Intestinal Samples Research Articles

Mechanical and ex-vivo assessment of functionalized surgical sutures for bacterial infection monitoring

Surgical sutures are long-established medical devices that play an important role closing and healing damaged tissues and organs postoperatively. However, current commercial sutures are not able to detect infections at the wound site, which are quite frequent after surgery. In this work, we present mechanically stable smart sutures for the real-time monitoring of bacterial growth and biofilm formation. For this purpose, a conducting polymer named poly(3,4-ethylenedioxythiophene) (PEDOT), which is able to detect bacteria metabolites, was implemented as a coating onto commercial biostable sutures. A protecting hydrogel layer with adhesive properties, which was made of polydopamine-polyacrylamide (PDA-PAM), was used to prevent the detachment of the sensing coating of PEDOT upon looping and knotting the suture. The protective hydrogel preserved not only the knot mechanical properties of the suture but also the electrochemical response of the PEDOT-coating and, therefore, its ability to detect NADH from bacteria respiration. Ex-vivo assays using sutured swine intestine samples demonstrated that the suture with the PDA-PAM hydrogel layer detects the growth of bacteria in real tissues. As a proof of concept, sutures coated with PEDOT and protected with PDA-PAM were used to inhibit the local growth of bacteria in sutured intestines by applying controlled electrostimuli. Results evidenced that smart electro-responsive sutures can be used as multi-task devices focused on fighting bacterial infections, meaning not only monitoring but also hampering bacteria growth.

Herb–drug interactions: Quantitative analysis of levofloxacin absorption and transporter expression in the rat intestine following combined treatment with Persicaria capitata (Buch.-Ham. ex D. Don) H. Gross

Persicaria capitata (Buch.-Ham. ex D. Don) H. Gross, a traditional Chinese medicinal plant, is often used to treat various urologic disorders in China. P. capitata extracts (PCE) have been used in combination with levofloxacin (LVFX) to treat urinary tract infections (UTIs) for a long time. However, little is known about the absorption of LVFX and transporter expression in the intestine after combined treatment with PCE, restricting the development and utilization of PCE. In view of this, a UPLC-MS/MS method was established for the determination of LVFX in intestinal sac fluid samples and in situ intestinal circulation perfusate samples to explore the effect of PCE on the intestinal absorption characteristics of LVFX ex vivo and in vivo. To further evaluate the interaction between LVFX and PCE, western blotting, immunohistochemistry, and RT-qPCR were utilized to determine the expression levels of drug transporters (OATP1A2, P-gp, BCRP, and MRP2) involved in the intestinal absorption of LVFX after combined treatment with PCE. Using the everted intestinal sac model, the absorption rate constant (Ka) and cumulative drug absorption (Q) of LVFX in each intestinal segment were significantly lower in groups treated with PCE than in the control group. Ka at 2 h decreased most in the colon segment (from 0.088 to 0.016 µg/h·cm2), and Q at 2 h decreased most in the duodenum (from 213.29 to 33.92 µg). Using the intestinal circulation perfusion model, the Ka value and percentage absorption rate (A) of LVFX in the small intestine decreased significantly when PCE and LVFX were used in combination. These results showed that PCE had a strong inhibitory effect on the absorption of LVFX in the rat small intestine (ex vivo and in vivo intestinal segments). In addition, PCE increased the protein and mRNA expression levels of efflux transporters (P-gp, BCRP, and MRP2) and decreased the expression of the uptake transporter OATP1A2 significantly. The effects increased as the PCE concentration increased. These findings indicated that PCE changed the absorption characteristics of levofloxacin, possibly by affecting the expression of transporters in the small intestine. In addition to revealing a herb–drug interaction (HDI) between PCE and LVFX, these results provide a basis for further studies of their clinical efficacy and mechanism of action.

Cold stress changes the composition and function of microbiota in the content and mucosa of the ileum and colon in piglets

Context Cold stress is a significant factor that contributes to the imbalance of energy in piglets during their early life. Recent studies have shown that gut microbiota plays a crucial role in maintaining energy homeostasis under cold-stress conditions. Aims This study aims to investigate the effects of cold stress on the microbiota and expression pathways in the colon and ileum of weaned piglets, providing new insights and methods for helping piglets resist cold stress. Methods In total, 10 piglets in the cold-stress group (n = 5) and room-temperature group (n = 5) were sacrificed. Intestinal contents and mucosa samples were collected for 16S rRNA analysis. Key results The results showed that cold stress increased the observed features and chao1 index in the colonic mucosa. The beta diversity of ileum, colon and ileum mucosa was significantly changed. Under cold stress, the relative abundance of Acholeplasma, Proteiniphilum, and Olsenella increased in the contents of the ileum and colon. The relative abundance of Ruminococcaceae sp., Butyricicoccus, and Lachnospiraceae FCS020 increased in the mucosa of the colon. Sphingomonas, Helicobacter, Cutibacterium, and Bradyrhizobium were significantly increased in the mucosa of the ileum. In predicted functions, after cold stress, the purine metabolism and degradation increased in the content and mucosa of the ileum and mucosa of the colon. The fat biosynthesis pathway increased in the content of the colon. Complex carbohydrate degradation increased in the mucosa of both. Conclusions These findings suggest that cold stress has a significant impact on the species richness, composition, and predicted functions of the microbiota in the ileum and colon of piglets, with these effects varying depending on the location within the gut. Implications Therefore, we can help piglets resist cold stress by modifying the structure of gut microbiota through the addition of probiotics or adjusting the composition of their diet.

Human fecal alpha-glucosidase activity and its relationship with gut microbiota profiles and early stages of intestinal mucosa damage

ObjectivesWe investigated potential relationships among initial lesions of the intestinal mucosa, fecal enzymatic activities and microbiota profiles. MethodsFecal samples from 54 volunteers were collected after recruitment among individuals participating in a colorectal cancer (CRC) screening program in our region (Northern Spain) or attending for consultation due to clinical symptoms; intestinal mucosa samples were resected during colonoscopy. Enzymatic activities were determined in fecal supernatants by a semi-quantitative method. The fecal microbiota composition was determined by 16S rRNA gene-based sequencing. The results were compared between samples from clinical diagnosis groups (controls and polyps), according with the type of polyp (hyperplastic polyps or conventional adenomas) and considering the grade of dysplasia for conventional adenomas (low and high grade dysplasia). ResultsHigh levels of α-glucosidase activity were more frequent among samples from individuals diagnosed with intestinal polyps, reaching statistical significance for conventional adenomas and for low grade dysplasia adenomas when compared to controls. Regarding the microbiota profiles, higher abundance of Christensenellaceae_R-7 group and Oscillospiraceae_UCG-002 were found in fecal samples displaying low α-glucosidase activity as compared with those with higher activity as well as in controls with respect to conventional adenomas. A relationship was evidenced among intestinal mucosal lesions, gut glucosidase activities and intestinal microbiota profiles. ConclusionsOur findings suggest a relationship among altered fecal α-glucosidase levels, the presence of intestinal mucosal lesions, which can be precursors of CRC, and shifts in defined microbial groups of the fecal microbiota.

NEWCASTLE DISEASE AND AVIAN INFLUENZA IN BROILER THAT VACCINATED ON ONE DAY OLD

Newcastle disease and Avian Influenza are highly contagious viral diseases that can affect poultry with high mortality rates, and are zoonotic diseases that endanger humans. In this case study, broiler chickens aged 12 days were vaccinated at hatching and came from Yangapi Village, Tembuku, Bangli showed clinical symptoms of drooping neck or torticollis, no appetite, and greenish white diluted feces. Examinations are continued at each laboratory to obtain a definite diagnosis. The methods used are anatomical pathology examination, histopathology preparations, HA-HI examination, bacterial identification, and qualitative examination for paracites. The results of anatomical pathology examination found pathognomonic lesions, like ptechie in the proventriculus and ventriculus, enlargement and congestion in the lungs, hemorrhagic trachea, and hyperemic intestines. The results of histopathological examination showed marked changes with single-core inflammatory cells or lymphoid suggesting an infectious agent in the form of a virus. The results of the virology laboratory examination with the HA and HI test showed that the case chicken was positively infected by Newcastle Disease and Avian Influenza viruses. Bacteriology laboratory by culture of lung and intestinal organ samples was carried out on Nutrient Agar, followed by selective media Eosin Methylene Blue Agar, primary tests, gram staining, biochemical tests and sugar tests until the results showed that Escherichia coli bacteria grew from the chicken intestines and were classified as normal flora. The results of the examination related to parasites in the feces of case chickens by qualitative methods, natif, sedimentation concentration, and floating were confirmed not to be found worm eggs or coccidia. From this case study, it can be concluded that the case chicken was infected with Newcastle Disease and Avian Influenza viruses.

Genotypic Characterization and Virulence Features of Bacteria Associated with Chicken Meat in Rivers State, Nigeria

Microbial investigation of live poultry birds were carried out in three different locations (Aluu, Elioparanwo and RSU) in Ikwerre, Obio-Akpor and Port Harcourt Local Government Areas, Rivers State, Nigeria. In this study a total of 125 samples of muscle (25), breast (25), intestine (25), wing (25) and leg (25) of poultry birds were analysed using molecular techniques and evaluating there virulence features. The birds were processed and microbiological analysis was done using standard procedures, molecular identification of the isolates was carried out. The mean total heterotrophic bacterial count of the muscle, breast, intestine, wing and leg in the three different locations ranged from 16.6±2.3 to 25.2±3.5, 1.6±0.2 to 24.8±8.6, 0.11±0.1 to 8.9±2.7, 1.1±0.8 to 10.6±1.8 and 3.4±0.1 to 4.9±0.9 CFU/g, respectively. The mean total coliform counts of the three locations ranged from 2.1±0.3 to 2.1±0.2, 1.8±0.2 to 0.9±0.6, and 0.32±2.5 to 0.32±0.25, 2.6±0.3 to 7.7±0.9 and 2.3±0.4 to 2.9±0.3 CFU/g, respectively. Virulence features of A. caviae (CP072326, OL45594) shows proteolytic activity, catalytic DNase, motility, starch hydrolysis and biofilm formation. A. baumanii (ON606300) showed Lipolytic activity, catalytic, starch hydrolysis, capsule formation and biofilm formation. C. braakii (ON557391) was positive for Motility, catalytic and proteolytic. JQ231164 C. freudii (motile, catalytic, proteolytic and biofilm formation. E. hormaechei (ON834331, ON834332) was positive for motility, starch hydrolysis and biofilm formation. MN733229 M. odoratiminus (Lipolytic, motile, catalytic, DNase, proteolytic, ureolytic, heomolytic, capsule formation and biofilm formation. The study revealed that microbial contamination of Chicken meat could arise from bacteria present in the environment and on birds. These findings, thus underscored the urgent need for improved biosecurity measures, enhanced hygiene practices to safeguard both poultry health and public health. There should therefore be stringent implementation of food safety measure throughout the poultry value chain from farm to table.

Lymphatic Uptake of a Highly Lipophilic Protease Inhibitor Prodrug from a Lipid-Based Formulation is Limited by Instability in the Intestine

Dabigatran etexilate (DABE) is a lipophilic double alkyl ester prodrug of dabigatran (DAB) which is a serine protease inhibitor used clinically as an anticoagulant. Recently, translocation of serine protease enzymes, including trypsin, from the gut into the mesenteric lymph and then blood has been associated with organ failure in acute and critical illnesses (ACIs). Delivery of DABE into mesenteric lymph may thus be an effective strategy to prevent organ failure in ACIs. Most drugs access the mesenteric lymph in low quantities following oral administration, as they are rapidly transported away from the intestine via the blood. Here, we examine the potential to deliver DABE into the mesenteric lymph by promoting association with lymph lipid transport pathways via co-administration with a lipid-based formulation (LBF). A series of self-emulsifying LBFs were designed and tested in vitro for their potential to form stable DABE loaded emulsions and keep DABE solubilised and stable over time in simulated gastrointestinal conditions. The LBFs were found to form fine emulsions with a droplet size of 214 ± 30 nm and DABE was stable in the formulation. The stability of DABE in vitro in simulated intestinal conditions, plasma and lymph samples was also evaluated to ensure stability in collected samples and to evaluate whether the prodrug is likely to release active DAB. Ultimately, a highly uniform and stable self-emulsifying Type III A LBF of DABE was chosen for progression into in vivo studies in male Sprague Dawley rats to confirm the lymphatic uptake and plasma pharmacokinetics. Both in vitro and in vivo in plasma and lymph, DABE was rapidly converted to an intermediate and DAB. The main species present in vivo in both plasma and lymph was DAB and mass transport of DABE and DAB in lymph was minimal (∼0.5 % of dose). Importantly, the concentration of DABE in lymph was substantially (20–176 fold) higher than in plasma, supporting that if the prodrug were stable and did not convert to DAB in the intestine, it would be lymphatically transported. Future studies will therefore focus on optimizing the design of the prodrug and formulation to improve stability during absorption and further promote lymphatic uptake.

Expression of Calretinin in the Cecal Muscularis Interna: Observation and Hypothetical Relevance to Appendicitis.

The unexpected observation of calretinin immunoreactivity in smooth muscle cells in the muscularis propria of the cecum led to a more detailed examination of calretinin expression and its possible relationship to propulsive contractile activity around the vermiform appendix. Immunohistochemistry and RNA in situ hybridization were performed to analyze calretinin expression in intestinal samples from 33 patients at ages ranging from mid-gestation fetuses to adults, as well as in some potentially relevant animal models. Dual immunolabeling was done to compare calretinin localization with markers of smooth muscle and interstitial cells of Cajal. Calretinin expression was observed consistently in the innermost smooth muscle layers of the muscularis interna in the human cecum, appendiceal base, and proximal ascending colon, but not elsewhere in the intestinal tract. Calretinin-positive smooth muscle cells did not co-express markers located in adjacent interstitial cells of Cajal. Muscular calretinin immunoreactivity was not detected in the ceca of mice or macaques, species which lack appendices, nor in the rabbit cecum or appendix. Localized expression of calretinin in cecal smooth muscle cells may reduce the likelihood of retrograde, calcium-mediated propulsive contractions from the proximal colon and suppress pro-inflammatory fecal stasis in the appendix.

Treatment of chickens with lactobacilli prior to challenge with Clostridium perfringens modifies innate responses and gut morphology

Necrotic enteritis caused by Clostridium perfringens (CP), is a common enteric disease of poultry that has been previously controlled by in-feed antibiotics. However, due to the rapid emergence of antimicrobial resistance, alternatives to antibiotics such as probiotics have received considerable attention because of their immunomodulatory and intestinal health benefits. The present study investigated the effects of probiotic lactobacilli on gut histomorphology and intestinal innate responses in chickens. Day-old male broiler chickens were treated with 1 × 107 or 1 × 108 colony-forming units (CFU) of a lactobacilli cocktail on days 1, 7, 14, and 20 post-hatch, while control groups were not treated with lactobacilli. On day 21, birds in all groups (except the negative control) were challenged with 3 × 108 CFU of CP for 3 days. Intestinal tissue samples were collected before and after the CP challenge to assess gene expression and for histomorphological analysis. Lactobacilli treatment at a dose of 1 × 108 CFU conferred partial protection against NE by lowering lesion scores, increasing villus height in the ileum and reducing crypt depth in the jejunum. In addition, 1 × 108 CFU of lactobacilli enhanced the expression of Toll-like receptor (TLR) 2, interferon-gamma (IFN-γ), interleukin (IL)-10, IL-12, and IL-13 in both the jejunum and ileum at different timepoints and subsequently decreased the expression of transforming growth factor beta (TGF-β) and IL-1β post-CP challenge. In conclusion, the results indicate that treatment with lactobacilli mitigated NE in a dose-dependent manner via improvement of intestinal morphology and modulation of innate immune response in chickens.

Comparative study of the gut microbial communities collected by scraping and swabbing in a fish model: a comprehensive guide to promote non-lethal procedures for gut microbial studies.

In the present study, we propose the use of swabs in non-lethal sampling procedures to collect the mucosa-adhered gut microbiota from the posterior intestine of fish, and therefore, we compare the bacterial communities collected by conventional scraping and by swabbing methods. For this purpose, samples of the posterior intestine of rainbow trout (Oncorhynchus mykiss) were collected first using the swabbing approach, and after fish euthanasia, by mucosa scraping. Finally, bacterial communities were compared by 16S rRNA gene Illumina sequencing. Results from the current study revealed that similar values of bacterial richness and diversity were found for both sampling procedures. Similarly, there were no differences between procedures when using qualitative metrics (Jaccard and unweighted UniFrac) for estimating inter-individual diversity, but the quantitative metrics (Bray-Curtis and weighted UniFrac) showed a higher dispersion when samples were obtained by swabbing compared to scraping. In terms of bacterial composition, there were differences in abundance for the phyla Firmicutes and Proteobacteria. The cause of these differential abundances may be the inability of the swab to access to certain areas, such as the basal region of the intestinal villi. Moreover, swabbing allowed a higher representation of low abundant taxa, which may also have an important role in host microbiome regardless of their low abundance. Overall, our results demonstrate that the sampling method is a factor to be considered in experimental design when studying gut bacterial communities to avoid potential biases in the interpretation or comparison of results from different studies. In addition, the advantages and disadvantages of each procedure (swabbing vs scraping) are discussed in detail, concluding that swabbing can be implemented as a reliable and non-lethal procedure for posterior gut microbiota studies, which is of particular interest for animal welfare and the 3Rs principle, and may offer a wide range of novel applications.

Regio-specific lipid fingerprinting of edible sea cucumbers using LC/MS

Sea cucumbers are a rich source of bioactive compounds and are gaining popularity as nutrient-rich seafood. They are consumed as a whole organism in Pacific regions. However, limited data are available on the comparison of their lipid composition and nutritional value. In this study, untargeted liquid chromatography/mass spectrometry was applied to comprehensively profile lipids in the skin, meat, and intestinal contents of three color-distinct edible sea cucumbers. Multivariate principal component analysis revealed that the lipid composition of the intestinal contents of red, black, and blue sea cucumbers differs from that of skin, and meats. Polyunsaturated fatty acids (PUFAs) are abundant in the intestinal contents, followed by meats of sea cucumber. Lipid nutritional quality assessments based on fatty acid composition revealed a high P:S ratio, low index of atherogenicity, and high health promotion indices for the intestinal contents of red sea cucumber, suggesting its potential health benefits. In addition, hierarchical cluster analysis revealed that the intestinal contents of sea cucumbers were relatively high in PUFA-enriched phospholipids and lysophospholipids. Ceramides are abundant in black skin, blue meat, and red intestinal content samples. Overall, this study provides the first insights into a comprehensive regio-specific profile of the lipid content of sea cucumbers and their potential use as a source of lipid nutrients in food and nutraceuticals.

Coinfection and nonrandom recombination drive the evolution of swine enteric coronaviruses

ABSTRACT Coinfection with multiple viruses is a common phenomenon in clinical settings and is a crucial driver of viral evolution. Although numerous studies have demonstrated viral recombination arising from coinfections of different strains of a specific species, the role of coinfections of different species or genera during viral evolution is rarely investigated. Here, we analyzed coinfections of and recombination events between four different swine enteric coronaviruses that infect the jejunum and ileum in pigs, including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), and a deltacoronavirus, porcine deltacoronavirus (PDCoV). Various coinfection patterns were observed in 4,468 fecal and intestinal tissue samples collected from pigs in a 4-year survey. PEDV/PDCoV was the most frequent coinfection. However, recombination analyses have only detected events involving PEDV/TGEV and SADS-CoV/TGEV, indicating that inter-species recombination among coronaviruses is most likely to occur within the same genus. We also analyzed recombination events within the newly identified genus Deltacoronavirus and found that sparrows have played a unique host role in the recombination history of the deltacoronaviruses. The emerging virus PDCoV, which can infect humans, has a different recombination history. In summary, our study demonstrates that swine enteric coronaviruses are a valuable model for investigating the relationship between viral coinfection and recombination, which provide new insights into both inter- and intraspecies recombination events among swine enteric coronaviruses, and extend our understanding of the relationship between coronavirus coinfection and recombination.

First molecular identification and phylogenetic illustration of Sarcocystis species infection in Red Sea shortfin mako shark (Isurus oxyrinchus Rafinesque, 1810)

Backgroundmembers of the genus Sarcocystis are intracellular obligate protozoan parasites classified within the phylum Apicomplexa and have an obligate heteroxenous life cycle involving two hosts. A more comprehensive understanding of the prevalence and geographic range of different Sarcocystis species in marine ecosystems is needed globally and nationally. Hence, the objective of this study was to document the incidence of Sarcocystis infection in sharks within the aquarium ecosystem of Egypt and to identify the species through the characterization of the SSU rDNA gene.MethodsAll organs of the mako shark specimen underwent macroscopic screening to detect the existence of a Sarcocystis cyst. Ten cysts were collected from the intestine and processed separately to extract the genomic DNA. The polymerase chain reaction (PCR) was accomplished by amplifying a specific 18S ribosomal RNA (rRNA) gene fragment. Subsequently, the resulting amplicons were subjected to purification and sequencing processes.ResultsMacroscopic examination of the mako shark intestinal wall sample revealed the presence of Sarcocystis cysts of various sizes and shapes, and sequencing of the amplicons from Sarcocystis DNA revealed a 100% nucleotide identity with the sequence of Sarcocystis tenella recorded from sheep in Iran; The mako shark sequence has been deposited in the GeneBank with the accession number OQ721979. This study presents the first scientific evidence demonstrating the presence of the Sarcocystis parasite in sharks, thereby documenting this specific marine species as a novel intermediate host in the Sarcocystis life cycle.ConclusionsThis is the first identification of Sarcocystis infection in sharks, and we anticipate it will be an essential study for future screenings and establishing effective management measures for this disease in aquatic ecosystems.

Comparative analysis of gut microbiome in Pangasionodon hypopthalmus and Labeo catla during health and disease.

The present study was conducted to study the composition of gut microbiome in the advanced fingerling and fingerling stage of striped pangasius catfish and catla during healthy and diseased conditions. Healthy pangasius and catla fishes were obtained from commercial farms and injected with the LD50 dose of A. hydrophila. The intestinal samples from the control and injected group were collected and pooled for 16s metagenomic analysis. Community analysis was performed by targeting the 16s rRNA gene to explore and compare the gut microbiota composition of these fishes. The operational taxonomic units (OTUs) consisted of four major phyla: Bacteroidia, Proteobacteria, Firmicutes, and Actinobacteria. Alpha and beta diversity indices were carried out to understand the diversity of microbes within and between a sample. While comparing the advanced fingerling and fingerling stage gut microbiome of Pangasius catfish, the dominance of Proteobacteria was found in fingerlings, whereas Firmicutes and Bacteroides were found in advanced fingerlings. In catla, Proteobacteria and Bacteroides were predominant. Taxonomic abundance of the microbiota in control and diseased Pangasius and catla fishes at phylum, class, order, family, genus, and species levels were also depicted. The present study is the first of its kind, and it will help to identify the diversity of novel potential bacterial species involved in disease protection of fishes. It can lead to the development of sustainable prophylactic measures against (re-)emerging bacterial diseases in aquaculture.

Detection of Sarcocystis albifronsi, Eimeria alpacae, and Cystoisospora felis in Eurasian lynx (Lynx lynx) in northwestern China

Eurasian lynx (Lynx lynx) is widely distributed in various habitats in Asia and Europe, and it may harbor multiple pathogens. Currently, the information on protozoan infection in Eurasian lynx is scarce. In this study, we performed nested polymerase chain reaction (nPCR) analysis to detect intestinal protozoan infection in three dead Eurasian lynxes, in northwestern China. Three dead Eurasian lynxes, an adult female (#1), an adult male (#2), and a cub male (#3), were sampled in West Junggar Mountain, the northwestern region of Xinjiang Uyghur Autonomous Region. The intestine samples were analyzed using nPCR. We used primers targeting the cytochrome C oxidase subunit I gene (COI) for detection of Sarcocystis and Eimeria species and targeting the small subunit 18 S ribosomal RNA gene (18S rRNA) for detection of Cystoisospora species. The nPCR-positive products were sequenced, aligned, and phylogenetically analyzed. Three intestinal protozoa, Sarcocystis albifronsi, Eimeria alpacae, and Cystoisospora felis, were found in three Eurasian lynxes. The intestine sample of Eurasian lynx #2 was detected with S. albifronsi and E. alpacae. In addition, C. felis was only found in the intestine sample of Eurasian lynx #3. To the best of our knowledge, S. albifronsi and E. alpacae were detected in Eurasian lynx for the first time. In addition, C. felis was firstly found in Eurasian lynx in China. These findings extend our knowledge of the geographical distribution and host range of intestinal protozoa.

Nanopore sequencing for identification and characterization of antimicrobial-resistant Escherichia coli and Salmonella spp. from tilapia and shrimp sold at wet markets in Dhaka, Bangladesh.

Wet markets in low-and middle-income countries are often reported to have inadequate sanitation resulting in fecal contamination of sold produce. Consumption of contaminated wet market-sourced foods has been linked to individual illness and disease outbreaks. This pilot study, conducted in two major wet markets in Dhaka city, Bangladesh during a 4-month period in 2021 aimed to assess the occurrence and characteristics of Escherichia coli and non-typhoidal Salmonella spp. (NTS) from tilapia (Oreochromis niloticus) and shrimp (Penaeus monodon). Fifty-four individuals of each species were collected. The identity of the bacterial isolates was confirmed by PCR and their susceptibility toward 15 antimicrobials was tested by disk diffusion. The whole genome of 15 E. coli and nine Salmonella spp. were sequenced using Oxford Nanopore Technology. E. coli was present in 60-74% of tilapia muscle tissue and 41-44% of shrimp muscle tissue. Salmonella spp. was found in skin (29%) and gills (26%) of tilapia, and occasionally in muscle and intestinal samples of shrimp. The E. coli had several Multilocus sequence typing and serotypes and limited antimicrobial resistance (AMR) determinants, such as point mutations on glpT and pmrB. One E. coli (BD17) from tilapia carried resistance genes for beta-lactams, quinolones, and tetracycline. All the E. coli belonged to commensal phylogroups B1 and A and showed no Shiga-toxin and other virulence genes, confirming their commensal non-pathogenic status. Among the Salmonella isolates, five belonged to Kentucky serovar and had similar AMR genes and phenotypic resistance patterns. Three strains of this serovar were ST198, often associated with human disease, carried the same resistance genes, and were genetically related to strains from the region. The two undetermined sequence types of S. Kentucky were distantly related and positioned in a separate phylogenetic clade. Two Brunei serovar isolates, one Augustenborg isolate, and one Hartford isolate showed different resistance profiles. This study revealed high fecal contamination levels in tilapia and shrimp sold at two main wet markets in Dhaka. Together with the occurrence of Salmonella spp., including S. Kentucky ST198, a well-known human pathogen, these results stress the need to improve hygienic practices and sanitation standards at markets to improve food safety and protect consumer health.

Development of a multiplex reverse transcription-quantitative PCR (qPCR) method for detecting common causative agents of swine viral diarrhea in China

BackgroundDiarrheal diseases caused by viral agents have led to a great morbidity, mortality, and economic loss in global pig industry. Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and group A porcine rotavirus (RVA) are main causative agents of swine viral diarrhea with similar clinical signs on Chinese farms and their co-infection is also common. However, it is still lack of a convenient method to detect these four agents.MethodsA TaqMan multiplex qPCR method was developed to detect PEDV, TGEV, PDCoV, and RVA, simultaneously. This method was then applied to investigate 7,342 swine fecal samples or rectal swabs, as well as 1,246 swine intestinal samples collected from 2075 farms in China in 2022.ResultsMinimum detection limits of this method were 3 copies/µL for PEDV, 4 copies/µL for TGEV, 8 copies/µL for RVA, and 8 copies/µL for PDCoV, suggesting a good sensitivity. No signals were observed by using this method detecting other viral agents commonly prevalent in pigs, which is suggestive of a good specificity. Application of this method on investigating clinical samples demonstrated a relatively high positive rate for PEDV (22.21%, 1907/8588) and RVA (44.00%, 3779/8588). In addition, co-infection between PEDV and RVA was observed on 360 investigated farms, accounting for 17.35% (360/2075) of the farms where co-infection events were screened.ConclusionsA TaqMan multiplex qPCR method targeting PEDV, TGEV, PDCoV, and RVA was developed in this study. This method demonstrated a good specificity and sensitivity on investigating these four common viruses responsible for viral diarrhea on Chinese pig farms, which represents a convenient method for the monitoring and differential diagnosis of swine viral diarrhea.

Parasites in Wildlife in the Federation of Bosnia and Herzegovina

Abstract Parasitic diseases of wild animals represent an important area of research. In addition to the significant impact on wildlife health and fitness, many parasitic diseases have zoonotic implications. Due to limited scientific information, this research aimed to investigate parasitic diseases in wildlife in Bosnia and Herzegovina (B&H), focusing on the Federation of Bosnia and Herzegovina (FB&H), emphasizing zoonotic species. In the period from April 2020 to November 2022, we conducted research on 9 wildlife species. We analyzed fecal samples to detect and identify diagnostic stages (eggs, larvae, cysts, and oocysts) of various animal endoparasites using coprological methods, such as sedimentation, flotation, and the Baermann technique. The MERIFLUOR® Cryptosporidium/Giardia test was also used for the detection of Cryptosporidium oocysts and Giardia cysts. In the case of red foxes, intestinal samples were examined using the intestinal scraping technique to detect adult helminths. All collected muscle samples were subjected to the artificial digestion method for Trichinella detection. From 1,278 samples, 70.9% were positive. Parasitic infections were confirmed in 15.9% (11/69) of bears; 83.7% (262/313) of red foxes; 67.6% (44/65) of wolves; 25% (1/4) of wildcats; 20% (1/5) of badger; 43.7% (7/16) of martens; 39.7% (76/191) of wild boars; 84.5% (350/414) of deer, and 77.1% (155/201) of hares. The finding of zoonotic parasites (Toxocara canis, Uncinaria spp., Trichinella spp., Echinococcus spp. etc.) is particularly important due to their potential detrimental effects on human health, which highlights the need for further investigations.

Sex-Dependent Changes to the Intestinal and Hepatic Abundance of Drug Transporters and Metabolizing Enzymes in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterized by death and dysfunction of motor neurons that result in a rapidly progressing loss of motor function. While there are some data on alterations at the blood-brain barrier (BBB) in ALS and their potential impact on CNS trafficking of drugs, little is reported on the impact of this disease on the expression of drug-handling proteins in the small intestine and liver. This may impact the dosing of the many medicines that individuals with ALS are prescribed. In the present study, a proteomic evaluation was performed on small intestine and liver samples from postnatal day 120 SOD1G93A mice (a model of familial ALS that harbors a human mutant form of superoxide dismutase 1) and wild-type (WT) littermates (n = 7/genotype/sex). Untargeted, quantitative proteomics was undertaken using either label-based [tandem mass tag (TMT)] or label-free [data-independent acquisition (DIA)] acquisition strategies on high-resolution mass spectrometric instrumentation. Copper chaperone for superoxide dismutase (CCS) was significantly higher in SOD1G93A samples compared to the WT samples for both sexes and tissues, therefore representing a potential biomarker for ALS in this mouse model. Relative to WT mice, male SOD1G93A mice had significantly different proteins (Padj < 0.05, |fold-change|>1.2) in the small intestine (male 22, female 1) and liver (male 140, female 3). This included an up-regulation of intestinal transporters for dietary glucose [solute carrier (SLC) SLC5A1] and cholesterol (Niemann-Pick c1-like 1), as well as for several drugs (e.g., SLC15A1), in the male SOD1G93A mice. There was both an up-regulation (e.g., SLCO2A1) and down-regulation (ammonium transporter rh type b) of transporters in the male SOD1G93A liver. In addition, there was both an up-regulation (e.g., phosphoenolpyruvate carboxykinase) and down-regulation (e.g., carboxylesterase 1) of metabolizing enzymes in the male SOD1G93A liver. This proteomic data set identified male-specific changes to key small intestinal and hepatic transporters and metabolizing enzymes that may have important implications for the bioavailability of nutrients and drugs in individuals with ALS.

Development of non-alcoholic steatohepatitis is associated with gut microbiota but not with oxysterol enzymes CH25H, EBI2, or CYP7B1 in mice

Liver steatosis is the most frequent liver disorder and its advanced stage, non-alcoholic steatohepatitis (NASH), will soon become the main reason for liver fibrosis and cirrhosis. The “multiple hits hypothesis” suggests that progression from simple steatosis to NASH is triggered by multiple factors including the gut microbiota composition. The Epstein Barr virus induced gene 2 (EBI2) is a receptor for the oxysterol 7a, 25-dihydroxycholesterol synthesized by the enzymes CH25H and CYP7B1. EBI2 and its ligand control activation of immune cells in secondary lymphoid organs and the gut. Here we show a concurrent study of the microbial dysregulation and perturbation of the EBI2 axis in a mice model of NASH.We used mice with wildtype, or littermates with CH25H−/−, EBI2−/−, or CYP7B1−/− genotypes fed with a high-fat diet (HFD) containing high amounts of fat, cholesterol, and fructose for 20 weeks to induce liver steatosis and NASH. Fecal and small intestinal microbiota samples were collected, and microbiota signatures were compared according to genotype and NASH disease state.We found pronounced differences in microbiota composition of mice with HFD developing NASH compared to mice did not developing NASH. In mice with NASH, we identified significantly increased 33 taxa mainly belonging to the Clostridiales order and/ or the family, and significantly decreased 17 taxa. Using an Elastic Net algorithm, we suggest a microbiota signature that predicts NASH in animals with a HFD from the microbiota composition with moderate accuracy (area under the receiver operator characteristics curve = 0.64). In contrast, no microbiota differences regarding the studied genotypes (wildtype vs knock-out CH25H−/−, EBI2−/−, or CYP7B1−/−) were observed.In conclusion, our data confirm previous studies identifying the intestinal microbiota composition as a relevant marker for NASH pathogenesis. Further, no link of the EBI2 – oxysterol axis to the intestinal microbiota was detectable in the current study.