Three highly sensitive monoclonal antibody-based serological assays for the detection of tomato mottle mosaic virus

In recent years, tomato mottle mosaic virus (ToMMV) has become one of the most important viral pathogens affecting solanaceous crop production in Yunnan, Hainan, and Shandong provinces of China, often causing huge yield reductions. To provide farmers and vegetable industry with reliable and easy-to-use ToMMV detection methods, we immunized BALB/c mice with purified ToMMV and obtained six hybridoma cell lines (i.e., 2D6, 9C12, 26A10, 3A4, 23A4 and 17B11) that secrete anti-ToMMV monoclonal antibodies (MAbs) through the hybridoma technology. Using these MAbs as the detection antibody, we developed three serological assays: antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA), dot enzyme-linked immunosorbent assay (dot-ELISA) and tissue print enzyme-linked immunosorbent assay (tissue print-ELISA) for ToMMV detection. Our test results showed that these three newly developed serological methods can be used to specifically detect ToMMV infection in plant samples, but not tobacco mosaic virus, tomato mosaic virus, cucumber green mottle mosaic virus and cucumber mosaic virus. Sensitivity analyses further showed that ACP-ELISA and dot-ELISA can be used to detect ToMMV infection in plant crude extracts diluted at 1:81,920 and 1:40,960 (weight/volume, g/mL), respectively. Surprisingly, the detection limit of the developed dot-ELISA was 26 times higher than that of traditional RT-PCR. Using field-collected plant samples, we have demonstrated that these three new serological methods are accurate and easy-to-use for large-scale detection of ToMMV in fields.

Immunoinformatic Study of Recombinant LigA/BCon1-5 Antigen and Evaluation of Its Diagnostic Potential in Primary and Secondary Binding Tests for Serodiagnosis of Porcine Leptospirosis

Leptospirosis is responsible for hampering the productivity of swine husbandry worldwide. The aim of this study was to assess the efficacy of bioinformatics tools in predicting the three-dimensional structure and immunogenicity of recombinant LigBCon1-5 (rLigBCon1-5) antigen. A battery of bioinformatics tools such as I-TASSER, ProSA and SAVES v6.0 were used for the prediction and assessment of the predicted structure of rLigBCon1-5 antigen. Bepipred-2.0, DiscoTope v2.0 and ElliPro servers were used to predict linear and conformational epitopes while T-cell epitopes were predicted using NetMHCpan 4.1 and IEDB recommended 2.22 method for MHC Class I and II peptides respectively. The results obtained using various in silico methods were then compared with wet lab experiments comprising of both primary (IgG Dot ELISA Dipstick test) and secondary-binding assays (Latex Agglutination Test [LAT]) to screen 1153 porcine serum samples. The three-dimensional structure of rLigA/BCon1-5 protein as predicted by I-TASSER was found to be reliable by Ramachandran Plot and ProSA. The ElliPro server suggested 10 and three potential linear and conformational B-cell-epitopes, respectively, on the peptide backbone of the rLigA/BCon1-5 protein. The DiscoTope prediction server suggested 47 amino acid residues to be part of B-cell antigen. Ten of the most efficient peptides for MHC-I and II grooves were predicted by NetMHCpan 4.1 and IEDB recommended 2.22 method, respectively. Of these, three peptides can serve dual functions as it can fit both MHC I and II grooves, thereby eliciting both humoral-and cell-mediated immune responses. The prediction of these computational approaches proved to be reliable since rLigBCon1-5 antigen-based IgG Dot ELISA Dipstick test and LAT gave results in concordance to gold standard test, the Microscopic Agglutination Test (MAT), for serodiagnosis of leptospirosis. Both the IgG Dot ELISA Dipstick test and LAT were serodiagnostic assays ideally suited for peripheral level of animal health care system as “point of care” tests for the detection of porcine leptospirosis.

First Report of Pea as a Natural Host of tomato mottle mosaic virus in China

Tomato mottle mosaic virus, a new species in the genus Tobamovirus and family Virgaviridae, was first reported on tomato in Mexico in 2013 (Li et al. 2013). Subsequently, tomato mottle mosaic virus (ToMMV) was found infecting pepper and eggplant in China (Li et al. 2014; Chai et al. 2018). ToMMV was thought to pose a serious risk to solanaceous crops due to its potential to break resistance and numerous transmission ways (Li et al. 2020), meanwhile, some plant species in the families of Amaranthaceae, Brassicaceae, Cucurbitaceae, and Verbenaceae, were found as the hosts of ToMMV under experimental condition (Sui et al. 2017; Li et al. 2020). To clarify the occurrence of ToMMV in Yunnan province of China, 395 plant samples, including tomato (Solanum lycopersicum L.), pepper (Capsicum annuum L.) and pea (Pisum sativum L.), exhibiting viral-like symptoms were collected in major crop plantations in Yunnan province in 2020. Total nucleic acids were extracted from the diseased samples using a CTAB method (Li et al. 2008), and tested by reverse transcription polymerase chain reaction (RT-PCR) with ToMMV specific primers, ToMMVMPF (5’-ATGGCTCTAACTGTTAGTGGT-3’) and ToMMVMPR (5’-TTAATACGAATCAGATCCCGCG-3’), which were designed based on the movement protein gene sequence of ToMMV YYMLJ isolate (KR824950). ToMMV was detected in 13 symptomatic samples (11 cherry tomato and 2 green pea plants) with a total detection rate of 3.29%. An 807-bp fragment was amplified from 2 out of 86 pea samples showing foliar chlorosis, mosaic, malformation and necrosis symptoms, and the amplicons were cloned and sequenced. Sequences obtained from the two pea samples were identical. Therefore, one sequence was deposited in the GenBank (accession no. MW561348). BLAST search result showed that the nucleotide sequence had the highest identity of 99.88% with the ToMMV TiLhaLJ isolate (KR824951). The ToMMV infection on the two pea samples was also verified by dot-enzyme immunoassay (Dot-ELISA) using ToMMV monoclonal antibody (kindly provided by Dr. Jianxiang Wu, Zhejiang University, China). To determine the pathogenicity of ToMMV on pea, ToMMV infectious cDNA clone was used to inoculate into 3-week-old healthy pea plants via an Agrobacterium-mediated method (Tu et al. 2021). Mottle or slight mosaic, chlorosis and malformation symptoms were observed on the upper leaves of the 8 out of 12 inoculated plants 8 days postinoculation, and ToMMV could be detected by RT-PCR from the 8 symptomatic plants but not from the asymptomatic and healthy control pea plants with the above described ToMMV specific primer pair. ToMMV has now only been detected in Mexico, China, Spain, USA, Israel and Australia on tomato, pepper and eggplant (Webster et al. 2014; Turina et al. 2016; Ambrós et al. 2017; Lovelock et al. 2020). To the best of our knowledge, this is the first report of natural infection of ToMMV in pea as well as the natural infection of ToMMV on plants outside of the family Solanaceae. The result also implies that ToMMV has a potential risk to more crops in the field. Since pea, pepper and tomato are economically important cash crops in China, proper virus management strategies for the cultivation of these crops should be adopted.

Three Highly Sensitive and High-Throughput Serological Approaches for Detecting Dickeya dadantii in Sweet Potato

Sweet potato stem and root rot is an important bacterial disease and often causes serious economic losses to sweet potato. Development of rapid and sensitive detection methods is crucial for diagnosis and management of this disease in field. Here, we report the production of four hybridoma cell lines (25C4, 16C10, 9B1, and 9H10) using Dickeya dadantii strain FY1710 as an immunogen. Monoclonal antibodies (MAbs) produced by these four hybridoma cell lines were highly specific and sensitive for D. dadantii detection. Indirect enzyme-linked immunosorbent assay (indirect-ELISA) results showed that the four MAbs 25C4, 16C10, 9B1, and 9H10 could detect D. dadantii in suspensions diluted to 4.89 × 104, 4.89 × 104, 9.78 × 104, and 9.78 × 104 CFU/ml, respectively. Furthermore, all four MAbs can react strongly and specifically with all four D. dadantii strains used in this study, not with the other seven tested bacterial strains. Using these four MAbs, three different serological approaches, triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-ELISA, and tissue-print-ELISA, were developed for detection of D. dadantii in crude extracts prepared from field-collected sweet potato plants. Among these three methods, TAS-ELISA and dot-ELISA were used to detect D. dadantii in suspensions diluted up to 1.23 × 104 and 1.17 × 106 CFU/ml, respectively, or in sweet potato crude extracts diluted up to 1:3,840 and 1:1,920 (wt/vol, grams per milliliter), respectively. Surprisingly, both TAS-ELISA and dot-ELISA serological approaches were more sensitive than the conventional PCR. Analyses using field-collected sweet potato samples showed that the newly developed TAS-ELISA, dot-ELISA, or tissue-print-ELISA were reliable in detecting D. dadantii in sweet potato tissues. Thus, the three serological approaches were highly valuable for diagnosis of stem and root rot in sweet potato production.

Comparative study between WIDAL and DOT ELISA in the diagnosis of Typhoid fever

Background: Typhoid fever, also known as enteric fever, is a communicable disease, found only in man and occurs due to systemic infection mainly by Salmonella typhi organisms. Blood culture is regarded as the gold standard for diagnosis and carry 70-75% diagnostic yield in the first week of illness. Aims and Objective: To compare the sensitivity and specificity of Widal test and dot ELISA with blood culture in the early diagnosis of Typhoid fever. Materials and Methods: A Cross-Sectional study was carried out in the Department of Microbiology, Era’s Lucknow Medical College and Hospital for a period of 18 months. Patients presenting with acute febrile illness suspicious of typhoid fever accompanied by clinical signs and symptoms of typhoid fever in the absence of any other known febrile illnesses, were included in the study. Widal and Dot ELISA was performed using serum samples and for blood culture aseptically collected blood was used. Results: Sensitivity, Specificity, PPV, NPV of DOT ELISA as compared to Blood culture for typhoid positivity was found to be 92.6%, 83.7%, 55.6% and 98.1% respectively. Diagnostic accuracy of DOT ELISA as compared to Blood culture was found to be 85.3%. Conclusion: For both early and late diagnosis of typhoid fever with high sensitivity as well as accuracy for identification of typhoid fever, the rapid diagnostic test (Dot Elisa) is better than the Widal test. However, it may be an increased burden to healthcare owing to a low positive predictive value in a low prevalence scenario.

Comparison of Dot ELISA Using GroEL Recombinant Protein as an Antigen and an Indirect Hemagglutination Assay for Serodiagnosis of Melioidosis

Background: Melioidosis is a disease caused by the Burkholderia pseudomallei bacterium. The mortality rate of infected patients is quite high because the symptoms are similar to those of various diseases, making it difficult to diagnose clinically and preventing the immediate treatment with effective antibiotics that is required for the management of acute infections. To provide appropriate treatment, accurate and rapid diagnosis is required. Objective: The aims of this study were to develop Dot ELISA using purified GroEL B. pseudomallei recombinant protein as an antigen and to compare the newly developed assay with an indirect hemagglutination assay (IHA) for the diagnosis of melioidosis. Methods: The GroEL recombinant protein was purified by immobilized metal affinity chromatography before being used as an antigen. The optimal conditions of the Dot ELISA were determined and used for subsequent experiments. A total of 291 serum samples were evaluated by the established Dot ELISA and IHA, using the bacterial culture method as the gold standard of melioidosis diagnosis. Results: The results from Dot ELISA and IHA revealed sensitivity, specificity, and accuracy of 85.7% (Dot ELISA)/64.3% (IHA), 94.4%/85.5%, and 93.1%/82.5%, respectively. Conclusion: These results indicate that the Dot ELISA developed is an efficient, simple, rapid and cost-effective technique for the early diagnosis of melioidosis and can be used in a local laboratory without specialized equipment.

Pixel-Based Machine Learning and Image Reconstitution for Dot-ELISA Pathogen Diagnosis in Biological Samples

Serological methods serve as a direct or indirect means of pathogen infection diagnosis in plant and animal species, including humans. Dot-ELISA (DE) is an inexpensive and sensitive, solid-state version of the microplate enzyme-linked immunosorbent assay, with a broad range of applications in epidemiology. Yet, its applicability is limited by uncertainties in the qualitative output of the assay due to overlapping dot colorations of positive and negative samples, stemming mainly from the inherent color discrimination thresholds of the human eye. Here, we report a novel approach for unambiguous DE output evaluation by applying machine learning-based pattern recognition of image pixels of the blot using an impartial predictive model rather than human judgment. Supervised machine learning was used to train a classifier algorithm through a built multivariate logistic regression model based on the RGB (“Red,” “Green,” “Blue”) pixel attributes of a scanned DE output of samples of known infection status to a model pathogen (Lettuce big-vein associated virus). Based on the trained and cross-validated algorithm, pixel probabilities of unknown samples could be predicted in scanned DE output images, which would then be reconstituted by pixels having probabilities above a cutoff. The cutoff may be selected at will to yield desirable false positive and false negative rates depending on the question at hand, thus allowing for proper dot classification of positive and negative samples and, hence, accurate diagnosis. Potential improvements and diagnostic applications of the proposed versatile method that translates unique pathogen antigens to the universal basic color language are discussed.