scholarly journals Development of Genotype-Specific Anti-Bovine Rotavirus A Immunoglobulin Yolk Based on a Current Molecular Epidemiological Analysis of Bovine Rotaviruses A Collected in Japan during 2017–2020

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1386
Author(s):  
Koki Odagiri ◽  
Nobuki Yoshizawa ◽  
Hisae Sakihara ◽  
Koji Umeda ◽  
Shofiqur Rahman ◽  
...  
Keyword(s):  
Passive Immunization ◽  
Epidemiological Survey ◽  
Neutralization Assay ◽  
Cross Reactivity ◽  
Economic Losses ◽  
Epidemiological Analysis ◽  
Beef Calves ◽  
Rotavirus A ◽  
Bovine Rotavirus ◽  
A Current

Bovine rotavirus A (RVA), a major causative pathogen of diarrhea in dairy and Japanese beef calves, has led to severe economic losses in numerous countries. A dual genotyping system based on genomic segments encoding VP7 (G genotype) and VP4 (P genotype), comprising the outer layer of the virion, has been used to understand the epidemiological dynamics of RVAs at the national and global levels. This study aimed to investigate occurrence frequency of G and P genotypes for multiple bovine RVAs from calf diarrheic samples collected in Japan from 2017 to 2020. After we produced anti-bovine RVA immunoglobulin yolks (IgYs) from hens immunized with the two RVAs with different genotypes (G6P[5] and G10P[11]) selected on the basis of the current epidemiological survey, we investigated cross-reactivity against bovine RVAs with different G and P combinations owing to establish a useful strategy to protect calves from RVA infections using the two IgYs. Consequently, the two produced anti-bovine IgYs showed strong cross-reactivity against bovine RVAs with the same G and/or P genotypes in neutralization assay, respectively. Therefore, our data suggest the possibility of a passive immunization to protect calves from a bovine RVA infections epidemic in Japan via oral administration of the two IgYs into calves. The findings presented herein will provide important information that IgY is one of the effective tools to prevent infections of various pathogens.

scholarly journals Phylogenetic Analyses of Rotavirus A from Cattle in Uruguay Reveal the Circulation of Common and Uncommon Genotypes and Suggest Interspecies Transmission

Pathogens ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 570
Author(s):  
Matías Castells ◽  
Rubén Darío Caffarena ◽  
María Laura Casaux ◽  
Carlos Schild ◽  
Samuel Miño ◽  
...  
Keyword(s):  
Dairy Products ◽  
Phylogenetic Analyses ◽  
Interspecies Transmission ◽  
Economic Losses ◽  
Cattle Production ◽  
Economic Sectors ◽  
Beef Calves ◽  
Rotavirus A ◽  
P Type ◽  
Neonatal Calf Diarrhea

Uruguay is one of the main exporters of beef and dairy products, and cattle production is one of the main economic sectors in this country. Rotavirus A (RVA) is the main pathogen associated with neonatal calf diarrhea (NCD), a syndrome that leads to significant economic losses to the livestock industry. The aims of this study are to determine the frequency of RVA infections, and to analyze the genetic diversity of RVA strains in calves in Uruguay. A total of 833 samples from dairy and beef calves were analyzed through RT-qPCR and sequencing. RVA was detected in 57.0% of the samples. The frequency of detection was significantly higher in dairy (59.5%) than beef (28.4%) calves (p < 0.001), while it did not differ significantly among calves born in herds that were vaccinated (64.0%) or not vaccinated (66.7%) against NCD. The frequency of RVA detection and the viral load were significantly higher in samples from diarrheic (72.1%, 7.99 log10 genome copies/mL of feces) than non-diarrheic (59.9%, 7.35 log10 genome copies/mL of feces) calves (p < 0.005 and p = 0.007, respectively). The observed G-types (VP7) were G6 (77.6%), G10 (20.7%), and G24 (1.7%), while the P-types were P[5] (28.4%), P[11] (70.7%), and P[33] (0.9%). The G-type and P-type combinations were G6P[11] (40.4%), G6P[5] (38.6%), G10P[11] (19.3%), and the uncommon genotype G24P[33] (1.8%). VP6 and NSP1-5 genotyping were performed to better characterize some strains. The phylogenetic analyses suggested interspecies transmission, including transmission between animals and humans.

scholarly journals Neonatal diarrhea and rotavirus A infection in beef and dairy calves, Brazil, 2006-2015

2020 ◽  
Vol 40 (1) ◽  
pp. 7-11
Author(s):  
Thais N.S. Medeiros ◽  
Elis Lorenzetti ◽  
Rodrigo P. Massi ◽  
Alice F. Alfieri ◽  
Amauri A. Alfieri
Keyword(s):  
Dairy Calves ◽  
Economic Losses ◽  
Viral Agent ◽  
Similar Frequency ◽  
Fecal Samples ◽  
Neonatal Diarrhea ◽  
Beef Calves ◽  
Rotavirus A ◽  
Geographical Regions ◽  
Cattle Herds

ABSTRACT: Calf diarrhea causes substantial economic losses in the cattle industry worldwide. Bovine rotavirus A (RVA) is the main viral agent that leads to enteric infection and diarrhea outbreaks in calves throughout the world. The aim of this retrospective (2006-2015) study was to determine the frequency of RVA detection in diarrheic fecal samples from beef and dairy calves from the three main cattle-producing regions of Brazil. Diarrheic fecal samples (n=1,498) of 124 beef and 56 dairy cattle herds from the Midwest, South, and Southeast geographical regions of Brazil were evaluated using the silver-stained polyacrylamide gel electrophoresis (ss-PAGE) technique. RVA double stranded-RNA was identified by the ss-PAGE technique in 410 (27.4%) fecal samples. The frequency of positive samples found in beef calves (31.9%; 328/1,027) was higher than the frequency found in diarrheic fecal samples from dairy calves (17.4%; 82/471). RVA infection was identified in calves from the three Brazilian geographical regions analyzed. However, the frequency of positive diarrheic calves in the Midwest region (39.4%), predominantly beef calves, was higher than in the South (19.4%) and Southeast (17.6%) regions. The temporal distribution of RVA-infected calves evaluated by two five-year periods (2006-2010, 24.5%; 2011-2015, 28.8%) demonstrated a very similar frequency of RVA in both periods. Considering the wide regional and temporal scope of this study, it can be concluded that RVA remains an important etiology of neonatal diarrhea in calves of Brazilian cattle herds.

scholarly journals To React or Not to React: The Dilemma of Fish Immune Systems Facing Myxozoan Infections

2021 ◽  
Vol 12 ◽  
Author(s):  
Astrid S. Holzer ◽  
M. Carla Piazzon ◽  
Damien Barrett ◽  
Jerri L. Bartholomew ◽  
Ariadna Sitjà-Bobadilla
Keyword(s):  
Immune Responses ◽  
Immune Evasion ◽  
B Lymphocyte ◽  
Control Strategies ◽  
Abiotic Factors ◽  
Passive Immunization ◽  
Life Cycles ◽  
Economic Losses ◽  
Special Focus ◽  
Host Immune System

Myxozoans are microscopic, metazoan, obligate parasites, belonging to the phylum Cnidaria. In contrast to the free-living lifestyle of most members of this taxon, myxozoans have complex life cycles alternating between vertebrate and invertebrate hosts. Vertebrate hosts are primarily fish, although they are also reported from amphibians, reptiles, trematodes, mollusks, birds and mammals. Invertebrate hosts include annelids and bryozoans. Most myxozoans are not overtly pathogenic to fish hosts, but some are responsible for severe economic losses in fisheries and aquaculture. In both scenarios, the interaction between the parasite and the host immune system is key to explain such different outcomes of this relationship. Innate immune responses contribute to the resistance of certain fish strains and species, and the absence or low levels of some innate and regulatory factors explain the high pathogenicity of some infections. In many cases, immune evasion explains the absence of a host response and allows the parasite to proliferate covertly during the first stages of the infection. In some infections, the lack of an appropriate regulatory response results in an excessive inflammatory response, causing immunopathological consequences that are worse than inflicted by the parasite itself. This review will update the available information about the immune responses against Myxozoa, with special focus on T and B lymphocyte and immunoglobulin responses, how these immune effectors are modulated by different biotic and abiotic factors, and on the mechanisms of immune evasion targeting specific immune effectors. The current and future design of control strategies for myxozoan diseases is based on understanding this myxozoan-fish interaction, and immune-based strategies such as improvement of innate and specific factors through diets and additives, host genetic selection, passive immunization and vaccination, are starting to be considered.

scholarly journals Combinations of Single Chain Variable Fragments From HIV Broadly Neutralizing Antibodies Demonstrate High Potency and Breadth

2021 ◽  
Vol 12 ◽  
Author(s):  
Rebecca T. van Dorsten ◽  
Kshitij Wagh ◽  
Penny L. Moore ◽  
Lynn Morris
Keyword(s):  
Neutralizing Antibodies ◽  
Passive Immunization ◽  
Neutralization Assay ◽  
Single Chain ◽  
Broadly Neutralizing Antibodies ◽  
High Potency ◽  
Hill Model ◽  
Mucosal Tissues ◽  
Action Model ◽  
Single Chain Variable Fragments

Broadly neutralizing antibodies (bNAbs) are currently being assessed in clinical trials for their ability to prevent HIV infection. Single chain variable fragments (scFv) of bNAbs have advantages over full antibodies as their smaller size permits improved diffusion into mucosal tissues and facilitates vector-driven gene expression. We have previously shown that scFv of bNAbs individually retain significant breadth and potency. Here we tested combinations of five scFv derived from bNAbs CAP256-VRC26.25 (V2-apex), PGT121 (N332-supersite), 3BNC117 (CD4bs), 8ANC195 (gp120-gp41 interface) and 10E8v4 (MPER). Either two or three scFv were combined in equimolar amounts and tested in the TZM-bl neutralization assay against a multiclade panel of 17 viruses. Experimental IC50 and IC80 data were compared to predicted neutralization titers based on single scFv titers using the Loewe additive and the Bliss-Hill model. Like full-sized antibodies, combinations of scFv showed significantly improved potency and breadth compared to single scFv. Combinations of two or three scFv generally followed an independent action model for breadth and potency with no significant synergy or antagonism observed overall although some exceptions were noted. The Loewe model underestimated potency for some dual and triple combinations while the Bliss-Hill model was better at predicting IC80 titers of triple combinations. Given this, we used the Bliss-Hill model to predict the coverage of scFv against a 45-virus panel at concentrations that correlated with protection in the AMP trials. Using IC80 titers and concentrations of 1μg/mL, there was 93% coverage for one dual scFv combination (3BNC117+10E8v4), and 96% coverage for two of the triple combinations (CAP256.25+3BNC117+10E8v4 and PGT121+3BNC117+10E8v4). Combinations of scFv, therefore, show significantly improved breadth and potency over individual scFv and given their size advantage, have potential for use in passive immunization.

scholarly journals Sensitive SYBR Green—Real Time PCR for the Detection and Quantitation of Avian Rotavirus A

2018 ◽  
Vol 6 (1) ◽  
pp. 2 ◽  
Author(s):  
David De la Torre ◽  
Claudete Astolfi-Ferreira ◽  
Ruy Chacon ◽  
Antonio Piantino Ferreira
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Sybr Green ◽  
Molecular Techniques ◽  
Economic Losses ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Rotavirus A ◽  
Avian Nephritis Virus ◽  
Polymerase Chain

Avian rotavirus A (ARtV-A) is a virus that affects young birds, causing acute diarrhea and economic losses in the poultry industry worldwide. The techniques used for the diagnosis of ARtV-A include electron microscopy, isolation in cell culture, and serology, as well as molecular techniques, such as the reverse transcription-polymerase chain reaction (RT-PCR). The objective of this work was to standardize a real-time RT-polymerase chain reaction (RT-qPCR) using SYBR Green chemistry for the rapid detection and quantification of ARtV-A from bird tissues and materials fixed on FTA cards on the basis of the nucleotide sequence of segment 6 (S6), which codes for the structural VP6 protein of ARtV-A. The results show the efficient amplification of the proposed target, with a limit of detection (LoD) of one copy gene (CG) per microliter of cDNA and a limit of quantification (LoQ) of 10 CGs per microliter. The efficiency of the primers was determined to be 95.66% using a standard curve, with an R2 value of 0.999 and a slope of −3.43. The specificity was determined using samples coinfected with ARtV-A, the chicken parvovirus, the chicken astrovirus, and the avian nephritis virus as positive controls and commercially available vaccines of the infectious bronchitis virus, infectious bursa disease virus, avian reovirus and healthy organs as negative controls. This technique, which lacks nonspecific PCR products and dimers, demonstrated greater sensitivity and specificity than conventional RT-PCR, and it reduced the analysis time by more than 50%.

scholarly journals 1089. Analytical Performance of an Ultrasensitive Immunoassay for Detection of Clostridium difficile Toxins in Stool

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S326-S326
Author(s):  
Amelita Bartolome ◽  
Anna Almazan ◽  
Salina Abusali ◽  
Stanley Tam ◽  
Eric Lee ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Room Temperature ◽  
High Sensitivity ◽  
Turnaround Time ◽  
Neutralization Assay ◽  
Cross Reactivity ◽  
Analytical Performance ◽  
Freeze Thaw ◽  
Gastrointestinal Pathogens ◽  
Sensitivity Specificity

Abstract Background Clostridium difficile infection (CDI) is the main cause for nosocomial diarrhea. Currently available assays for the diagnosis of CDI show deficits in sensitivity, specificity, and/or turnaround time. The Singulex Clarity® C. diff toxins A/B assay, in development for the Singulex Clarity® system, was designed to provide an accurate and automated detection of C. difficile toxins A (TcdA) and B (TcdB) in stool. Here, the analytical performance of the assay is reported. Methods Limits of detection (LoD) for TcdA and TcdB in stool and buffer was determined, and a preliminary cutoff, as compared with cell cytotoxicity neutralization assay (CCNA), was established. Analytical reactivity against 38 toxigenic and nontoxigenic C. difficile strains of eight different toxinotypes was determined. Cross-reactivity against 53 other gastrointestinal pathogens and potential interference by 11 endogenous and exogenous substances were determined. Reproducibility was tested with triplicate samples (n = 85), and stability was evaluated in samples stored at room temperature, refrigerated, and frozen conditions, and subjected to three freeze-thaw cycles. Results The LoDs for TcdA and TcdB were 0.8 and 0.3 pg/mL in buffer, and 2.0 and 0.7 pg/mL in stool, respectively. Using a preliminary cutoff, the assay demonstrated 96.3% sensitivity and 96.1% specificity compared with CCNA. The Singulex Clarity® C. diff toxins A/B assay detected toxins from all tested strains and toxinotypes. No cross-reactivity or interference were detected. The repeatability was 99%, and samples for C. difficile toxin testing were stable up to 8 hours in room temperature, 1 week in 2–8°C, 6 months in −70°C, and up to three freeze–thaw cycles. Conclusion The Singulex Clarity C. diff toxins A/B assay (in development) can detect TcdA and TcdB at very low concentrations and it has high sensitivity and specificity compared with CCNA. The assay demonstrates reactivity to common C. difficile strains, does not show cross-reactivity to common gastrointestinal pathogens, is robust against common interferents, allows for toxin detection in both fresh and frozen stool samples and up to three freeze–thaw cycles, and provides results with high reproducibility. Disclosures A. Bartolome, Singulex, Inc.: Employee, Salary. A. Almazan, Singulex, Inc.: Employee, Salary. S. Abusali, Singulex, Inc.: Employee, Salary. S. Tam, Singulex, Inc.: Employee, Salary. E. Lee, Singulex, Inc.: Employee, Salary. A. Changavi, Singulex, Inc.: Employee, Salary. W. Trinh, Singulex, Inc.: Employee, Salary. K. Chau, Singulex, Inc.: Employee, Salary. J. Estis, Singulex, Inc.: Employee, Salary. B. Noland, Singulex, Inc.: Employee, Salary. J. Bishop, Singulex, Inc.: Employee, Salary.

scholarly journals Neutralization Assay for Zika and Dengue Viruses by Use of Real-Time-PCR-Based Endpoint Assessment

2017 ◽  
Vol 55 (10) ◽  
pp. 3104-3112 ◽  
Author(s):  
Heather L. Wilson ◽  
Thomas Tran ◽  
Julian Druce ◽  
Myrielle Dupont-Rouzeyrol ◽  
Michael Catton
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
High Sensitivity ◽  
Neutralization Assay ◽  
Cross Reactivity ◽  
Health Concern ◽  
Confirmatory Test ◽  
Virus Neutralization Assay ◽  
History Of ◽  
Infective Complications

ABSTRACTThe global spread and infective complications of Zika virus (ZKV) and dengue virus (DENV) have made them flaviviruses of public health concern. Serological diagnosis can be challenging due to antibody cross-reactivity, particularly in secondary flavivirus infections or when there is a history of flavivirus vaccination. The virus neutralization assay is considered to be the most specific assay for measurement of anti-flavivirus antibodies. This study describes an assay where the neutralization endpoint is measured by real-time PCR, providing results within 72 h. It demonstrated 100% sensitivity (24/24 ZKV and 15/15 DENV) and 100% specificity (11/11 specimens) when testing well-characterized sera. In addition, the assay was able to determine the correct DENV serotype in 91.7% of cases. The high sensitivity and specificity of the real-time PCR neutralization assay makes it suitable to use as a confirmatory test for sera that are reactive in commercial IgM/IgG enzyme immunoassays. Results are objective and the PCR-based measurement of the neutralization endpoint lends itself to automation so that throughput may be increased in times of high demand.

Disruption of PF4/Hep Multimolecular Complex Assembly Using a Minimally Anticoagulant Heparin (ODSH)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 723-723
Author(s):  
Manali Joglekar ◽  
Pedro Quintana ◽  
Stephen Marcus ◽  
Jian Liu ◽  
Gowthami M. Arepally
Keyword(s):  
Complex Formation ◽  
Electrostatic Interactions ◽  
Conflicts Of Interest ◽  
Anticoagulant Activity ◽  
Neutralization Assay ◽  
Cross Reactivity ◽  
Antibody Binding ◽  
Fixed Amount ◽  
Platelet Factor ◽  
Molar Ratios

Abstract Abstract 723 Recent studies indicate that multimolecular complexes of platelet factor 4 (PF4) and heparin (H) are central to the pathogenesis of Heparin-Induced Thrombocytopenia (HIT). PF4/H multimolecular complexes are recognized preferentially by HIT antibodies (Rauova, Blood 2005) and are potently immunizing in a murine immunization model (Suvarna, Blood 2005). Because PF4/H multimolecular complexes assemble through non-specific electrostatic interactions, we hypothesized that disruption of PF4/H charge-dependent interactions could reduce immune mediated complications. To test this hypothesis, we employed a minimally anticoagulant compound (2-O, 3-O desulfated heparin, or ODSH, ParinGenix, Inc.) and characterized the charge-dependent interactions of murine PF4 (mPF4), ODSH and unfractionated heparin (UFH). In chromogenic assays of thrombin (IIa) generation, UFH was >80-fold more potent than ODSH in inactivating heparin (IC50 of residual IIa generation for UFH=3.1 nM v. ODSH= 259 nM, (Figure 1A). However, when equimolar amounts of UFH or ODSH (1.7 mM) were tested in a PF4 neutralization assay (Saggin, Thrombosis and Haemostasis 1992), the amount of mPF4 required to neutralize 50% of the anticoagulant activity of ODSH (IC50) was 25μg/mL, as compared to 73μg/mL for UFH (~3-fold difference), indicating that charge-dependent interactions, but not anticoagulant activity, were preserved between PF4 and ODSH (Figure 1B). When ODSH was added at 2.5, 5 or 10 fold molar excess to a fixed amount of UFH (6nM) in the PF4 neutralization assay, a proportionate increase in the amount of PF4 was needed to neutralize UFH, indicating that ODSH promotes the anticoagulant effect of UFH through preferential binding of PF4. To further characterize the biophysical interactions of PF4, ODSH and UFH, we used spectrophotometry and zeta potential to study the multimolecular complex formation (Suvarna, Blood 2007). We noted that mPF4 and ODSH formed multimolecular complexes at molar ratios of 2:1, whereas mPF4 and UFH complexes occurred at molar ratios of 1:1. When increasing concentrations of ODSH were added to pre-formed PF4/H multimolecular complexes, we noted a decrease in absorbance with increasing amounts of ODSH, indicating disruption of PF4/H multimolecular complexes (Figure 1C). However, when increasing amounts of UFH was added to preformed PF4/ODSH multimolecular complexes, a plateau in signal was noted, suggesting a higher affinity of ODSH for PF4. In PF4/H immunoassays, incubation of ODSH (1μg/mL) with HIT antibodies was effective in reducing antibody binding by >50% as compared to wells without ODSH. HIT antibodies did not recognize hPF4 (10mg/mL) in complex with ODSH (0.4-3.2 mg/mL), indicating minimal cross-reactivity of HIT antibodies with PF4/ODSH complexes (Figure 1D). In summary, we show that ODSH, a minimally anticoagulant heparin, can disrupt PF4/H multimolecular complex formation through charge dependent interactions and interfere with HIT antibody binding. These studies suggest that manipulation of PF4:H charge interactions can be a potential therapeutic strategy in the management of HIT. Disclosures: No relevant conflicts of interest to declare.

Epidemiological survey of classical swine fever in Tibetan pigs in Nyingchi, Tibet China

2016 ◽  
Author(s):  
Jia-Kui Li ◽  
Hui Zhang ◽  
Peng Shang ◽  
Yangzom Chamba
Keyword(s):  
Classical Swine Fever ◽  
Epidemiological Survey ◽  
Economic Losses ◽  
Serum Samples ◽  
Immune Effect ◽  
Tibetan Pigs ◽  
Significant Difference ◽  
Long Time ◽  
The Government

Classical swine fever (CSF) is a major hazardous disease to the pigs and as a dangerous epidemic; it causes a serious economic losses to the pig industry. Though, a national compulsory immunization of CSF vaccines had been carried out for a long time, scarce information can be got about the immune effect of CSF in Tibetan pigs. The present study was to investigate the seroprevalence of CSF in Tibetan pigs in Nyingchi area of Tibet, China. A total 454 samples were collected from November 2014 to January 2015 and were investigated through enzyme-linked immuno sorbent assay (ELISA). The results showed that 241 (53.1%, 95% CI 48.4-57.8) pigs were found to be positive for CSF with the further distribution of 53.3% (95% CI 46.8–59.6), 49.5% (95% CI 42.2-56.8) and 93.8% (95% CI 69.8-99.8) in Tibetan counties of Nyingchi, Mainling and Gongbo'gyamda, respectively. There was no significant difference in male (52.8%, 95% CI 46.4-59.1) and female pigs (50.0%, 95% CI 42.6-57.4). Though, 53.1% of the serum samples were tested out positive to CSF, only the seroprevalence of CSF in Tibetan pigs in Gongbo'gyamda were higher than 70% which was ruled by the government. The low seroprevalence of CSF in Tibetan pigs should arise a serious concern and effective methods should be taken, in order to prevent CSF infection effectively.

Epidemiological analysis of porcine rotavirus A genotypes in Germany

2018 ◽  
Vol 214 ◽  
pp. 93-98 ◽  
Author(s):  
Oliver Wenske ◽  
Antje Rückner ◽  
Daniel Piehler ◽  
Bernd-Andreas Schwarz ◽  
Thomas W. Vahlenkamp
Keyword(s):  
Epidemiological Analysis ◽  
Rotavirus A ◽  
Porcine Rotavirus
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