Search Results (21)

Background: The minimized pan-coronavirus (CoV) vaccine-1 developed by our laboratory contained pDNA sequences of feline coronavirus serotype-1 (FCoV1) and SARS-CoV2 (SCoV2) spike B-cell epitopes plus FCoV/SCoV2-conserved, CoV-specific polymerase cytotoxic T-lymphocyte (CTL) epitopes formulated in lipid nanoparticle (LNP). Only FCoV2 infects feline cell lines needed for developing native challenge inoculum that causes feline infectious peritonitis (FIP). Hence, Pilot Study 1 evaluated the therapeutic efficacy and safety of the pan-CoV vaccine-1 in feline immunodeficiency virus (FIV)-infected cats, with or without FCoV1 coinfection. Pilot Study 2 evaluated the cross-protective effect of pan-CoV vaccines in specific-pathogen-free (SPF) cats against intranasal challenge with FIP virus serotype 2 (FIPV2). Methods: In Study 1, we vaccinated two FIV-infected cats (one negative and another positive for FCoV1 coinfection) intramuscularly twice with CTL epitopes-LNP vaccine and later twice with pan-CoV vaccine-1. Controls included two unvaccinated FIV-infected cats with or without FCoV1 coinfection. Study 2 assessed the sequential vaccinations of three pan-CoV vaccines in four SPF cats. The first two vaccinations were with pan-CoV vaccine-2, followed by pan-CoV vaccine-3 (twice), and lastly with pan-CoV vaccine-1 (once). Three SPF controls included two cats immunized with LNP and one lacking any immunization. Pan-CoV vaccine-2 contained pDNAs with modified FCoV1/SCoV2 B-cell epitopes plus CTL epitopes in LNP. Pan-CoV vaccine-3 contained only pDNAs with FCoV1 B-cell epitopes plus CTL epitopes in LNP. Results: Study 1 demonstrated no adverse effect with 25 μg and 50 μg CTL epitopes-LNP vaccine and 50 μg pan-CoV vaccine-1. However, 100 μg pan-CoV vaccine-1 caused fever 24 h later, which was resolved by a single Meloxicam treatment. Both vaccinees developed cross-FCoV2 neutralizing antibodies (XNAbs), immunoblot binding antibodies (bAbs) to FCoV1 receptor-binding domain (RBD), and T-cell responses to FCoV1 RBD, whereas one vaccinee also developed bAbs to SCoV2 RBD. Study 2 demonstrated no adverse effects after each vaccination. Three vaccinees developed low-titer XNAbs and bAbs to FCoV2 spike-2 by the fourth vaccination. Upon challenge, all cats developed FCoV2 NAbs and bAbs to FCoV2 nucleocapsid and RBD. High vaccine-induced T-cell responses to FCoV1 RBD and T-cell mitogen responses declined with an increase in responses to FCoV2 RBD at three weeks post-challenge. Two of the three controls died from FIP, whereas one vaccinee, with the lowest vaccine-induced immunity, died from skin vasculitis lesions and detection of FIPV2 infection by semi-nested RT-snPCR in feces. Conclusions: In Pilot Study 1, the pan-CoV vaccine-LNP dose of 50 μg had no adverse effects, but adverse effects were observed at 100 μg dose. In Pilot Study 2, the FCoV1-based B-cell vaccine(s) induced low levels of XNAbs against FIPV2 and delayed challenge infection against high-dose FIPV2. The high-dose FIPV2 infections in the vaccinated and control cats started to clear, by single housing at 23–26 weeks post-challenge, whereas two cats in Pilot Study 1 cleared natural FCoV1 transmission by 26 weeks post-infection. Full article

Soybean oil has recently emerged as the most widely consumed genetically modified (GM) vegetable oil globally. DNA-based methods offer considerable advantages for monitoring GM-derived products; however, their efficacy strongly depends on the quality and quantity of extracted DNA. Owing to intensive processing, refined oils typically contain extremely low concentrations of severely fragmented DNA, making DNA extraction highly challenging. To address this issue, we introduce an innovative magnetic bead-based DNA extraction protocol specifically tailored to refined soybean oils. Optimal DNA adsorption was achieved using 300 nm carboxyl (-COOH)-modified magnetic beads under optimized conditions, including 1 M guanidine isothiocyanate (GITC) buffer at pH 6.0, combined with ethanol at a 1:1 ratio. Subsequently, we developed a cetyltrimethylammonium bromide (CTAB)-magnetic bead method in which DNA was efficiently transferred from the oil phase to the aqueous phase, concentrated via precipitation, resuspended in GITC buffer, and finally purified using magnetic beads. Comparative evaluations using nested polymerase chain reaction (PCR) and real-time PCR confirmed that this method significantly outperformed traditional CTAB-based methods (CTAB alone, CTAB-hexane) and two representative silica membrane-based extraction kits. Spike recovery experiments further demonstrated its superior efficacy, achieving a DNA recovery rate of 76.37%. The proposed protocol is simple, user-friendly, cost-effective, and highly efficient, markedly reducing reliance on large volumes of organic solvents (e.g., hexane and chloroform) and minimizing the required centrifugation steps. This novel method established an effective approach for DNA extraction from refined vegetable oils, facilitating the development of rapid and reliable GM detection. Full article

Cryptosporidiosis is a gastrointestinal disease affecting terrestrial and aquatic vertebrates worldwide. This study investigated molecularly and microscopically the prevalence and the diversity of Cryptosporidium spp. in goats across the Bouira communes, Algeria. A total of 559 fecal samples were collected from 70 farms, representing 16.6% of the regional goat population. Samples were analyzed using microscopy (modified Ziehl-Neelsen staining) and molecular methods (i.e., qPCR and nested PCR targeting the 18S rRNA gene, followed by sequencing). Microscopy detected Cryptosporidium in 6.1% of samples, while qPCR revealed a significantly higher prevalence of 13.6% (p < 0.00001), confirming the superior sensitivity of molecular diagnostics. Spatial analysis identified significant clustering (Moran’s I = 0.330, p = 0.0003), with communes-level prevalence ranging from 6.7% to 45.7%. Infection rates correlated positively with humidity and rainfall but negatively with temperature. Phylogenetic analysis confirmed Cryptosporidium xiaoi as the sole species circulating, showing 100% genetic similarity to global caprine isolates. Despite C. xiaoi’s host adaptation, a GenBank review highlighted six other zoonotic species infecting goats worldwide, underscoring potential cross-species transmission risks. The study emphasizes the need for PCR-based surveillance to assess true prevalence and zoonotic threats, while climatic findings support targeted interventions in high-risk areas. Full article

Rodents are recognized as reservoirs of a wide range of pathogens, including microsporidia. The presence of microsporidia in the environment of mainland Spain and its islands has become increasingly known, as the number of studies has multiplied over time. The present study was conducted to determine the occurrence and diversity of microsporidia in three rodent species (Mus musculus, Rattus norvegicus, and Rattus rattus) in the Canary Islands, Spain. Ninety-three fecal samples were obtained from wild rodents on La Gomera and Gran Canaria Islands. Each sample was tested using Weber’s modified trichrome staining and immunofluorescence antibody tests (IFATs) against the Encephalitozoon genus and Enterocytozoon bieneusi. The microscopy-positive samples were subsequently analyzed using a nested polymerase chain reaction (PCR) followed by Sanger sequencing. The staining technique showed 38.7% (36/93) positivity, whereas the IFATs for Encephalitozoon spp. and Ent. bieneusi revealed 3.2% (3/93) and 6.5% (6/93) positivity, respectively. Finally, the nested PCR and nucleotide sequence analysis confirmed a 9.7% (9/93) occurrence of Ent. bieneusi and 17.2% occurrence (16/93) of different undetermined microsporidia species, whereas no Encephalitozoon spp. were detected. Seven different Ent. bieneusi genotypes were detected as follows: three known (AAE1, D, and SBM1) and four novel (GRE1, GRE2, LGE1, and LGE2), all of which belonged to Group 1. The results demonstrate, for the first time, that microsporidia are present in the rodent populations of the Canary Islands. Further studies are needed to determine the impact of the presence of microsporidia in rodents on the zoonotic transmission of these parasites. Full article

Pinna nobilis, an ecologically significant and critically endangered bivalve endemic to the Mediterranean Sea, has been classified as “Critically Endangered” by IUCN due to habitat degradation, climate change, and mass mortality events caused by the protozoan parasite Haplosporidium pinnae. Effective conservation efforts require robust molecular tools for species identification and genetic monitoring, necessitating the development of optimized DNA extraction and amplification protocols for a non-invasive sampling protocol. In this study, we evaluated multiple DNA extraction methods—Chelex-100, the sodium chloride (NaCl) method, a modified CTAB protocol, and a commercial kit, NucleoSpin Tissue Kit—using minute shell fragments from both ethanol-preserved and air-dried (dead) samples. We optimized key parameters, including incubation times, temperatures, and sample preparation, to determine the most effective protocol for obtaining high-quality DNA suitable for downstream applications. Additionally, we assessed different PCR strategies, including nested and semi-nested approaches targeting the COI gene marker, to enhance species identification. To further refine the methodology, we evaluated novel specific primers for nested PCR, improving sensitivity and specificity in detecting P. nobilis DNA from minute and degraded samples. Our results provide an optimized, cost-effective, and time-efficient workflow for non-invasive molecular identification of P. nobilis, with broad implications for conservation genetics, biodiversity monitoring, and species recovery programs. Full article

Reliable detection of sunflower (Helianthus annuus) in edible and used cooking oil (UCO) is crucial for the sustainable production of food and biodiesel. In this study, a variety of sunflower oils (crude, cold pressed, extra virgin, refined, and UCO) were examined using different methods of DNA extraction and PCR amplification to develop an efficient technology for the identification of sunflower in oils. DNA extraction kits such as NucleoSpin Food, DNeasy mericon Food, and Olive Oil DNA Isolation as well as modified CTAB method were found to be able to isolate amplifiable genomic DNA from highly processed oils. Novel uniplex, double, and nested PCR systems targeting the sunflower-specific helianthinin gene were developed for efficient identification of sunflower. New sunflower DNA markers were revealed by uniplex PCRs. The combination of modified CTAB and nested PCR was demonstrated as a reliable, rapid, and cost-effective technology for detecting traces of sunflower in 700 μL of highly processed oil, including refined and used cooking oil. The study will contribute to both the food industry and the energy sector as developed methods can be used for oil authenticity testing in food and biodiesel production. Full article

(1) Background: The detection of methylated SEPT9 (mSEPT9) in plasma is a promising approach to non-invasive colorectal cancer (CRC) screening. Traditional approaches have limitations in sensitivity and cost-effectiveness, particularly in resource-limited settings. (2) Methods: We developed a semi-nested realtime PCR assay utilizing extendable blocking probes (ExBP) to enhance the detection of low-level mSEPT9 based on DNA melting. This assay allows for the discrimination of mSEPT9 in the presence of high concentrations of non-methylated SEPT9 (up to 100,000 times higher). (3) Results: The assay demonstrated a sensitivity of 73.91% and specificity of 80%, showcasing its ability to detect very low levels of methylated DNA effectively. The innovative use of ExBP without costly modified probes simplifies the assay setup and reduces the overall costs, enhancing its applicability in diverse clinical settings. (4) Conclusions: This novel assay significantly improves the detection of mSEPT9, offering a potential advance in CRC screening and monitoring. Its cost-efficiency and high sensitivity make it particularly suitable for the early detection and management of CRC, especially in settings with limited resources. Future studies are encouraged to validate this assay in larger populations to establish its clinical benefits and practical utility. Full article

Background: Efforts on a global scale for combating malaria have achieved substantial progress over the past twenty years. Two Central American nations have accomplished their goal of eliminating malaria: El Salvador and Belize. Honduras has decreased the incidence of malaria and now reports fewer than 4000 malaria cases annually, aspiring to reach elimination by 2030. To accomplish this goal, it is essential to assess the existing strategies employed for malaria control and to address the task of incorporating novel intervention strategies to identify asymptomatic reservoirs. Methods: A survey for detecting asymptomatic cases was carried out in the community of Kaukira, in Gracias a Dios, Honduras, focusing on malaria transmission during 2023. Asymptomatic community members were recruited as participants, malaria screening was performed through a rapid diagnostic test in situ, and a blood sample was collected on filter paper. Highly sensitive molecular assays based on photo-induced electron transfer PCR (PET-PCR) were performed to detect the two species of Plasmodium circulating in Honduras: Plasmodium vivax and Plasmodium falciparum. In addition, the identification of the parasite species was verified by amplifying three genetic markers (Pvmsp3α, Pvmsp3ß, and Pfmsp1). Results: A total of 138 participants were recruited, mostly adult women. All individuals tested negative on the rapid diagnostic test. Positive results for malaria were detected by PET-PCR in 17 samples (12.3%). Most samples (12 out of 17) were amplified with a Ct value between 37 and 42, indicating very low parasitemias. Out of the 17 samples, 16 of them also showed amplification in the species assays. There were nine cases of P. falciparum infections and seven cases of P. vivax infections that were further confirmed by nested PCR (nPCR) of Pvmsp3 and Pfmsp1. Parasitemias ranged from 100 p/μL to less than 0.25 p/μL. One sample showed mixed infection. Conclusions: The existence of asymptomatic malaria reservoirs in Honduras can contribute to disease transmission and pose a challenge that may hinder elimination efforts, requiring public health authorities to modify surveillance strategies to identify the disease and treat this population accordingly. Full article

Leptospirosis is a zoonotic disease of significant concern for human and animal health, with domestic animals, including dogs, acting as reservoirs for human infection. Serology is widely used for leptospirosis diagnosis, even though the standard microscopic agglutination test (MAT) using a panel of serovars lacks specificity and can lead to detection limitations in certain regions. In this study, we aimed to develop an antibody detection tool for dogs using an indirect enzyme-linked immunosorbent assay (ELISA) with a set of local serovar isolates, including Paidjan, Dadas, and Mini, to enhance the accuracy of leptospirosis surveillance in our region. The specificity and sensitivity of various antigen preparations, namely leptospiral whole-cell protein (WCP), total membrane protein (TMP), and outer membrane protein (OMP), were assessed using sera from infected and non-infected dogs, as well as negative puppy sera. Leptospirosis diagnosis was supported using a genus-specific nested polymerase chain reaction test on all collected sera. Protein preparations were validated using SDS-PAGE and Western blotting analysis. In the results, the standard MAT failed to detect antibodies in any of the dogs confirmed as being infected using PCR and isolation, highlighting its limitations. In contrast, the OMP-based ELISAs using local isolates of Leptospira serovars gave positive results with sera from all infected dogs, and negative results with sera from all dogs from non-endemic areas. IgG titres of infected and unvaccinated dogs from endemically affected areas were significantly higher than those in non-endemic regions. Using the OMP-based IgG/ELISAs with the local serovar Dadas resulted in higher specificity and lower sensitivity than when using the WCP- and TMP-based IgG/ELISAs. Agreement analysis revealed fair and moderate concordance between OMP-based IgG/ELISAs and PCR results, whereas slight and fair agreement was observed between OMP-based ELISAs and the MAT. Overall, the modified OMP-based IgG/ELISAs, utilising relevant local serovar isolates from dogs, demonstrated improved accuracy in detecting leptospirosis in the study area, overcoming the limitations of the MAT. This study highlights the importance of identifying and incorporating these local circulating serovar isolates into serological techniques for leptospirosis diagnosis and surveillance. Full article

Hepatitis E virus (HEV) infection has been demonstrated in various animal species; those recognized as potential zoonotic reservoirs pose a considerable risk to public health. In Brazil, HEV-3 is the only genotype identified in humans and swine nationwide, in a colony-breeding cynomolgus monkey and, recently, in bovines and capybara. There is no information regarding HEV exposure in the equine population in Brazil. This study aimed to investigate anti-HEV antibodies and viral RNA in serum samples from horses slaughtered for meat export and those bred for sport/reproduction purposes. We used a commercially available ELISA kit modified to detect species-specific anti-HEV, using an anti-horse IgG-peroxidase conjugate and evaluating different cutoff formulas and assay precision. Serum samples (n = 257) were tested for anti-HEV IgG and HEV RNA by nested RT-PCR and RT-qPCR. The overall anti-HEV seroprevalence was 26.5% (68/257) without the detection of HEV RNA. Most municipalities (53.3%) and farms (58.8%) had positive horses. Animals slaughtered for human consumption had higher risk of HEV exposure (45.5%) than those bred for sports or reproduction (6.4%) (p < 0.0001). The statistical analysis revealed sex and breeding system as possible risk-associated factors. The first serological evidence of HEV circulation in Brazilian equines reinforces the need for the surveillance of HEV host expansion in a one-health approach. Full article

Cockatiels (Nymphicus hollandicus) are among the most commonly sold psittacines pets. The aim of this study was to evaluate the occurrence of Cryptosporidium spp. in domestic N. hollandicus and identify risk factors for this infection. We collected fecal samples from 100 domestic cockatiels in the city of Araçatuba, São Paulo, Brazil. Feces from birds of both genders and older than two months were collected. Owners were asked to complete a questionnaire to identify how they handle and care for their birds. Based on nested PCR targeting the 18S rRNA gene, the prevalence of Cryptosporidium spp. in the cockatiels sampled was 9.00%, 6.00% based on Malachite green staining, 5.00% based on modified Kinyoun straining, and 7.00% when the Malachite green was combined with Kinyoun. Applying multivariate logistic regression to test the association between Cryptosporidium proventriculi positivity and potential predictors showed that gastrointestinal alterations was a significant predictor (p < 0.01). Amplicons from five samples were sequenced successfully and showed 100% similarity with C. proventriculi. In summary, this study demonstrates the occurrence of C. proventriculi in captive cockatiels. Full article

The efficacy of the available genome-walking methods is restricted by low specificity, high background, or composite operations. We herein conceived bridging PCR, an efficient genome-walking approach. Three primers with random sequences, inner walker primer (IWP), bridging primer (BP), and outer walker primer (OWP), are involved in bridging PCR. The BP is fabricated by splicing OWP to the 5′-end of IWP’s 5′-part. A bridging PCR set is constituted by three rounds of amplification reactions, sequentially performed by IWP, BP plus OWP, and OWP, respectively pairing with three nested sequence-specific primers (SSP). A non-target product arising from IWP alone undergoes end-lengthening mediated by BP. This modified non-target product is a preferentially formed hairpin between the lengthened ends, instead of binding with shorter OWP. Meanwhile, a non-target product, triggered by SSP alone or SSP plus IWP, is removed by nested SSP. As a result, only the target DNA is accumulated. The efficacy of bridging PCR was validated by walking the gadA/R genes of Levilactobacillus brevis CD0817 and the hyg gene of rice. Full article

The recent surge in cutaneous leishmaniasis (CL) in Sri Lanka has rendered clinical diagnosis difficult; thus, laboratory confirmation is indispensable. A modified (two novel inner primers to detect CL caused by Leishmania donovani) nested Internal Transcribed Spacer-1 (ITS1) PCR-Restriction Fragment Length Polymorphism (RFLP) method was developed and tested. The sensitivity of the modified nested PCR was tested using serial dilutions (10³ to 10⁻²) of the DNA extract of a cultured L. donovani DD8 strain. Patients (n = 194) from Southern Sri Lanka were examined clinically, microscopically (Slit Skin Smear-SSS) and using the modified nested PCR. The modified nested PCR detected 2.55 fg of parasite DNA compared to ITS1 PCR (25 fg) and detected more cases than SSS (94.3% vs. 77.3%; p < 0.01). The RFLP pattern was L. donovani in all cases. The modified nested PCR performed well in clinically doubtful lesions (95% by PCR vs. 60% by SSS; p < 0.01), ulcerated nodules (91% vs. 71.8%; p < 0.01) and plaques (100% vs. 66.7%; p < 0.01). SSS demonstrated sensitivity (80.9%), specificity (81.8%), PPV (98.7%) and NPV (20.5%) against modified PCR. Low parasite loads and atypical lesions can be diagnosed by the proposed method with higher accuracy. Full article

Electro-Wetting-On-Dielectric (EWOD) based digital operations have demonstrated outstanding potential in actuating and manipulating liquid droplets. Here, we adapted the EWOD for extracting femtogram quantities of cell-free DNA (cf-DNA) from 1 μL of KSOM mouse embryo culture medium. Our group extracted the femtogram quantity of cf-DNA from 1 μL of mouse embryo culture medium in our previous work. Here, we initially explain a modification from our previous extraction protocol, which improves the extraction percentage to 36.74%. Though the modified extraction protocol improves the extraction percentage from our previously reported work, the quantity is still in the femtogram range. The cf-DNA in femtogram quantity is in subcritical/subthreshold concentration for any further analysis, such as sequencing. To the best of our knowledge, we need a minimum of picogram/nanogram DNA quantities for further analysis. We demonstrated a ground-breaking mechanism of this subcritical concentration of cf-DNA amplification to the nanogram range and performed DNA sequencing. Basic Local Alignment Search Tool (BLAST) is used as a sequence similarity search program to confirm the identity percentage between query and subject. More than 97% of nucleotide identities between query and subject sequences have been obtained from the sequencing result. Hence, we can use the methodology to amplify the subcritical concentration of extracted DNA for further analytics. Moreover, as we extract the cf-DNA from the embryo culture medium, the natural growth of the embryo has not been disrupted. This entire mechanism will pave a new path towards the lab-on-a-chip (LOC) concept. Full article

Glycogen storage disease type Ia (GSDIa) is an autosomal recessive disorder caused by glucose-6-phosphatase (G6PC) deficiency. GSDIa causes not only life-threatening hypoglycemia in infancy, but also hepatocellular adenoma as a long-term complication. Hepatocellular adenoma may undergo malignant transformation to hepatocellular carcinoma. New treatment approaches are keenly anticipated for the prevention of hepatic tumors. Gene replacement therapy (GRT) is a promising approach, although early treatment in infancy is essential for its safety and efficiency. Thus, GRT requires screening systems for early disease detection. In this study, we developed a screening system for GSDIa using dried blood spots (DBS) on filter paper, which can detect the most common causative mutation in the East-Asian population, c.648G>T in the G6PC gene. Our system consisted of nested PCR analysis with modified competitive oligonucleotide priming (mCOP)-PCR in the second round and melting curve analysis of the amplified products. Here, we tested 54 DBS samples from 50 c.648G (wild type) controls and four c.648T (mutant) patients. This system, using DBS samples, specifically amplified and clearly detected wild-type and mutant alleles from controls and patients, respectively. In conclusion, our system will be applicable to newborn screening for GSDIa in the real world. Full article