Search Results (51)

Kittens are born with some maternally derived antibodies (MDA) that, together with maternal gut microbiota, bring some form of early defense against pathogens. However, this protection declines and the rapid changes and challenges the kitten typically encounters can adversely affect their health. Nutrition can modulate the immune system and gut microbiota, offering a boost to protection following weaning. This controlled, randomized, double-blinded study tests a dietary supplement containing nucleotides, oligosaccharides, vitamin E and β-carotene in 50 domestic short-haired kittens. Control kittens were fed standard, dry kitten diet whilst test kittens received the same diet with supplement for 52 weeks. Kittens received routine vaccinations and blood samples were collected throughout the study for standard hematology, serum antibodies and cytokine expression. Rectal fecal samples were analyzed for gut microbiota via 16S rRNA gene sequencing. Supplemented kittens showed significantly improved antibody response to Chlamydia vaccine at various timepoints versus controls, with no consistent effects of supplementation on responses to other vaccines. Several cytokines were significantly influenced by the supplement, which also significantly altered gut microbiota diversity. In conclusion, a dietary supplement was shown to promote immune response and gut microbiota changes in kittens and may help to support a healthy transition into adulthood. Full article

Acute gastroenteritis (AGE) is a major cause of illness and death in children under five, especially in low- and middle-income countries, and rotavirus A (RVA) and norovirus are the leading viral agents. The present study aimed to describe the development of a commercial multiplex TaqMan-based RT-qPCR assay to detect those viruses to enhance surveillance and public health responses in Brazil. The assay validation involved optimizing primers and probes for multiplex RT-qPCR, assessing analytical sensitivity, and confirming specificity. A multicenter pilot study across Brazil’s AGE surveillance network assessed the assay’s performance. The IBMP NAT assay demonstrated high specificity and sensitivity for detecting RVA and norovirus GI and GII. No cross-reactivity was observed. LoD95 values were low: 18.6 (GI), 71.2 (GII), and 12.3 (RVA) copies/reaction. In 379 clinical samples, diagnostic sensitivity and specificity exceeded 96% for all targets. The assay showed strong reproducibility across operators and instruments. Stability tests confirmed consistent performance under freeze–thaw, transport, and storage conditions. Compared to in-house RT-qPCR, the IBMP NAT test yielded lower Ct values, indicating improved detection of low viral loads. The IBMP NAT Kit significantly advances molecular diagnostics, enabling rapid, sensitive, and reliable detection of RVA and norovirus in fecal specimens. It strengthens public health surveillance and supports timely responses to AGE outbreaks, helping reduce disease burden in vulnerable populations. Full article

Canine Kobuvirus (CaKoV) has been identified as an agent involved in gastrointestinal diseases among dogs worldwide, with a particular impact on young individuals. This study reports the first molecular detection of CaKoV in Ecuadorian dogs with gastroenteritis, using a sensitive SYBR Green-based RT-qPCR assay. The assay demonstrated high sensitivity, with detection limits approaching a single copy of genetic material (1 copy/μL), with a standard efficiency curve of 100.6% and a correlation coefficient of 0.997, facilitating accurate CaKoV detection even at a minimal number of gene copies; it was also highly specific for CaKoV genome amplification, as no amplification was shown for other canine enteric viruses [Canine Parvovirus (CPV-2), Canine Astrovirus (CaAstV), Canine Coronavirus (CCoV), and Canine Distemper Virus (CDV)], and demonstrated strong reproducibility across different runs. A total of 250 fecal samples were used to validate the assay and detect the presence of CaKoV, with 91 samples testing positive for CaKoV, confirming the virus’ presence across multiple provinces in Ecuador, with Pichincha reporting the highest number of positive samples. Phylogenetic analysis of the partial 3D gene sequence exhibited a nucleotide identity ≥ 90% with sequences of CaKoV strains from different countries around the world. Co-infections with other enteric viruses such as CPV-2, CCoV, and CaAstV were observed in 61.3% of CaKoV-positive samples from dogs with gastroenteritis, with triple co-infections (CPV-2, CaAstV, and CaKoV) being the most frequently detected combination in the study. The present study showed that CaKoV is circulating in domestic dogs affected with gastroenteric disease and in apparently healthy dogs. This work establishes CaKoV as a possible contributor to canine gastroenteritis in Ecuador, in addition to the typical viruses such as CPV-2 and CCoV; moreover, this study illustrates a molecular assay that is both rapid and reliable for the diagnosis of CaKoV. Full article

Water treatment systems in swimming ponds support the natural self-cleaning capabilities of water based on the functions of repository macrophytes in their regeneration zone and the regulation of the internal metabolism of the reservoirs. As part of the project, a functional modular filtration chamber with system multiplication capabilities was designed and created. This element is dedicated to water treatment systems in natural swimming ponds. The prototype system consisted of modular filtration chambers and pump sections, as well as equipment adapted to the conditions prevailing in the eco-pool. An innovative solution for selective shutdown of the filtration chamber without closing the circulation circuit was also used, which forms the basis of a patent application. A verified high-performance adsorbent, Rockfos^® modified limestone, was used in the filtration chamber. In order to determine the effective filtration rate for three small test ponds with different flow rates (5 m/h, 10 m/h and 15 m/h), the selected physicochemical parameters of water (temperature, pH, electrolytical conductivity, oxygen saturation, total hardness, nitrites, nitrates, and total phosphorus, including adsorption efficiency and bed absorption capacity) were researched before and after filtration. Tests were also carried out on the composition of fecal bacteria and phyto- and zooplankton. Based on high effective phosphorus filtration efficiency of 32.65% during the operation of the bed, the following were determined: no exceedances of the standards for the tested parameters in relation to the German standards for eco-pools (FLL—Forschungsgesellschaft Landschaftsentwicklung Landschaftsbau e. V., 2011); lower number of fecal pathogens (on average 393—coliform bacteria; 74—Escherichia coli; 34—fecal enterococci, most probably number/100 mL); the lowest share of problematic cyanobacteria in phytoplankton (<250,000 individuals/dm³ in number and <0.05 µg/dm³—biomass); low chlorophyll a content (2.2 µg/dm³—oligotrophy) and the presence of more favorable smaller forms of zooplankton, an effective filtration speed of 5 m/h. This velocity was recommended in the FLL standards for swimming ponds, which were adopted in this study as a reference for rapid filters. In testing the functional efficiency of a dedicated filtration system for a Type II test pond (50 m²—area and 33 m³—capacity), at a filtration rate of 5 m/h, an average effective phosphorus adsorption efficiency of 18.28–53.98% was observed under the bed work-in-progress conditions. Analyses of other physicochemical water parameters, with appropriate calculations and statistical tests, indicated progressive functional efficiency of the system under bathing conditions. Full article

Giardiasis is a globally prevalent waterborne zoonosis. Rapid enrichment and detection technologies for this disease are essential. Cyst outer wall proteins are ideal targets for the enrichment and detection of cysts in the environment, but there are few available targets with suboptimal effectiveness. In this study, Giardia duodenalis (G. duodenalis) cysts were purified, and outer wall proteins were biotinylated, followed by streptavidin magnetic bead purification and mass spectrometry. Sixty-three novel cyst wall proteins were identified, and their functions were annotated through Gene Ontology (GO) and KEGG analyses. The β-giardin and α-1 giardin were among the newly identified and predicted to be located on the outer wall of G. duodenalis cysts. For the characterization of these two targets, we applied sequence analysis, prokaryotic expression, preparation of polyclonal antibodies, and determination of subcellular localization. Finally, based on β-giardin immunomagnetic beads were prepared using the polyclonal antibodies and tested for their enrichment efficiency. Immunomagnetic beads targeting β-giardin achieved 65% cyst enrichment efficiency in fecal samples, comparable to conventional methods. Clinical evaluation across 163 multi-host fecal samples (ferrets, Siberian tigers, red-crowned cranes) demonstrated concordance with nested PCR, successfully enriching cysts from PCR-positive specimens. The immunomagnetic beads method targeting β-giardin demonstrated effective G. duodenalis cyst enrichment in multi-host fecal samples. These results provide a proteomic framework for the cyst wall proteins of G. duodenalis, expanding the detection targets for G. duodenalis cysts. It also establishes a theoretical foundation for subsequent research on the composition and function of G. duodenalis cysts. Full article

Cryptosporidium is a genus of protozoa that infects the gastrointestinal and respiratory epithelium of various host species. The aim of this study was to perform experimental infection in conventional mice with three Cryptosporidium species isolated from humans. The three Cryptosporidium species, namely Cryptosporidium canis, Cryptosporidium parvum, and Cryptosporidium ryanae, were obtained from fecal samples collected from patients hospitalized in an infectious disease hospital. The mice, from 10-day-old litters kept with their mother, were divided into three groups and orally infected with one of the Cryptosporidium species. The first oocysts were identified in the feces of the mice four days post-infection. The infection was successful with all three Cryptosporidium species, but the infection level (expressed as the number of oocysts per microscopic field) was low. The infection was detected using a rapid immunochromatographic test 40 days post-infection. Furthermore, starting on the 17th day after infection, the mothers also tested positive on the rapid immunochromatographic test, having been negative until that point. It was concluded that mice could represent a source of infection for the three Cryptosporidium species in other susceptible species, including humans. No behavioral changes or diarrhea were observed in any of the experimental cases. Full article

Cryptosporidium is a major waterborne parasite that causes gastrointestinal illness. Conventional assays, including microscopy and immunological identification, often suffer from false positives or negatives due to non-specific binding or morphological differences between Cryptosporidium species. We developed a novel qPCR assay based on a Cryptosporidium-specific Conserved Signature Protein (CSP) to address the limitations of testing complex samples, including those from recreational waters. The CSP (hypothetical protein (cgd2_3830)) was identified as taxonomically unique to Cryptosporidium species. The CSP sequence and designed qPCR assay primers/probe demonstrated high specificity for the targeted Cryptosporidium species when tested against NCBI RefSeq databases. qPCR assay efficiency was determined as 95% and an R² value of 0.99, with a slope and intercept of −3.4 and 40.1, respectively. Additionally, the Lower Limit of Detection (ALLOD) was determined as three gene copies, suggesting the potential to detect even a single oocyst. No non-specific amplification products or primer dimers were observed when the qPCR assay was evaluated using recreational water, fecal solution, and wastewater, while spike-in-control tests indicated minimal interference with the sensitivity of the assay, highlighting application for testing complex environmental DNA extracts. These findings highlight the application of the novel CSP-based qPCR assay for the rapid and sensitive detection of Cryptosporidium sp., thereby circumventing the sequence variability and multi-copy limitations associated with existing molecular markers. This proof-of-concept study presents a diagnostic framework utilizing CSP-based markers for developing water quality monitoring strategies, with scope for expansion to other microbial pathogens and potential applications in clinical and food safety settings. Full article

Effective species identification is crucial for the conservation and management of marine mammals, particularly in regions such as the Mediterranean Sea, where several cetacean populations are endangered or vulnerable. In this study, we developed and validated a High-Resolution Melting (HRM) analysis protocol for the rapid, cost-effective, and reliable identification of the four representative marine cetacean species that occur in the Mediterranean Sea: the bottlenose dolphin (Tursiops truncatus), the striped dolphin (Stenella coeruleoalba), the sperm whale (Physeter macrocephalus), and the fin whale (Balaenoptera physalus). Species-specific primers targeting mitochondrial DNA regions (cytochrome b and D-loop) were designed to generate distinct melting profiles. The protocol was tested on both tissue and fecal samples, demonstrating high sensitivity, reproducibility, and discrimination power. The results confirmed the robustness of the method, with melting curve profiles clearly distinguishing the target species and achieving a success rate > 95% in identifying unknown samples. The use of HRM offers several advantages over traditional sequencing methods, including reduced cost, speed, portability, and suitability for degraded samples, such as those from the stranded individuals. This approach provides a valuable tool for non-invasive genetic surveys and real-time species monitoring, contributing to more effective conservation strategies for cetaceans and enforcement of regulations against illegal trade. Full article

Drosophila suzukii, commonly known as spotted-wing drosophila, has emerged as a highly destructive pest in global fruit and wine production. The effectiveness of chemical control is significantly compromised by rapid resistance development and a limited range of insecticide options. Biological control presents a promising sustainable alternative. Our previous work suggested the La Jolla Virus (LJV) as a suitable candidate for the development of an insect virus-based control option. Here, we characterized the natural transmission and pathology of the virus. We tested various modes of horizontal transmission, including airborne, venereal and oral, and fecal routes. To understand LJV pathology in infected flies, we studied feeding behavior and demonstrated changes in food absorption compared to non-infected flies. We also investigated the impact on fecundity and egg-to-adult success rate. Altogether, these results collectively improve our understanding of LJV transmission in natural populations and the implication of infected flies in food ingestion and overall fitness. Full article

Rotavirus group A (RVA) is a major cause of pediatric acute gastroenteritis (AGE). Vaccination is an effective public health strategy and Angola implemented it in 2014. This hospital-based study aimed to estimate the prevalence of RVA infection and the severity of AGE in children under five years of age treated at six hospitals in Luanda Province. Between April 2021 and May 2022, 1251 fecal samples were screened by an immunochromatographic rapid test (SD Bioline). Data on socio-demographic profile, nutritional status, and clinical assessment were obtained. The association of RVA infection and AGE severity with possible risk factors was evaluated with a binary logistic regression model. Overall, the detection rate was 57.8% and girls tend to be more often infected than boys (55.2%). Infection was more common in the youngest group (1 to 6 months, 60.3%). Important sources of RVA infection were drinking water kept in tanks (57.9%) and private sanitary facilities with piped water (61%). Surprisingly, according to the Vesikari Scale score, the most severe symptoms were observed in children vaccinated with two doses (80.7%). RVA prevalence remains high despite vaccination, and further studies should address the association between infection sources and disease severity, as well as the causes underlying vaccine (un)effectiveness. Full article

Background/Objectives: The emergence of the mobilized colistin resistance 1 (mcr-1) gene, which causes colistin resistance, is a serious concern in animal husbandry, particularly in pigs. Although antibiotic regulations in many countries have prohibited the use of colistin in livestock, the persistence and dissemination of this plasmid-mediated gene require effective and rapid monitoring. Therefore, a rapid, sensitive, and specific method combining recombinase polymerase amplification (RPA) with an in-house lateral flow assay (LFA) for the mcr-1 gene detection was developed. Methods: The colistin agar test and broth microdilution were employed to screen 152 E. coli isolates from pig fecal samples of five antibiotic-used farms. The established RPA-in-house LFA was validated with PCR for mcr-1 gene detection. Results: The RPA-in-house LFA was completed within 35 min (20 min of amplification and 5–15 min on LFA detection) at 37 °C. The sensitivity, specificity, and accuracy were entirely 100% in concordance with PCR results. No cross-reactivity was detected with seven common pathogenic bacteria or other mcr gene variants. Conclusions: Therefore, the in-house RPA-LFA serves as a point-of-care testing tool that is rapid, simple, and portable, facilitating effective surveillance of colistin resistance in both veterinary and clinical settings, thereby enhancing health outcomes. Full article

Sepsis is a complex clinical syndrome characterized by an uncontrolled inflammatory response to an infection that may result in septic shock and death. Recent research has revealed a crucial link between sepsis and alterations in the gut microbiota, showing that the microbiome could serve an essential function in its pathogenesis and prognosis. In sepsis, the gut microbiota undergoes significant dysbiosis, transitioning from a beneficial commensal flora to a predominance of pathobionts. This transformation can lead to a dysfunction of the intestinal barrier, compromising the host’s immune response, which contributes to the severity of the disease. The gut microbiota is an intricate system of protozoa, fungi, bacteria, and viruses that are essential for maintaining immunity and metabolic balance. In sepsis, there is a reduction in microbial heterogeneity and a predominance of pathogenic bacteria, such as proteobacteria, which can exacerbate inflammation and negatively influence clinical outcomes. Microbial compounds, such as short-chain fatty acids (SCFAs), perform a crucial task in modulating the inflammatory response and maintaining intestinal barrier function. However, the role of other microbiota components, such as viruses and fungi, in sepsis remains unclear. Innovative therapeutic strategies aim to modulate the gut microbiota to improve the management of sepsis. These include selective digestive decontamination (SDD), probiotics, prebiotics, synbiotics, postbiotics, and fecal microbiota transplantation (FMT), all of which have shown potential, although variable, results. The future of sepsis management could benefit greatly from personalized treatment based on the microbiota. Rapid and easy-to-implement tests to assess microbiome profiles and metabolites associated with sepsis could revolutionize the disease’s diagnosis and management. These approaches could not only improve patient prognosis but also reduce dependence on antibiotic therapies and promote more targeted and sustainable treatment strategies. Nevertheless, there is still limited clarity regarding the ideal composition of the microbiota, which should be further characterized in the near future. Similarly, the benefits of therapeutic approaches should be validated through additional studies. Full article

Current diagnostic methods for detecting foodborne pathogens are time-consuming, require sophisticated equipment, and have a low specificity and sensitivity. Magnetic nanoparticles (MNPs) and plasmonic/colorimetric biosensors like gold nanoparticles (GNPs) are cost-effective, high-throughput, precise, and rapid. This study aimed to validate the use of MNPs and GNPs for the early detection of Escherichia coli O157:H7, Salmonella enterica spp., Campylobacter jejuni, and Listeria monocytogenes in bovine fecal samples. The capture efficiency (CE) of the MNPs was determined by using Salmonella Typhimurium (ATCC_13311) adjusted at an original concentration of 1.5 × 10⁸ CFU/mL. One (1) mL of this bacterial suspension was spiked into bovine fecal suspension (1 g of fecal sample in 9 mL PBS) and serially diluted ten-fold. DNA was extracted from Salmonella Typhimurium to determine the analytical specificity and sensitivity/LOD of the GNPs. The results showed that the CE of the MNPs ranged from 99% to 100% and could capture as little as 1 CFU/mL. The LOD of the GNPs biosensor was 2.9 µg/µL. The GNPs biosensor was also tested on DNA from 38 naturally obtained bovine fecal samples. Out of the 38 fecal samples tested, 81.6% (31/38) were positive for Salmonella enterica spp., 65.8% (25/38) for C. jejuni, 55.3% (21/38) for L. monocytogenes, and 50% (19/38) for E. coli O157:H7. We have demonstrated that MNP and GNP biosensors can detect pathogens or their DNA at low concentrations. Ensuring food safety throughout the supply chain is paramount, given that these pathogens may be present in cattle feces and contaminate beef during slaughter. Full article

Recent studies suggest that the dietary intake of human milk oligosaccharides (HMOs) provides health benefits from infancy up to adulthood. Thus far, beneficial changes in the adult gut microbiome have been observed at oral doses of 5–20 g/day of HMOs. Efficacy of lower doses has rarely been tested. We assessed four HMO molecular species—2′Fucosyllactose (2′FL), Lacto-N-neotetraose (LNnT), 3′Sialyllactose (3′SL), and 6′Sialyllactose (6′SL)—at predicted doses from 0.3 to 5 g/day for 6-year-old children and adults (n = 6 each), using ex vivo SIFR^® technology (Cryptobiotix, Ghent, Belgium). This technology employing bioreactor fermentation on fecal samples enables us to investigate microbial fermentation products that are intractable in vivo given their rapid absorption/consumption in the human gut. We found that HMOs significantly increased short-chain fatty acids (SCFAs), acetate, propionate (in children/adults), and butyrate (in adults) from predicted doses of 0.3–0.5 g/day onwards, with stronger effects as dosing increased. The fermentation of 6′SL had the greatest effect on propionate, LNnT most strongly increased butyrate, and 2′FL and 3′SL most strongly increased acetate. An untargeted metabolomic analysis revealed that HMOs enhanced immune-related metabolites beyond SCFAs, such as aromatic lactic acids (indole-3-lactic acid/3-phenyllactic acid) and 2-hydroxyisocaproic acid, as well as gut–brain-axis-related metabolites (γ-aminobutyric acid/3-hydroxybutyric acid/acetylcholine) and vitamins. The effects of low doses of HMOs potentially originate from the highly specific stimulation of keystone species belonging to, for example, the Bifidobacteriaceae family, which had already significantly increased at doses of only 0.5 g/day LNnT (adults) and 1 g/day 2′FL (children/adults). Full article

Giardia duodenalis and Cryptosporidium spp. are important zoonotic protozoan pathogens that infect the gastro-intestinal tract of numerous vertebrates, including humans, and both parasites are responsible for water- or food-borne outbreaks of disease worldwide. Although, globally, both parasites are highly prevalent, particularly in developing countries, epidemiological data from Romania are scarce, and genotyping has rarely been performed. The aims of the present study were to investigate the occurrence and genetic diversity of G. duodenalis and Cryptosporidium spp. in patients hospitalized in Northwestern Romania in relation to clinical and paraclinical presentation and to identify the relative frequency of non-specific symptoms and potential risk factors. Between June 2022 and January 2024, 426 fecal samples were screened for gastro-intestinal parasites by rapid tests and microscopical examination, further confirmed by PCR and sequencing. Giardia duodenalis was detected and characterized in 12 samples (2.82%), while Cryptosporidium parvum was confirmed in four samples (0.94%). A majority of positive patients were symptomatic and reported nausea and vomiting with a significantly higher frequency compared to negative ones. This study provides new insights into the epidemiological status and clinical implications of gastro-intestinal parasite species and genospecies in Romania that are necessary for an in-depth understanding of the potential zoonotic transmission and improvement of patient care. Full article