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Operational taxonomic unit saturation profiles at 99% sequence similarity level, for the. Antarctic samples collected. Hangar Cove (HC), Islands (I), Rothera Point (RP) and South Cove (SC), where 1-3 represent each sample replicate.
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Foot-and-mouth disease virus (FMDV) is one of the most devastating viral pathogens of cloven-hoofed animals. The detection of antibodies (Ab) against FMDV structural proteins (SP) using virus neutralization test (VNT) and liquid-phase blocking ELISA (LPBE) is the standard procedure in use for monitoring seroconversion in animals post vaccination, t...
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... Echinodermata, Cnidaria, Gastrotricha and Bryozoa were grouped as BECGB with a total of 9 OTUs where 11% of which had 100% identity matches to previously annotated sequence data. Sampling saturation profiles showed that the sequencing effort was not sufficient to determine the full extent of the diversity for any of the four sampled sites (Fig. 2). The slope of the OTU rarefaction curves did not approach saturation at 97% cut-off for all the meiobenthic phyla and more specifically for the nematodes, arthropods and even for the platyhelminthes which comprised a low abundance phylum where rarefaction curves tend to converge and reach an asymptote 28 (Supplementary Figure S1) and ...
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... (Kruskal-Wallis, p = 0.494), OTUs and the annelids with ca. 3-9 OTUS (Kruskal-Wallis, p = 0.110), found in the Antarctic meiobenthic samples (Supplementary Figure S2). In fact, the majority of the samples showed that 90-100% of the OTUs were shared between sites, with the exception of one of the triplicates of the Islands sample that had approximately 30% of unique OTUs (Fig. 3). ...
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... studies in the Southern Ocean 1, 2 have described a greater number of species than presented in this data set here (for example 524 nematode species compared with our estimate of 140 OTUs). However the fact that we identified such a number of OTUs in shallow waters at four sampling sites, some of which are geographically close (rather than the whole of the Southern Ocean for the 524 species 2 ) (Supplementary Figure S2) validates the conclusion that there is still much to discover, especially in the sediments. ...
Citations
... Foot-and-mouth disease virus (FMDV) is a transboundary animal disease that contributes to the emergence of diverse viral strains across different continents, posing significant challenges for control [30][31][32][33]. In Egypt, the virus is characterized by its high genetic variability, which complicates vaccine development and necessitates continuous surveillance [14]. ...
Foot-and-mouth disease virus (FMDV) remains a major threat to livestock in Egypt, with ongoing outbreaks involving serotypes A, O, and SAT2. This study aimed to improve the understanding of these circulating FMDV strains to improve control measures. Between 2022 and 2023, 134 cattle samples from across Egypt were analyzed, revealing a 67.9% positivity rate for Pan FMDV. Of these positive samples, 64 were identified as serotype A and 27 as serotype O. Genetic analysis indicated that serotype O strains clustered within the EA-3 topotype, suggesting endemic persistence and potential vaccine evasion, while serotype A strains were associated with the African topotype and linked to regions such as Ethiopia, Kenya, and Sudan. Notable amino acid mutations in the VP1 protein of both serotypes highlighted potential challenges to vaccine effectiveness. These findings underscore the need for enhanced surveillance, timely vaccine updates, and regional cooperation to effectively manage FMD outbreaks in Egypt and neighboring countries.
... Chemical treatments are generally effective in combatting RKN, but their detrimental effects on the environment and human health, and instances of resistance developing among nematodes, have caused global restrictions on nematicidal chemicals. Although crop rotation is a common method of controlling RKN, their broad host range minimizes the efficacy of this approach as a nematode management method (Salem et al. 2019a). ...
Biocontrol microorganisms are important tools for the control of root knot nematodes ( Meloidogyne spp.). Endophytic fungi have shown great potential as biocontrol agents in such applications. We here isolated an endophytic fungus from tomato root galls infected with M. incognita and identified the isolate as Acremonium sclerotigenum based on morphology and the internal transcribed spacer sequence. The biocontrol potential of this fungus was evaluated both in vitro and in vivo. Specifically, in vitro analyses were conducted to determine the potential of A. sclerotigenum to increase Meloidogyne incognita juvenile (J2 stage) mortality and decrease M. incognita egg hatching rates. The results revealed that A. sclerotigenum culture filtrates caused high J2 mortality rates (up to 95.5%) and significantly inhibited egg hatching (by up to ~ 43%). Furthermore, eggs treated with the culture filtrate were disaggregated and could not develop into nematodes. An in vivo experiment showed that treatment of tomato plants with A. sclerotigenum suppressed root knot nematode populations and significantly reduced the galling index. Both A. sclerotigenum treatment and exposure to the nematicide abamectin had good control effects, with efficacy rates of 55.43% and 70.58%, respectively. In summary, the endophytic fungus A. sclerotigenum here showed excellent potential for biocontrol of M. incognita . Further studies should be conducted to identify the nematicidal compounds produced by this fungus and to establish the molecular mechanism of action associated with the observed biocontrol effects.
... Moreover, trade movement affects the spread of the virus because there are no limitations for animal trading among the governorates, which contributes to the emergence of different viral epicenters and devastating outbreaks. Further, the virus is a transboundary animal disease, affecting the emergence of different types of viruses of intercontinental origin [1,34]. In Egypt, three serotypes were isolated and identified (A, O, and SAT2). ...
Background and Aim: Foot-and-mouth disease (FMD) virus causes continuous outbreaks, leading to serious economic consequences that affect animal productivity and restrict trade movement. The potential influence of the disease was due to the emergence of new strains or re-emergence of local strains with major antigenic variations due to genetic mutations. This study aims to evaluate circulating virus in samples collected from infected animals during an outbreak using antigenic characterization and identify whether there is an emergence of a new strain or mutation.
Materials and Methods: Reverse-transcription polymerase chain reaction (RT-PCR) was used to screen 86 samples. Viral protein 1 (VP1) codon sequencing was performed. The virus was isolated from the samples inoculated on the baby-hamster kidney cell line and Enzyme-linked immunosorbent assay was performed for serotyping and antigen detection.
Results: Based on the RT-PCR screening results, 10 positive samples were selected for sequencing. The sequences belonged to the FMD serotype A African topotype originating from the ancestor prototype Sudan/77, with which it shared 98.48% ± 1.2% similarity. The divergence with local isolates from 2020 was 9.3%. In addition, the sequences were 96.84% ± 1.01% and 95.84% ± 0.79% related to Egyptian-Damietta type 2016 and Sudanese-2018, respectively. Divergence with vaccinal strains ranged from 10% to 17%. Amino acid sequence analysis revealed that the isolates had variation in the most prominent antigenic regions (residues 35–75) and the immunogenic determinants of the G-H loop of VP1 (residues 100–146 and 161–175).
Conclusion: The current isolates should be included in the locally produced vaccine to provide broader immunogenic coverage against serotype A African topotypes.
... The coding sequence of 6-Histidine residues and an enterokinase recognition sequence were inserted at the 3´ end of N and RBD coding sequences. The synthesized sequences were cloned into the BssHII/ PstI sites of the pFastBac cloning vector and expression was derived by the Ppol promoter (Salem et al. 2019b;Elmenofy et al. 2020;Sheikhzadeh et al. 2020). ...
Serological assays for SARS-CoV-2 are being utilized at an exponential rate for surveillance programs. This enterprise was designed to develop and validate a qualitative immunochromatographic test, via the Lateral Flow Assay (LFA), for detection of immunoglobulins M and G (IgM and IgG) against both nucleocapsid (N) and the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. Both targeted proteins were cloned and expressed in baculovirus expression system utilizing insect cells Sf9. The recombinant RBD and N proteins were purified and conjugated with gold nanoparticles (AuNPs) to set up the coating antigens pad. Both anti-human IgG and IgM were dispensed on nitrocellulose membrane to capture human antibodies in serum samples. A home-made dispensing system was developed to draw identical test and control lines. The validity of the developed LFA was verified by testing serum samples from 103 convalescent COVID-19 patients who were PCR positive for SARS-CoV-2 along with 28 control serum samples. The developed strips showed distinctive bands for IgM and IgG of both proteins (RBD and N) in positive samples. The sensitivity of RBD-based LFA was 70.9% and 39.8% for IgG and IgM, respectively, with a specificity of 100% for both. The N-based LFA exhibited a sensitivity of 73.8% and 35.9% for IgG and IgM, respectively, while its specificity was 75% and 100% for IgG and IgM, respectively. Our developed LFA could afford a tool for surveillance programs in low-resource countries. Moreover, it might be functional for rapid and inexpensive monitoring of the anti-SARS-CoV-2 antibodies in the sera of vaccinated individuals.
... Later on, polyclonal antibodies were replaced by MAbs as the tracer and capture for large-scale serology [18], and VLPs replaced inactivated viruses, whose manufacture was limited to BSL3 laboratories [18,19]. A recombinant VP2 subunit protein from the Egyptian SAT2 isolate and a P1 capsid polyprotein of serotype O were also chosen as the diagnostic antigens [20,21]. The rP1-SPCE system paired guinea pig anti-serum as the tracer and demonstrated excellent agreement with in-house liquid-phase blocking ELISA [21]. ...
The serum neutralization (SN) test has been regarded as the “gold standard” for seroconversion following foot-and-mouth disease virus (FMDV) vaccination, although a high-level biosafety laboratory is necessary. ELISA is one alternative, and its format is constantly being improved. For instance, standard polyclonal antisera have been replaced by monoclonal antibodies (MAbs) for catching and detecting antibodies, and inactive viruses have been replaced by virus-like particles (VLPs). To the best of current knowledge, however, no researchers have evaluated the performances of different MAbs as tracers. In previous studies, we successfully identified site 1 and site 2 MAbs Q10E and P11A. In this study, following the established screening platform, the VLPs of putative escape mutants from sites 1 to 5 were expressed and used to demonstrate that S11B is a site 3 MAb. Additionally, the vulnerability of VLPs prompted us to assess another diagnostic antigen: unprocessed polyprotein P1. Therefore, we established and evaluated the performance of blocking ELISA (bELISA) systems based on VLPs and P1, pairing them with Q10E, P11A, S11B, and the non-neutralizing TSG MAb as tracers. The results indicated that the VLP paired with S11B demonstrated the highest correlation with the SN titers (R2 = 0.8071, n = 63). Excluding weakly positive serum samples (SN = 16–32, n = 14), the sensitivity and specificity were 95.65% and 96.15% (kappa = 0.92), respectively. Additionally, the P1 pairing with Q10E also demonstrated a high correlation (R2 = 0.768). We also discovered that these four antibodies had steric effects on one another to varying degrees, despite recognizing distinct antigenic sites. This finding indicated that MAbs as tracers could not accurately detect specific antibodies, possibly because MAbs are bulky compared to a protomeric unit. However, our results still provide convincing support for the application of two pairs of bELISA systems: VLP:S11B-HRP and P1:Q10E-HRP.
... To assess the immunoreactivity of purified rRBD against SARS-CoV-2 antibodies, an indirect ELISA was exploited to test serum samples from infected and vaccinated human sera. As a positive control, inactivated SARS-CoV-2 was used as a coating antigen, and unrelated recombinant purified protein (FMDV 1B, Salem et al., 2019b) was used as a control negative coating antigen. Clear signals indicating the immunoreactivity of purified rRBD against SARS-CoV-2 antibodies, were appeared with serum samples either from convalescent or vaccinated sera (Fig. 4A). ...
As the second wave of COVID-19 launched, various variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have emerged with a dramatic global spread amongst millions of people causing unprecedented case fatalities and economic shut-downs. That initiated a necessity for developing specific diagnostics and therapeutics along with vaccines to control such a pandemic. This endeavor describes generation of murine derived recombinant single-chain variable fragments (scFvs) as a monoclonal antibody (MAb) platform targeting the receptor binding domain (RBD) of Spike protein of SARS-CoV-2. A specific synthesized RBD coding sequence was cloned and expressed in Baculovirus expression system. The recombinant RBD (rRBD) was ascertained to be at the proper encoding size of ∼ 600bp and expressed protein of the molecular weight of ∼ 21KDa. Purified rRBD was proved genuinely antigenic and immunogenic, exhibiting specific reactivity to anti-SARS-CoV-2 antibody in an indirect enzyme-linked immunosorbent assay (ELISA), and inducing strong seroconversion in immunized mice. The scFv phage display library against rRBD was successfully constructed, revealing ∼ 90% recombination frequency, and great enriching factor reaching 88% and 25% in polyclonal Ab-based and MAb-based ELISAs, respectively. Typically, three unique scFvs were generated, selected, purified and molecularly identified. That was manifested by their: accurate structure, close relation to the mouse immunoglobulin (Ig) superfamily, right anchored six complementarily-determining regions (CDRs) as three within variable heavy (vH) and variable light (vL) regions each, and proper configuration of the three-dimensional (3D) structure. Besides, their expression downstream in a non-suppressive amber codon of E. coli strain SS32 created a distinct protein band at an apparent molecular weight of ∼ 27KDa. Moreover, the purified scFvs showed authentic immunoreactivity and specificity to both rRBD and SARS-CoV-2 in western blot and ELISA. Accordingly, these developed scFvs platform might be a functional candidate for research, inexpensive diagnostics and therapeutics, mitigating spread of COVID-19.
... It is necessary to produce Abs (polyclonal and/or monoclonal) for FMDV SP ELISA tests against each of newly emerged, genotypically crucial, exotic strains of FMDV just like the effort of a new primer-probe design for the molecular differentiation of novel FMDV with PCR. However, the period for the production of polyclonal Abs for FMDV serology takes a long time and laborious work (Parida 2009;Lavoria et al. 2012;Sala et al. 2018;Salem et al. 2019). ...
... Besides, cross-reactions were detected among the FMDV serotypes in ELISA (Figure 3). Cross-reactions between the FMDV Abs is a possible problem due to the common sharing epitopes between the FMDV serotypes strains (Parida 2009;Bari et al. 2014;Ascoli and Aggeler 2018;Salem et al. 2019). Here, additional blocking steps were performed to alleviate cross-reactions. ...
Antibodies(Abs) have been always a major place in diagnostic laboratories. Many diagnostic techniques like Enzyme-Linked Immuno Sorbent Assays (ELISA), immunofluorescence, Ab-microarray platforms, immunoblots, X-ray crystallography require the Abs. ELISA is the main test that used Abs for Foot and Mouth Disease (FMDV) serology. It needs polyclonal or monoclonal Abs to detect FMDV antigen or Abs. For this purpose, solid-phase competitive ELISA (SPCE) or liquid phase blocking ELISA (LPBE) and non-structural protein (NSP) ELISA are used. SPCE and LPBE have mainly used FMDV structural protein (SP)-Ab detection. In this study, it was aimed to produce a polyclonal Ab against FMDV ANep84 (Genotype VII) and OTUR07 (OPanAsia II), ATUR11 (A Iran05) strains for LPBE, FMDV SP-antibody detection. For this purpose, 4 guinea pigs and 6 rabbits were used for each serotype of FMDV. After producing Abs, checkerboard ELISA titration was performed to determine the optimal test dilution of Abs. Backgrounds, cross-reactions against three strains of FMDV were also checked. In conclusion, polyclonal Abs were produced against FMDV ANep84 (Genotype VII) and O Tur07 (O Pan Asia II), ATUR11 (A Iran05) strains, and standardized for LPBE test.
... Purified r3AB protein (50 ng/ well) was coated in ELISA plates. As a recombinant VP2 protein developed in our lab [27] has been effectively used earlier for anti-FMDV Ab detection, it was used herein by the same concentration in parallel with r3AB as a coating antigen. Sera from naïve calves infected with FMDV O, A, and SAT 2 serotypes, have been separately mixed in PBS containing 3% BSA, plated at 100 μL per well in duplicate (four times) and incubated for 2 h at 37 °C. ...
During an ongoing outbreak of Foot-and-Mouth Disease Virus (FMDV), it is crucial to distinguish naturally infected from vaccinated seropositive animals. This would support clinical assessment and punctual vigilance. Assays based on 3ABC non-structural protein as an antigen are reliable for this intention. However, the insolubility and degradation of recombinant 3ABC during expression and purification are serious challenges. In this study, alternatively to expressing the recombinant 3ABC (r3ABC), we expressed the 3AB coding sequence (~672 bp) as a recombinant protein (r3AB) with a molecular mass of ~26 KDa. Analytical data from three-dimensional structure, hydrophilicity, and antigenic properties for 3ABC and 3AB exhibited the 3C protein as a hydrophobic, while 3AB as a hydrophilic and highly antigenic protein. The expressed r3AB was recovered as a completely soluble matter after merely native purification, unlike the full expressed r3ABC. Immunoreactivity of r3AB to anti-FMDV antibody in infected sera with different FMDV serotypes was confirmed by the western blot and indirect ELISA. Besides, the authentic antigenicity of purified r3AB was demonstrated through its ability to induce specific seroconversion in mice. Summarily, the removal of 3C: has influenced neither 3D structure nor antigenic properties of the purified r3AB, overcame insolubility and degradation of the r3ABC, and generated a potential superior antigen (r3AB) for herd screening of animals to any FMDV serotype.
... Anti-mouse-Alkaline Phosphatase Sigma-Aldrich (St. Louis, MO) was used as a secondary antibody. Nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3′-indolyphosphate p-toluidine (BCIP) were used as substrates for detection (Salem et al. 2018;Salem et al. 2019b;Elmenofy et al. 2020b). ...
The search for effective and bioactive antimicrobial molecules to encounter the medical need for new antibiotics is an encouraging area of research. Plant defensins are small cationic, cysteine-rich peptides with a stabilized tertiary structure by disulfide-bridges and characterized by a wide range of biological functions. The heterologous expression of Egyptian maize defensin (MzDef) in Escherichia coli and subsequent purification by glutathione affinity chromatography yielded 2 mg/L of recombinant defensin peptide. The glutathione-S-transferase (GST)-tagged MzDef of approximately 30 kDa in size (26 KDa GST + ~ 4 KDa MzDef peptide) was immunodetected with anti-GST antibodies. The GST-tag was successfully cleaved from the MzDef peptide by thrombin, and the removal was validated by the Tris-Tricine gel electrophoresis. The MzDef induced strong growth inhibition of Rhizoctonia solani, Fusarium verticillioides, and Aspergillus niger by 94.23%, 93.34%, and 86.25%, respectively, whereas relatively weak growth inhibitory activity of 35.42% against Fusarium solani was recorded. Moreover, strong antibacterial activities were demonstrated against E. coli and Bacillus cereus and the moderate activities against Salmonella enterica and Staphylococcus aureus at all tested concentrations (0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 µM). Furthermore, the in vitro MTT assay exhibited promising anticancer activity against all tested cell lines (hepatocellular carcinoma, mammary gland breast cancer, and colorectal carci-noma colon cancer) with IC 50 values ranging from 14.85 to 29.85 µg/mL. These results suggest that the recombinant peptide MzDef may serve as a potential alternative antimicrobial and anticancer agent to be used in medicinal application.
... Several previous studies (37,38) proposed the N-terminal end of VP2 to be inter-serotypically conserved, which we showed in our study by multiple bioinformatics analysis (protein variability analysis, mutational study, and identification of B cell epitopes). Salem et al. designed and developed an indirect ELISA using the N-terminal conserved regions of VP2, which provided higher sensitivity than VNT and LPBE (37). ...
... Several previous studies (37,38) proposed the N-terminal end of VP2 to be inter-serotypically conserved, which we showed in our study by multiple bioinformatics analysis (protein variability analysis, mutational study, and identification of B cell epitopes). Salem et al. designed and developed an indirect ELISA using the N-terminal conserved regions of VP2, which provided higher sensitivity than VNT and LPBE (37). However, that study used one Egyptian SAT2 isolate (gb|AAZ83686) as model for the development of a VP2-based type-independent indirect ELISA approach and used only the O, A, and SAT2 antisera to evaluate the sensitivity and the specificity of their kit. ...
... These findings suggest that the designed protein can be a suitable candidate for the development of a serotype-independent diagnostic tool using ELISA-based approaches. Similar approaches were developed by Salem et al. using one Egyptian SAT2 isolate of FMDV (gb|AAZ83686), which showed the expected level of sensitivity in FMD detection (37). No such strategy is available in the Asian region until now. ...
Foot-and-mouth disease (FMD) is an economically devastating disease of the livestock worldwide and caused by the FMD virus (FMDV), which has seven immunologically distinct serotypes (O, A, Asia1, C, and SAT1–SAT3). Studies suggest that VP2 is relatively conserved among three surface-exposed capsid proteins (VP1–VP3) of FMDV, but the level of conservation has not yet been reported. Here we analyzed the comparative evolutionary divergence of VP2 and VP1 to determine the level of conservation in VP2 at different hierarchical levels of three FMDV serotypes (O, A, and Asia1) currently circulating in Asia through an in-depth computational analysis of 14 compiled datasets and designed a consensus VP2 protein that can be used for the development of a serotype-independent FMDV detection tool. The phylogenetic analysis clearly represented a significant level of conservation in VP2 over VP1 at each subgroup level. The protein variability analysis and mutational study showed the presence of 67.4% invariant amino acids in VP2, with the N-terminal end being highly conserved. Nine inter-serotypically conserved fragments located on VP2 have been identified, among which four sites showed promising antigenicity value and surface exposure. The designed 130 amino acid long consensus VP2 protein possessed six surface-exposed B cell epitopes, which suggests the possible potentiality of the protein for the development of a serotype-independent FMDV detection tool in Asia. Conclusively, this is the first study to report the comparative evolutionary divergence between VP2 and VP1, along with proposing the possible potentiality of a designed protein candidate in serotype-independent FMDV detection.
