the continent.

A Highly Sensitive Indirect Competitive Enzyme-Linked Immunosorbent Assay (ic-ELISA) by Antigen Coating for Diethyl Phthalate Analysis in Foods

As we have known, with the plasticizer disturbance in 2011 in Taiwan, long-term exposure to diethyl phthalate (DEP), one of the widely used phthalate esters, can lead to serious health problems. Therefore, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) by antigen-coated plate format for DEP in foods was proposed in this paper. The polyclonal antibodies were raised against diethyl 4-aminophthalate (4-DEAP) conjugated to bovine serum albumin by the amino diazotization linkage method. Coating antigen was prepared with 4-DEAP conjugated to ovalbumin using the same procedure. Under the optimal experimental conditions, the ic-ELISA has a linear working range of 0.005–18.6 ng/mL (R 2 = 0.9921), with a limit of detection of 0.0049 ng/mL. Low cross-reactivity (<9 %) to structurally related phthalates was observed. The method was successfully applied to the determination of DEP in fruit juice, milky tea, pure milk, and sour milk, without purification or preconcentration. Satisfactory recoveries were obtained ranging from 91.1 to 109.3 %. The results suggested that the developed ic-ELISA is a simple, sensitive, and specific method for the rapid monitoring of DEP in food samples.

Development of ELISA test for determination of the level of antibodies against Rhodococcus equi in equine serum and colostrum

Rhodococcus equi infection occurs worldwide and is one of the major causes of losing foals in the first six months of life. The application of serological tests in the diagnostics of rhodococcosis is limited, however they play a crucial role in immunological studies.The objective of this study was to develop and standardize ELISA test for the determination of the level of antibodies against Rhodococcus equi in equine serum and colostrum.Bacterial cell lysate was used as antigen. The test was standardized on 175 sera obtained from adult horses kept on rhodococcosis-free and endemic farms. Positive and negative control sera were used. The test detected IgG antibodies mainly against VapA protein, which was confirmed by Western blot analysis.The test was easy to perform, did not require inactivation of sera and had low well-to-well variation. The shelf life of antigen-coated ELISA plates was 21 days.The test allowed to reveal significant increase of R. equi-specific antibodies in both serum and colostrum in response to the vaccination (p<0.001). Therefore it can be applied to the evaluation of efficacy of immunization. Moreover, no statistically significant difference in the baseline antibody level in adult horses from rhodococcosis-free and endemic farm was revealed (α=0.05).

Production and characterization of antibodies against crosslinked gelatin nanoparticles and first steps toward developing an ELISA screening kit

Nanotechnologies are finding a growing range of applications in the food sector. Nanoparticles are used notably to add vitamins and other nutrients to foods and beverages without affecting taste and color. They are also used to develop new tastes, preserve food texture, control the release of flavors, improve the bioavailability of compounds such as antioxidants and vitamins, and monitor freshness with nanosensors. Crosslinked gelatin nanoparticles are a component of nano-sized carriers for nutrient and supplement delivery in foods and related products. This paper describes the production and characterization of polyclonal antibodies against gelatin nanoparticles. Two immunization schemes were investigated: subcutaneous injection with and without a first intravenous injection. Two enzyme-linked immunosorbent assay formats were used to characterize the antibodies: an inhibition format with an antigen-coated plate for detection of the immune response and a sandwich format for development of the method. The antibodies showed good sensitivity with an IC50 equal to 0.11 ng mL(-1) using indirect ELISA format and a good specificity for the nanomaterials, without significant cross-reactivity against native gelatin. The limit of detection was determined-0.42, 0.27, 0.26, and 0.24 μg mL(-1) for apple, orange juice, milk, and soft drink matrices, respectively. ELISA technology offers rapid, low-cost assays for screening foods, feeds, and beverages. We have studied a prototype ELISA for detection of gelatin-based nanocarrier systems. Fruit juices, milk, and a soft drink were the matrices selected for assay development.

Insights into the antibacterial and immunomodulatory functions of tilapia hepcidin (TH)2-3 against Vibrio vulnificus infection in mice

The antimicrobial and immunomodulatory functions of the antimicrobial peptide, tilapia hepcidin (TH)2-3, against a bacterial endotoxin under in vitro conditions was previously reported. In this study, we investigated the antibacterial and immunomodulatory functions of TH2-3 in mice infected with the pathogen, Vibrio vulnificus. A TH2-3 injection in V. vulnificus-infected mice produced an increased survival rate compared to mice injected with V. vulnificus only. In addition, a TH2-3 injection increased the bacteriostatic property against V. vulnificus in mice. Gene expressions examined using a microarray demonstrated that TH2-3 modulated several V. vulnificus-responsive genes in the host. A neutralizing antibody assay of mice serum against inactivated V. vulnificus antigen-coated plates demonstrated the induction of an immune response by TH2-3 against the pathogen. Taken together, TH2-3 enhanced the survival rate of mice against the bacterial pathogen V. vulnificus through both antimicrobial and immunomodulatory functions. These properties make the TH2-3 peptide a good candidate for development as a new antimicrobial drug and suggest that TH2-3 can underpin the design of adjuvants for further development of vaccines.

Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus

BackgroundCucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, can be transmitted by seeds and infects many cucurbit species, causing serious yield losses in cucumber and watermelon plants. In this paper, five serological methods including antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), Dot-immunobinding assay (DBIA), direct tissue blot immunoassay (DTBIA) and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) were described for detection and diagnosis of CGMMV.ResultsUsing the purified CGMMV particles as immunogens, six murine monoclonal antibodies (MAbs) were produced. Five serological methods were established using the MAb 4H1 and detection sensitivity was compared using purified preparations and infected-plant tissue extracts. The detection sensitivity of ACP-ELISA was 0.16 ng of purified CGMMV, whereas TAS-ELISA was more sensitive than ACP-ELISA with a minimum detection of 0.04 ng of purified CGMMV. The sensitivities of TAS-ELISA and DBIA were similar for detecting CGMMV in infected-plant tissue extracts, and were four times higher than ACP-ELISA. The IC-RT-PCR was the most sensitive method, which could detect as little as 0.1 pg of purified virus. The detection sensitivity of IC-RT-PCR for CGMMV-infected plant tissues was about 400 times higher than that of TAS-ELISA and DBIA.ConclusionsThe established ACP-ELISA, TAS-ELISA, DBIA and DTBIA are suitable for routine CGMMV detection of large-scale samples in the field survey, while IC-RT-PCR is more sensitive and suitable for acquiring information about the viral genome.

Investigation of an epidemic of Hepatitis E in Nellore in south India

To determine the incidence of acute hepatitis because of hepatitis E virus (HEV) and the source of the epidemic in Nellore in south India in 2008. Anti-HEV IgM ELISA and RT-PCR for HEV-RNA were carried out on blood and stool samples from patients with acute hepatitis presenting to different hospitals in the city. The city was divided into 33 clusters, and 20 families from each cluster were systematically interviewed to determine the incidence of hepatitis E in the city. The survey was conducted on 2685 residents of 673 households from 24th November to 4th December 2008. The overall incidence of hepatitis was 5.7% (152/2685), i.e. an estimated 23,915 persons in the city were affected. There were two deaths because of acute hepatitis in the population surveyed translating to an estimated 315 deaths. Men had higher attack rates than women (7.8%vs. 3.5%) and young adults compared to children under 5 years (6.9%vs. 2.9%). Families drinking water from the pumping station at Bujjamarevu had the highest attack rate of 54.5% (39.8-69.2%). HEV IgM antibodies were present in 80/100 plasma samples tested. HEV-RNA was detected in 43/100 individuals tested, and isolates were characterized as genotype 1 by sequencing. Sewage draining into the river close to the pumping stations and broken pipelines crossing sewage drains may have triggered this large outbreak.

Simple method of ‘on-plate’ growth for improved antibody screening

Antibody phage display is a powerful biomolecular selection technology now routinely used for refining antibody diversity towards analytes of both therapeutic and diagnostic interest. Post selection, automated robotic systems can be utilised to pick, express and analyse large numbers of putative analyte-specific clones allowing the parallel screening of thousands of antibodies in less time. Most screening techniques involve a spatial addressing process whereby the selected antibodies are extracted from the cells and analysed to verify specificity. Using a simple ‘on-plate’ growth and screening approach, we show that antibody-expressing clones can be simultaneously cultured and analysed rapidly in antigen-coated ELISA plate wells yielding high binding signals and saving valuable selection time, while also eliminating the necessity for antibody extraction. The utilisation of the ‘on-plate’ technique for the screening of Fab and scFv antibodies, and a comparative analysis with commonly used antibody extraction processes, are described.

An Indirect Competitive Fluorescence Immunoassay for Determination of Dicyclohexyl Phthalate in Water Samples

A sensitive and specific indirect competitive fluorescence immunoassays (FIA) has been developed for the quantitative determination of dicyclohexyl phthalate (DCHP) using an antigen-coated plate format. The polyclonal antibodies raised against dicyclohexyl 4-amino phthalate conjugated to bovine serum albumin (BSA) by the amino diazotization linkage method. Antiserum with a sufficiently high titer was generated in rabbits and fluorescein isothiocyanate (FITC) was used as sensitive labels to construct the fluorescence immunoassay (FIA) for measurement of targeted compounds. Under optimized FIA condition, the quantitative working range was from 0.1 to 200 μg L−1 with a limit of detection of 0.05 μg L−1. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique, and the cross-reactivity rates were less than 10%. Four kinds of water samples (tap water, lake water, river water and leachate) had been detected in this assay, the recovery was 91.3–107.8%. The proposed fluorescence immunoassay turned out to be a powerful tool for monitoring of dicyclohexyl phthalate in water samples at trace level.

First Report of Malva vein clearing virus Naturally Occurring in Hollyhock in Germany.

Hollyhocks are popular garden plants and selected cultivars of Alcea rosea (family Malvaceae) are widespread in Germany. In spring 2009, dozens of A. rosea plants displaying strong vein clearing and veinal yellowing symptoms were found in private gardens in Hannover, Lower Saxony. Electron microscopic examinations of negatively stained adsorption preparations of five randomly selected samples of symptomatic plants or their offshoots revealed flexuous filamentous particles resembling those of potyviruses. Sap extracts also reacted strongly positive in an antigen coated plate (ACP)-ELISA with the broad-spectrum potyvirus antiserum AS-0573/I (DSMZ, Braunschweig, Germany). RNA extracts (RNeasy Kit, Qiagen, Valencia, CA) of the above mentioned leaf samples were used as templates in reverse transcription (RT)-PCR assays with potyvirus specific primers (2) that have been shown to amplify the 3' terminus of the genome of many potyvirus species. For extracts from symptomatic samples, this resulted in a consistent amplification of an ~1.6-kbp fragment, whereas no products were obtained from RNA extracts of asymptomatic plants. From one positive sample, the amplified fragment was cloned and one clone was partially sequenced. The nucleotide (nt) and amino acid sequences showed the highest identities (81 to 83% and 87 to 90%, respectively) to GenBank sequences FJ539084, FM212972, EU884405, and FJ561293 of the potyvirus Malva vein clearing virus (MVCM). On the basis of these identity values and according to the species demarcation criteria in the genus Potyvirus, the virus can be regarded as a German isolate of the recently sequenced MVCV (3,4). Direct sequencing of the 5'-end of the amplified RT-PCR fragment revealed sequences of only one potyvirus species. The virus isolate has been submitted to the DSMZ Plant Virus Collection (Braunschweig, Germany) under accession PV-0963 and the sequence obtained from the cloned cDNA is deposited in GenBank (GQ856544). In addition, sap from affected leaves was mechanically inoculated onto sets of herbaceous indicator plants (Chenopodium quinoa, C. foliosum, C. murale, C. amaranticolor, Datura stramonium, Nicotiana benthamiana, N. hesperis, Petunia hybrida, and Solanum lycopersicum) of which only C. quinoa plants became infected. Symptoms of weak chlorosis along and beside veins of inoculated leaves, but not systemic leaves, became visible 2 weeks postinoculation. Symptomatic leaves contained flexuous filamentous particles and ACP-ELISA and RT-PCR confirmed virus presence. The partially sequenced amplicon showed 99% nt identity to the sequence from the cloned cDNA. To our knowledge, this is the first report of a MVCV isolate naturally occurring in A. rosea and C. quinoa is the first host identified that does not belong to the plant family Malvaceae. In contrast, the MVCV isolate used in the host range study of Lunello et al. (4) did not infect A. rosea and C. quinoa, confirming previous host range descriptions by Brunt et al. (1). Since MVCV infections of hollyhocks seem to cause only leaf symptoms and do not noticeably affect growth or flowering of the plants, this will hopefully not impair the usability of this popular garden plant.

Occurrence of latent virus infection in visually-rated cowpea (Vigna unguiculata L. Walp) seedlings

The presence of latent infections was studied in five cowpeas varieties. Seeds of the varieties were planted and the seedlings inoculated with antigens from Cucumber mosaic virus (CMV) genus Cucumovirus, Bean common mosaic virus (BCMV) genus Potyvirus (Blackeye cowpea mosaic virus strain), Southern bean mosaic virus (SBMV) genus Sobemovirus and Cowpea mottle virus (CPMoV) genus Carmovirus seven days after planting. Seedlings expressing symptoms were rouged at two weeks after inoculation, while asymptomatic ones were subjected to serological indexing to detect the presence/absence of latent infection. Protein A-sandwich enzyme-linked immunosorbent assay (PAS ELISA) was employed for the serological detection of CMV, SBMV and CPMoV, while antigen-coated plate (ACP) ELISA was used to detect BCMV in the asymptomatic plants. Cowpea seedlings without virus symptoms but with positive serological reactions were considered as being latently infected. All of the inoculated TVu 1272 and SuVita-2 plants showed symptoms consistent with CMV and CPMoV infections, respectively. The rate of CMV latent infection was high in TVu 1179 (14.5%), low in SuVita-2 (1.3%) but not recorded in TVu 1272.

Structural and Functional Analysis of a C3b-specific Antibody That Selectively Inhibits the Alternative Pathway of Complement

Amplification of the complement cascade through the alternative pathway can lead to excessive inflammation. Targeting C3b, a component central to the alternative pathway of complement, provides a powerful approach to inhibit complement-mediated immune responses and tissue injury. In the present study, phage display technology was employed to generate an antibody that selectively recognizes C3b but not the non-activated molecule C3. The crystal structure of C3b in complex with a Fab fragment of this antibody (S77) illustrates the structural basis for this selectivity. Cleavage of C3 to C3b results in a plethora of structural changes within C3, including the rearrangement of macroglobulin domain 6 enabling binding of S77 to the adjacent macroglobulin domain 7 domain. S77 blocks binding of factor B to C3b inhibiting the first step in the formation of the alternative pathway C3 convertase. In addition, S77 inhibits C5 binding to C3b. This results in significantly reduced formations of anaphylatoxins and membrane-attack complexes. This study for the first time demonstrates the structural basis for complement inhibition by a C3b-selective antibody and provides insights into the molecular mechanisms of alternative pathway complement activation.

First report of Shallot virus X in shallot in New Zealand

In January 2005, mild mosaic and chlorosis were observed on leaves of shallot ( Allium cepa var. aggregatum ) cv. Red Prisma growing in Marlborough, New Zealand. Leaves from 100 plants were collected and bulked into groups of ten. Three composite samples tested positive for Shallot mite-borne latent virus (ShMbLV) using polyclonal antiserum (supplied by Dr. E. Barg, Biologische Bundesanstalt, Germany) in an antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA). Similar symptoms were seen in shallot cv. Jermor in the same region in December 2007. Leaves from these plants also tested positive for ShMbLV by ACP-ELISA. To confirm the identification of the samples from 2007, dried tissue of the type isolate of ShMbLV was obtained (Van Dijk & van der Vlugt, 1994). RNA was extracted from diseased shallot samples and the ShMbLV type isolate and tested by RT-PCR using primers that amplify a ca . 750 bp fragment between the coat protein (CP) and ORF6 region of allexiviruses (Chen et al ., 2004). For both samples, amplicons of the expected size were obtained and sequenced directly. Analysis showed a 93 to 95% nucleotide identity with Shallot virus X (ShVX) (GenBank Accession No. M97264). RT-PCR was then done using specific primers designed to amplify a 912 bp fragment of ShVX including the CP gene (ShVX-CPF: 5 '?-ATTTAGGGGTGAAGGTCTGT- 3 '?; ShVX-CPR: 5 '?-GAGTTTTGAGGTCGTTGG-3 '?). Amplicons of the correct size were obtained from both diseased shallot samples and the ShMbLV type isolate. Subsequently, one-step immunocapture RT-PCR was performed using the ShMbLV antiserum and the ShVXspecific primers, and bands of the correct size were obtained for both samples. The amplicons were cloned and sequenced. A BLAST search showed that the sequence from shallot (EU835197) and that of the type isolate of ShMbLV (EU835196) showed 93 and 95% nucleotide identity, respectively, with ShVX (M97264). According to the criteria demarcating species in the genus (Adams et al ., 2004), it is suggested that ShMbLV should be considered a synonym of ShVX as previously proposed (Van Dijk & van der Vlugt, 1994). This is the first report of ShVX in New Zealand and until these findings it was considered a regulated pest. However, no phytosanitary measures will be imposed to eradicate the virus and it is likely to spread in the future, especially as the vector, Aceria tulipae , is present in New Zealand. Allexiviruses, including ShVX, often infect allium crops in combination with carlaviruses and/or potyviruses and cause significant yield losses (Van Dijk & van der Vlugt, 1994; Chen et al ., 2004)

First Report of Ranunculus mild mosaic virus on Ranunculus asiaticus in Yunnan Province, China.

In June 2007, a new viral disease occurred in commercial fields of Ranunculus asiaticus in the Yunnan Province of China. Infected plants exhibited mosaic symptoms and growth abnormalities. Viral disease incidence for this ornamental crop host in the Yunnan Province was estimated to range from 10 to 20%. Electron microscopic examination of negatively stained leaf-dip preparations from symptomatic plants identified long, flexuous linear particles (approximately 800 nm). The samples were tested using indirect antigen-coated plate (ACP)-ELISA. ACP-ELISA results showed that the leaf samples from symptomatic plants reacted positively to the potyvirus group antibody (Agdia Inc., Eklhart, IN). Total nucleic acid extracted from symptomatic plants was tested using reverse transcription (RT)-PCR with primers (S 5'-GGNAAAAYAGYGGNCARCC-3'; M4: 5'-GTTTTCCCAGTCACGAC-3' [N = A, G, C, or T; Y = C or T; and R = A or G]) designed to amplify the 3' terminal region of genomic RNA of the genus Potyvirus (1). RT-PCR produced a 1,650-bp amplification product that was cloned and sequenced (GenBank Accession No. EU684747). The sequenced portion showed 90 and 99% identity with the Ranunculus mild mosaic virus (RMMV) isolates (GenBank Accession Nos. DQ152191 and EF445546) from Italy and Israel, respectively (2). To our knowledge, this is the first report of RMMV in China. Infection from this virus may cause losses for cut-flower production of Ranunculus asiaticu and it is also a potential threat for international trade of Ranunculus germplasm.

Capsicum chlorosis virus (Genus Tospovirus) Infecting Chili Pepper (Capsicum annuum) in India.

Chili pepper (Capsicum annuum L.) is one of the most important spice crops in India. In October of 2006, symptoms indicative of tospovirus infection were noticed in several commercial fields of chili pepper near Bangalore in Karnataka State. Chlorotic and necrotic spots and rings on leaves, apical necrosis, and leaf distortion were observed. Disease incidence was more than 20%. Mechanical inoculation with sap extracts from these symptomatic plants showed that the host range and symptomatology of the virus was similar to those described for Capsicum chlorosis virus (CaCV) (1,3). The virus reacted with antisera specific to Groundnut bud necrosis virus (GBNV) and Watermelon silver mottle virus of serogroup IV tospoviruses in antigen-coated plate ELISA. It did not react with antisera specific to Tomato spotted wilt virus, Impatiens necrotic spot virus, or Iris yellow spot virus in double-antibody sandwich-ELISA. Immunosorbent electron microscopy of infected sap using GBNV antiserum revealed the presence of strongly decorated quasi-spherical virus particles. Reverse transcription (RT)-PCR was conducted to further identify the virus. No amplification was observed from extracts of symptomatic plants (n = 10) by RT-PCR using GBNV-specific primers (4), indicating that the diseased chili was not infected with GBNV. However, a DNA fragment of approximately 850 bp was amplified by using primers specific to the nucleocapsid (N) gene of CaCV (CaCF 5'-CTATAGAWGTACTAGGCTTTGAGC-3' and CaCR 5'-CATGTCTAACGTCAGGCAACTTAC-3'). Direct sequencing of the amplicon (GenBank Accession No. EF625227) revealed a nucleotide sequence identity ranging from 85.5% with isolates from Thailand (GenBank Accession Nos. AY846366, AY647437, and AF134400) and China (GenBank Accession No. DQ355974) to 98.1% with isolates from Australia (GenBank Accession Nos. AY036057 and AY036058). Phylogenetic analysis of the N protein sequences showed that the chili pepper isolate from India formed a cluster with those from Australia. This cluster was distinct from the one formed by the peanut isolates from Thailand and a tomato isolate from India (2). To our knowledge, this is the first report of CaCV infection of chili pepper in India. The potential impact of CaCV on tomato and chili pepper production in India remains to be seen.

Development of an enzyme-linked immunosorbent assay for quantification of Salmonella enteritidis-specific antibodies in egg yolk

The present study aims at developing an indirect ELISA to quantify yolk antibodies specific to all surface proteins of the invasive Salmonella Enteritidis (SE), which acquired the 1.8, 14.1, and ~ 50 Kb plasmids. An ELISA checkerboard was used in four different experiments to account for the different parameters included in the preliminary ELISA procedure, and consequently to maximize the difference in Optical Density (OD) values between control positive and negative yolk samples. The first experiment aimed at studying the impact of 5% Bovine Serum Albumin (BSA) dissolved in distilled water as a blocking reagent on a 28 µg/well SE antigen-coated plate, while applying the positive and negative control yolk samples to different concentrations of Phosphate-Buffered Saline (PBS).Conjugate application was maintained constant at a dilution of 1:500 in PBS. The second experiment was similar to the first one, but the positive and negative control yolk samples were diluted in PBS-Tween 20, and the conjugate dilution was changed to 1:1500 in PBS-Tween 20. In the third experiment, the conjugate was diluted at 1:1500 in 5% BSA/PBS-Tween 20 diluent or PBS-Tween 20 diluent with no 5% BSA. The objective of the fourth experiment was to study the impact of four different concentrations of SE-coated antigen levels (28µg/well, 56µg/well, 84µg/well, and 112µg/well), while fixing the blocking step with 5% BSA in distilled water, and the conjugate dilution set at 1:1000 in 5% BSA/PBSTween 20, and fixing the control yolk samples dilution at 1% in PBS-Tween 20. This last experimental procedure allowed the highest difference in mean absorbance OD values of the positive control minus the negative control samples, which was equivalent to 0.381. In addition, the final protocol for this ELISA was applied on individual egg yolk samples of two groups of chicken layers: one challenged in the esophagus at 11 days with 5.4 x 1010 CFU/ml/bird of SE, and the second group was not challenged. The mean OD values of the egg yolk of antibodies specific against SE of the two groups were significantly different (0.8578 versus 0.5250; p<0.05), which indicates the possible application of the developed ELISA for screening SE infection by examining egg yolks produced by commercial layers.