Evaluation of different media compositions promoting hepatocyte differentiation in the canine liver organoid model.

Organoids are 3-dimensional (3D) self-assembled structures capable of replicating the microanatomy and physiology of the epithelial components of their organ of origin. Adult stem cell (ASC) derived organoids from the liver have previously been shown to differentiate into primarily mature cholangiocytes, and their partial differentiation into functional hepatocytes can be promoted using specific media compositions. While full morphological differentiation of mature hepatocytes from ASCs has not yet been reported for any species, the functional differentiation can be approximated using various media compositions. Six differentiation media formulations from published studies on hepatic organoids were used for the differentiation protocol. Target species for these protocols were humans, mice, cats, and dogs, and encompassed various combinations and concentrations of four major hepatocyte media components: Bone morphogenetic protein 7 (BMP7), Fibroblast Growth Factor 19 (FGF19), Dexamethasone (Dex), and Gamma-Secretase Inhibitor IX (DAPT). Additionally, removing R-spondin from basic organoid media has previously been shown to drive the differentiation of ASC into mature hepatocytes. Differentiation media (N = 20) were designed to encompass combinations of the four major hepatocyte media components. The preferred differentiation of ASC-derived organoids from liver tissue into mature hepatocytes over cholangiocytes was confirmed by albumin production in the culture supernatant. Out of the twenty media compositions tested, six media resulted in the production of the highest amounts of albumin in the supernatant of the organoids. The cell lines cultured using these six media were further characterized via histological staining, transmission electron microscopy, RNA in situ hybridization, analysis of gene expression patterns, immunofluorescence, and label-free proteomics. The results indicate that preferential hepatocyte maturation from canine ADC-derived organoids from liver tissue is mainly driven by Dexamethasone and DAPT components. FGF19 did not enhance organoid differentiation but improved cell culture survival. Furthermore, we confirm that removing R-spondin from the media is crucial for establishing mature hepatic organoid cultures.

Adult Animal Stem Cell-Derived Organoids in Biomedical Research and the One Health Paradigm.

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that do not offer the same complexity as living animals but do offer financial affordability and more experimental freedom; therefore, they are commonly used. Traditional 2D cell lines cannot fully simulate the complexity of the epithelium of healthy organs and limit scientific progress. The One Health Initiative was established to consolidate human, animal, and environmental health while also tackling complex and multifactorial medical problems. Reverse translational research allows for the sharing of knowledge between clinical research in veterinary and human medicine. Recently, organoid technology has been developed to mimic the original organ's epithelial microstructure and function more reliably. While human and murine organoids are available, numerous other organoids have been derived from traditional veterinary animals and exotic species in the last decade. With these additional organoid models, species previously excluded from in vitro research are becoming accessible, therefore unlocking potential translational and reverse translational applications of animals with unique adaptations that overcome common problems in veterinary and human medicine.

Changes of Enterocyte Morphology and Enterocyte: Goblet Cell Ratios in Dogs with Protein-Losing and Non-Protein-Losing Chronic Enteropathies.

This study aimed to assess the morphometry of enterocytes as well as the goblet cell-to-enterocyte ratio in different intestinal segments of dogs with chronic enteropathies (CE). Histopathological intestinal samples from 97 dogs were included in the study (19 healthy juveniles, 21 healthy adults, 24 dogs with protein-losing enteropathy (PLE), and 33 CE dogs without PLE). Healthy adult small intestinal enterocytes showed progressively reduced epithelial cell height in the aboral direction, while juvenile dogs showed progressively increased epithelial cell height in the aboral direction. CE dogs had increased epithelial cell height in the duodenum, while PLE dogs had decreased epithelial cell heights compared to healthy adult dogs. Both the CE and PLE dogs showed decreased enterocyte width in the duodenal segment, and the ileal and colonic enterocytes of CE dogs were narrower than those of healthy adult dogs. CE dogs had a lower goblet cell-to-enterocyte ratio in the colon segment compared to healthy dogs. This study provides valuable morphometric information on enterocytes during canine chronic enteropathies, highlighting significant morphological enterocyte alterations, particularly in the small intestine, as well as a reduced goblet cell-to-enterocyte ratio in the colon of CE cases compared to healthy adult dogs.

Diagnostic investigation of Mycoplasma hyorhinis as a potential pathogen associated with neurological clinical signs and central nervous system lesions in pigs.

Mycoplasma hyorhinis (M. hyorhinis) is a commensal of the upper respiratory tract in swine with the typical clinical presentations of arthritis and polyserositis in postweaning pigs. However, it has also been associated with conjunctivitis and otitis media, and recently has been isolated from meningeal swabs and/or cerebrospinal fluid of piglets with neurological signs. The objective of this study is to evaluate the role of M. hyorhinis as a potential pathogen associated with neurological clinical signs and central nervous system lesions in pigs. The presence of M. hyorhinis was evaluated in a clinical outbreak and a six-year retrospective study by qPCR detection, bacteriological culture, in situ hybridization (RNAscope®), and phylogenetic analysis and with immunohistochemistry characterization of the inflammatory response associated with its infection. M. hyorhinis was confirmed by bacteriological culture and within central nervous system lesions by in situ hybridization on animals with neurological signs during the clinical outbreak. The isolates from the brain had close genetic similarities from those previously reported and isolated from eye, lung, or fibrin. Nevertheless, the retrospective study confirmed by qPCR the presence of M. hyorhinis in 9.9% of cases reported with neurological clinical signs and histological lesions of encephalitis or meningoencephalitis of unknown etiology. M. hyorhinis mRNA was confirmed within cerebrum, cerebellum, and choroid plexus lesions by in situ hybridization (RNAscope®) with a positive rate of 72.7%. Here we present strong evidence that M. hyorhinis should be included as a differential etiology in pigs with neurological signs and central nervous system inflammatory lesions.

Evaluation of colonization, variable lipoprotein-based serological response, and cellular immune response of Mycoplasma hyorhinis in experimentally infected swine.

Mycoplasma hyorhinis (Mhr) is a commensal of the upper respiratory tract that can be shed by nasal secretions and transmitted by direct contact in neonatal and nursery pigs. Lesions associated with Mhr infection include polyserositis and arthritis; however, systemic Mhr disease pathogenesis is not well characterized. This study aimed to investigate the immunopathogenesis and bacterial dissemination pattern of Mhr using single and multiple inoculation approaches in a caesarian-derived colostrum-deprived (CDCD) pig model. Animals in three treatment groups were inoculated once (Mhr 1; n = 12) or four (Mhr 2; n = 8) times with Mhr or sham-inoculated (NC group; n = 3) nasally and by tonsillar painting. Inoculum consisted of a triple cloned Mhr field isolate (4.5 × 107 CFU/mL) in Friis medium. Clinical signs were evaluated daily during the study. Serum and oral fluid antibody (IgA and IgG) response and cellular immune response were assessed using a recombinant chimeric VlpA-G-based indirect ELISA and by ELISpot, respectively. The presence of Mhr in oral fluids, nasal and oropharyngeal swabs were evaluated by qPCR. At 6 wpi, pigs were euthanized and evaluated for gross lesions consistent with Mhr and bacterial colonization in tonsils by qPCR. No clinical signs or gross lesions consistent with Mhr-associated disease were observed throughout the study. For Mhr 2 group, the presence of IgA and IgG in serum and oral fluids were detected at 2 and 4 weeks post-inoculation (wpi), respectively, while in Mhr 1, only IgA was detected in oral fluids at 6 wpi. The proportion of animals shedding Mhr in nasal secretions varied from 20 to 40 % in the Mhr 1 and 62.5-100% in the Mhr 2 group. However, the proportion of animals shedding Mhr in oropharyngeal swabs was consistent through the study (60 %) in Mhr 1 and fluctuated from 20 % to 87.5 % in Mhr 2 group. The lack of clinical signs and the presence of Mhr specific humoral response and bacterial colonization indicates that the multiple inoculation experimental model may mimic subclinical natural infection in the field. In addition, the humoral and transient cellular response did not result in bacterial clearance. Based on these results, animals would have to be exposed multiple times to mount a detectable immune response.

Performance of Commercial Mycoplasma hyopneumoniae Serum Enzyme-Linked Immunosorbent Assays under Experimental and Field Conditions.

Mycoplasma hyopneumoniae is an economically significant pathogen of swine. M. hyopneumoniae serum antibody detection via commercial enzyme-linked immunosorbent assays (ELISAs) is widely used for routine surveillance in commercial swine production systems. Samples from two studies were used to evaluate assay performance. In study 1, 6 commercial M. hyopneumoniae ELISAs were compared using serum samples from 8-week-old cesarean-derived, colostrum-deprived (CDCD) pigs allocated to the following 5 inoculation groups of 10 pigs each: (i) negative control, (ii) Mycoplasma flocculare (strain 27399), (iii) Mycoplasma hyorhinis (strain 38983), (iv) Mycoplasma hyosynoviae (strain 34428), and (v) M. hyopneumoniae (strain 232). Weekly serum and daily oral fluid samples were collected through 56 days postinoculation (dpi). The true status of pigs was established by PCR testing on oral fluids samples over the course of the observation period. Analysis of ELISA performance at various cutoffs found that the manufacturers' recommended cutoffs were diagnostically specific, i.e., produced no false positives, with the exceptions of 2 ELISAs. An analysis based on overall misclassification error rates found that 4 ELISAs performed similarly, although one assay produced more false positives. In study 2, the 3 best-performing ELISAs from study 1 were compared using serum samples generated under field conditions. Ten 8-week-old pigs were intratracheally inoculated with M. hyopneumoniae Matched serum and tracheal samples (to establish the true pig M. hyopneumoniae status) were collected at 7- to 14-day intervals through 98 dpi. Analyses of sensitivity and specificity showed similar performance among these 3 ELISAs. Overall, this study provides an assessment of the performance of current M. hyopneumoniae ELISAs and an understanding of their use in surveillance.

Early detection and differential serodiagnosis of Mycoplasma hyorhinis and Mycoplasma hyosynoviae infections under experimental conditions.

Mycoplasma hyorhinis (MHR) and Mycoplasma hyosynoviae (MHS) are common opportunistic pathogens in the upper respiratory tract and tonsils of swine. The identification of the specific species involved in clinical cases using conventional diagnostic methods is challenging. Therefore, a recombinant chimeric polypeptide based on the seven known variable lipoproteins (A-G) specific of MHR and a cocktail of surface proteins detergent-extracted from MHS cultures were generated and their suitability as antemortem biomarkers for serodiagnosis of MHR- and MHS-infection were evaluated by ELISA. M. hyorhinis and MHS ELISA performance, evaluated using serum samples collected over a 56-day observation period from pigs inoculated with MHR, MHS, M. hyopneumoniae, M. flocculare, or Friis medium, varied by assay, targeted antibody isotype, and cutoffs. The progressions of MHR and MHS clinical diseases were evaluated in relation to the kinetics of the isotype-specific antibody response in serum and bacterial shedding in oral fluids during the observation period. In pigs inoculated with MHR, bacterial DNA was detected in one or more of the 5 pens at all sampling points throughout the study, IgA was first detected at DPI 7, one week before the first clinical signs, with both IgA and IgG detected in all samples collected after DPI 14. The peak of MHS shedding (DPI 8) coincided with the onset of the clinical signs, with both IgA and IgG detected in all serum samples collected ≥ DPI 14. This study demonstrated, under experimental conditions, that both ELISAs were suitable for early detection of specific antibodies against MHR or MHS. The diagnostic performance of the MHR and MHS ELISAs varied depending on the selected cutoff and the antibody isotype evaluated. The high diagnostic and analytical specificity of the ELISAs was particularly remarkable. This study also provides insights into the infection dynamics of MHR-associated disease and MHS-associated arthritis not previously described.

Preoptimización de consultas en fuentes de datos con capacidades limitadas / Query preoptimization in data sources with limited capabilities

Tecnologías emergentes tales como Web Semántica, P2P y Grid Semántico han hecho accesible una gran cantidad de fuentes de datos, caracterizadas por propiedades que impactan en la complejidad de la optimización tradicional de consultas y en las tareas de procesamiento. En este trabajo se describe el efecto de las capacidades de consulta limitadas en el proceso de encontrar un buen plan para evaluar una consulta. Este problema es NP-Completo y diferentes enfoques han sido utilizados para encontrar soluciones aproximadas. Se considera el proyecto WebSrcMed, que implementa una estrategia de optimización de dos fases para encontrar planes de ejecución buenos en términos de bajo costo y seguros ya que, al menos, una combinación de fuentes de datos puede responder la consulta. En la primera fase, las descripciones de capacidades limitadas son consideradas para particionar el espacio de búsqueda en conjuntos de preplanes donde las capacidades de la consulta son satisfechas y se selecciona un buen preplán entre ellos. En la fase, el espacio de planes inducidos por el preplán es explorado y se produce un buen plan de ejecución. Debido a que espacio de búsqueda de los preplanes puede ser grande, se requieren técnicas eficientes para explorarlo adecuadamente. Se presenta un algoritmo evolutivo que encuentra eficientemente preplanes seguros y buenos. En un estudio experimental se observó que la solución encuentra buenos preplanes para consultas complejas. La eficiencia del algoritmo utilizado (número de soluciones diferentes respecto al número de soluciones encontradas) es superior a la eficiencia mostrada en técnicas aleatorias existentes.