Tick-borne pathogens Ehrlichia, Hepatozoon, and Babesia co-infection in owned dogs in Central Thailand.

Tick-borne pathogens are transmitted by a wide range of tick species and affect both human and animal health. Understanding the diversity of these pathogens and their co-infection rates in domesticated animals in urban areas is crucial for effective disease management and prevention. In this study, a total of 565 owned dogs in the central region of Thailand were investigated for the infection rate of three genera of Ehrlichia, Hepatozoon, and Babesia infection using multiplex PCR. The results revealed an overall infection rate of 19.1%, with Ehrlichia having the highest infection rate (12.2%), followed by Babesia (2.5%) and Hepatozoon (1.4%). The rate of co-infection was 3%, with mixed infections involving two or three genera. Male dogs exhibited a slightly higher infection rate compared to females, although not statistically significant. Young adult dogs (1-3 years) showed the highest infection rate of both single infections and co-infections. Monthly infection rate indicated variations throughout the year, with co-infection rate significantly associated with overall infection rate. Clinical manifestations in three genera of infected dogs included thrombocytopenia and eosinopenia. The results of this study are useful to design strategies for the management and prevention of tick-borne diseases in the study area.

Diversity and genetic characterization of Chlamydia isolated from Siamese crocodiles (Crocodylus siamensis).

Chlamydiosis, an infection caused by several Chlamydia species, has been reported in Nile, saltwater, and Siamese crocodiles. Despite its widespread reports in various countries, including Thailand, genetic information on Chlamydia species remains limited. This study presents a whole-genome-based characterization of Siamese crocodile-isolated Chlamydia. The results showed that Siamese crocodile Chlamydia contained a single circular chromosome with a size of 1.22-1.23 Mbp and a plasmid with a size of 7.7-8.0 kbp. A plasmid containing eight coding sequences (CDSs) was grouped in a ß lineage. A chromosome sequence had approximately 1,018-1,031 CDSs. Chlamydial factors involving virulence were documented in terms of the presence of cytotoxins and several virulence factors in the chromosomes of Siamese crocodile Chlamydia. The analysis of antimicrobial resistance genes in the Chlamydia genome revealed that the most common resistance genes were associated with aminoglycosides, fluoroquinolones, macrolides, tetracyclines, and cephalosporins, with loose matching (identities between 21.12 % and 74.65 %). Phylogenetic analyses, encompassing the assessments of both whole proteome and nine taxonomic markers, revealed that Siamese crocodile Chlamydia was separated into three lineages (lineages I-III) with high bootstrapping statistic support. Interestingly, isolate 12-01 differed genetically from the others, suggesting that it is a new member of Chlamydia. The study findings indicate that Siamese crocodiles are susceptible hosts to Chlamydia, involving more than one species. This study is the first employing the highest number of whole-genome data on Siamese crocodile Chlamydia and provides better insights into pathogen genetics.

Transcriptional Profiling of the Effect of Coleus amboinicus L. Essential Oil against Salmonella Typhimurium Biofilm Formation.

Salmonella enterica serovar Typhimurium cause infections primarily through foodborne transmission and remains a significant public health concern. The biofilm formation of this bacteria also contributes to their multidrug-resistant nature. Essential oils from medicinal plants are considered potential alternatives to conventional antibiotics. Therefore, this study assessed the antimicrobial and antibiofilm activities of Coleus amboinicus essential oil (EO-CA) against S. Typhimurium ATCC 14028. Seventeen chemical compounds of EO-CA were identified, and carvacrol (38.26%) was found to be the main constituent. The minimum inhibitory concentration (MIC) of EO-CA for S. Typhimurium planktonic growth was 1024 µg/mL while the minimum bactericidal concentration was 1024 µg/mL. EO-CA at sub-MIC (≥1/16× MIC) exhibited antibiofilm activity against the prebiofilm formation of S. Typhimurium at 24 h. Furthermore, EO-CA (≥1/4× MIC) inhibited postbiofilm formation at 24 and 48 h (p < 0.05). Transcriptional profiling revealed that the EO-CA-treated group at 1/2× MIC had 375 differentially expressed genes (DEGs), 106 of which were upregulated and 269 were downregulated. Five significantly downregulated virulent DEGs responsible for motility (flhD, fljB, and fimD), curli fimbriae (csgD), and invasion (hilA) were screened via quantitative reverse transcription PCR (qRT-PCR). This study suggests the potential of EO-CA as an effective antimicrobial agent for combating planktonic and biofilm formation of Salmonella.

Epidemiology of Chlamydia sp. infection in farmed Siamese crocodiles (Crocodylus siamensis) in Thailand.

BACKGROUND: Although Chlamydia sp. causes widespread disease outbreaks in juvenile crocodiles in Thailand, data regarding the epidemiology, and risk factors of such infections are limited. The aim of this study was to investigate the prevalence and possible risk factors associated with Chlamydia sp. infections on Siamese crocodile (Crocodylus siamensis) farms in Thailand. A cross-sectional study was conducted from July to December 2019. Samples were collected from 40 farms across six regions in Thailand. Conjunctival, pharyngeal, and cloacal swab samples were analyzed for Chlamydiaceae nucleic acids using semi-nested PCR followed by phylogenetic analysis based on the ompA gene fragment. Risk factors of infection were analyzed using chi-square and univariate regression to calculate odds ratios. RESULTS: The prevalence of Chlamydia sp. infection across all regions was 65%. The ompA phylogenetic analysis showed that Chlamydia sp. detected in this study was genetically closely related to Chlamydia crocodili and Chlamydia caviae. The risk factors for infection were water source, reusing treated wastewater from the treatment pond, not disposing of leftover food, low frequency of water replacement in the enclosure of juvenile crocodiles, and lack of water replacement after the death of a crocodile. CONCLUSION: The prevalence of Chlamydia sp. infection in farmed crocodiles in Thailand was 65% during the study period. Cloacal swabs were superior to conjunctival and pharyngeal swabs due to their higher sensitivity in detecting Chlamydia sp., as well as their lower invasiveness. Good management and biosecurity in crocodile farming can reduce the risk of Chlamydia sp.

Plant molecular farming-derived epidermal growth factor revolutionizes hydrogels for improving glandular epithelial organoid biofabrication.

Epidermal growth factor (EGF) is a known signaling cue essential towards the development and organoid biofabrication particularly for exocrine glands. This study developed an in vitro EGF delivery platform with Nicotiana benthamiana plant-produced EGF (P-EGF) encapsulated on hyaluronic acid/alginate (HA/Alg) hydrogel to improve the effectiveness of glandular organoid biofabrication in short-term culture systems. Primary submandibular gland epithelial cells were treated with 5 - 20 ng/mL of P-EGF and commercially available bacteria-derived EGF (B-EGF). Cell proliferation and metabolic activity were measured by MTT and luciferase-based ATP assays. P-EGF and B-EGF 5 - 20 ng/mL promoted glandular epithelial cell proliferation during 6 culture days on a comparable fashion. Organoid forming efficiency and cellular viability, ATP-dependent activity and expansion were evaluated using two EGF delivery systems, HA/Alg-based encapsulation and media supplementation. Phosphate buffered saline (PBS) was used as a control vehicle. Epithelial organoids fabricated from PBS-, B-EGF-, and P-EGF-encapsulated hydrogels were characterized genotypically, phenotypically and by functional assays. P-EGF-encapsulated hydrogel enhanced organoid formation efficiency and cellular viability and metabolism relative to P-EGF supplementation. At culture day 3, epithelial organoids developed from P-EGF-encapsulated HA/Alg platform contained functional cell clusters expressing specific glandular epithelial markers such as exocrine pro-acinar (AQP5, NKCC1, CHRM1, CHRM3, Mist1), ductal (K18, Krt19), and myoepithelial (α-SMA, Acta2), and possessed a high mitotic activity (38-62% Ki67 cells) with a large epithelial progenitor population (∼70% K14 cells). The P-EGF encapsulation strikingly upregulated the expression of pro-acinar AQP5 cells through culture time when compared to others (B-EGF, PBS). Thus, the utilization of Nicotiana benthamiana in molecular farming can produce EGF biologicals amenable to encapsulation in HA/Alg-based in vitro platforms, which can effectively and promptly induce the biofabrication of exocrine gland organoids.

Natural Antioxidant, Antibacterial, and Antiproliferative Activities of Ethanolic Extracts from Punica granatum L. Tree Barks Mediated by Extracellular Signal-Regulated Kinase.

The nonedible parts of the pomegranate plant, such as tree barks and fruit peels, have pharmacological properties that are useful in traditional medicine. To increase their value, this study aimed to compare the antioxidative and antibacterial effects of ethanolic extracts from pomegranate barks (PBE) and peels (PPE). The antiproliferative effects on HeLa and HepG2 cells through the extracellular signal-regulated kinase pathway were also evaluated. The results indicated that the total amounts of phenolics and flavonoids of PBE and PPE were 574.64 and 242.60 mg equivalent gallic acid/g sample and 52.98 and 23.08 mg equivalent quercetin/g sample, respectively. Gas chromatography−mass spectrometry revealed that 5-hdroxymethylfurfural was the major component of both PBE (23.76%) and PPE (33.19%). The 2,2-diphenyl-1-picryl-hydrazyl-hydrate free radical scavenging capacities of PBE and PPE, in terms of the IC50 value, were 4.1 and 9.6 µg/mL, respectively. PBE had a greater potent antibacterial effect against Escherichia coli, Staphylococcus aureus, Salmonella Enteritidis, and S. Typhimurium. PBE and PPE (1000 µg/mL) had exhibited no cytotoxic effects on LLC-MK2. PBE and PPE (250 and 1000 µg/mL, respectively) treatments were safe for BHK-21. Both extracts significantly inhibited HepG2 and HeLa cell proliferations at 10 and 50 µg/mL, respectively (p < 0.001). The results indicated that PBE and PPE have remarkable efficiencies as free radical scavengers and antibacterial agents, with PBE exhibiting greater efficiency. The inhibitory effects on HepG2 might be through the modulation of the ERK1/2 expression. PBE and PPE have the potential for use as optional supplementary antioxidative, antibacterial, and anticancer agents.

Coding-Complete Genome Sequence of a Lumpy Skin Disease Virus Isolated during the 2021 Thailand Outbreak.

Lumpy skin disease (LSD) is an economically devastating and transboundary disease in cattle. Here, we report the coding-complete genome sequence of the LSDV72/PrachuapKhiriKhan/Thailand/2021 strain, which was isolated from an affected cow during the first LSD outbreak in Thailand in 2021. The sequence will be beneficial for future genomic studies of the virus.

Magnetic bioassembly platforms towards the generation of extracellular vesicles from human salivary gland functional organoids for epithelial repair.

Salivary glands (SG) are exocrine organs with secretory units commonly injured by radiotherapy. Bio-engineered organoids and extracellular vesicles (EV) are currently under investigation as potential strategies for SG repair. Herein, three-dimensional (3D) cultures of SG functional organoids (SGo) and human dental pulp stem cells (hDPSC) were generated by magnetic 3D bioassembly (M3DB) platforms. Fibroblast growth factor 10 (FGF10) was used to enrich the SGo in secretory epithelial units. After 11 culture days via M3DB, SGo displayed SG-specific acinar epithelial units with functional properties upon neurostimulation. To consistently develop 3D hDPSC in vitro, 3 culture days were sufficient to maintain hDPSC undifferentiated genotype and phenotype for EV generation. EV isolation was performed via sequential centrifugation of the conditioned media of hDPSC and SGo cultures. EV were characterized by nanoparticle tracking analysis, electron microscopy and immunoblotting. EV were in the exosome range for hDPSC (diameter: 88.03 ± 15.60 nm) and for SGo (123.15 ± 63.06 nm). Upon ex vivo administration, exosomes derived from SGo significantly stimulated epithelial growth (up to 60%), mitosis, epithelial progenitors and neuronal growth in injured SG; however, such biological effects were less distinctive with the ones derived from hDPSC. Next, these exosome biological effects were investigated by proteomic arrays. Mass spectrometry profiling of SGo exosomes predicted that cellular growth, development and signaling was due to known and undocumented molecular targets downstream of FGF10. Semaphorins were identified as one of the novel targets requiring further investigations. Thus, M3DB platforms can generate exosomes with potential to ameliorate SG epithelial damage.

Molecular detection and characterization of lumpy skin disease viruses from outbreaks in Thailand in 2021.

Lumpy skin disease (LSD) is one of the most important transboundary and emerging diseases in cattle. The disease causes significant economic losses in animal production and trade worldwide. The first LSD outbreak was recorded in March 2021, at Roi-Et province in the northeastern region of Thailand. Thereafter, the disease had rapidly spread into neighbouring provinces and throughout the country. The aim of the present study was to provide information regarding to the molecular detection and characterization of LSD viruses from outbreaks in Thailand in 2021. There were 1,748,112 susceptible and 604,404 affected animals (n = 588,512 [36.30%], beef cattle; n = 12,367 [15.74%], dairy cattle and n = 3524 [7.35%], buffaloes). The morbidity and mortality rates were 34.57% and 3.47%, respectively, and the case fatality rate was 10.05% (60,713 deaths). Based on real-time polymerase chain reaction results, the p32 gene of LSD virus (LSDV) was detected more frequently in skin nodule samples (54/77, 70.13%) than in nasal swabs (26/55, 42.57%) and EDTA blood (16/77, 20.78%) samples. Moreover, the copy number of the p32 gene was higher in skin nodule samples than in nasal swab and EDTA blood samples (cycle threshold value = 21.94 ± 0.62 vs. 31.52 ± 0.66 and 34.27 ± 0.32, respectively). Furthermore, 29 (53.70%) of 54 capripoxvirus-positive skin nodule samples were successfully isolated from Madin-Darby bovine kidney cells, and the cytopathic effect was observed 72 h after inoculation. Based on the phylogenetic trees of the GPCR, ANK and RPO30 gene sequences, the LSDV isolates from Thailand were distinct from both the LSDV-field and LSDV-vaccine groups and were closely correlated with the LSDV strains isolated from mainland China, Hong Kong territory and Vietnam in 2020. Additionally, they could be a potential virulent vaccine-recombinant LSDV strain.

Zoonotic pathogens survey in free-living long-tailed macaques in Thailand.

Long-tailed macaques (Macaca fascicularis) are known to harbour a variety of infectious pathogens, including zoonotic species. Long-tailed macaques and humans coexist in Thailand, which creates potential for interspecies pathogen transmission. This study was conducted to assess the presence of B virus, Mycobacterium spp., simian foamy virus (SFV), hepatitis B virus (HBV), and Plasmodium spp. in 649 free-living Thai long-tailed macaques through polymerase-chain reaction. DNA of SFV (56.5%), HBV (0.3%), and Plasmodium spp. (2.2%) was detected in these macaques, whereas DNA of B virus and Mycobacterium spp. was absent. SFV infection in long-tailed macaques is broadly distributed in Thailand and is correlated with age. The HBV sequences in this study were similar to HBV sequences from orangutans. Plasmodium spp. DNA was identified as P. inui. Collectively, our results indicate that macaques can carry zoonotic pathogens, which have a public health impact. Surveillance and awareness of pathogen transmission between monkeys and humans are important.

Evidence of Influenza A Virus Infection in Cynomolgus Macaques, Thailand.

Little is known about the ecology of influenza A virus (IAV) in nonhuman primates (NHPs). We conducted active surveillance of IAV among 672 cynomolgus macaques (Macaca fascicularis) living in 27 free-ranging colonies in Thailand between March and November 2019. A hemagglutination inhibition (HI) assay was employed as the screening test against 16 subtypes of avian influenza virus (AIV) and two strains of the H1 subtype of human influenza virus. The serum samples with HI titers ≥20 were further confirmed by microneutralization (MN) assay. Real-time RT-PCR assay was performed to detect the conserved region of the influenza matrix (M) gene. The seropositive rate for subtypes of IAV, including AIV H1 (1.6%, 11/672), AIV H2 (15.2%, 102/672), AIV H3 (0.3%, 2/672), AIV H9 (3.4%, 23/672), and human H1 (NP-045) (0.9%, 6/672), was demonstrated. We also found antibody against more than one subtype of IAV in 15 out of 128 positive tested sera (11.7%). Moreover, influenza genome could be detected in 1 out of 245 pool swab samples (0.41%). Evidence of IAV infection presented here emphasizes the role of NHPs in the ecology of the virus. Our findings highlight the need to further conduct a continuous active surveillance program in NHP populations.

Overview of avian influenza virus in urban feral pigeons in Bangkok, Thailand.

This survey assessed the presence of avian influenza virus (AIV) in urban feral pigeons (UFPs) in Bangkok, Thailand. A total of 485 UFPs were collected from eight study sites, and blood, tracheal, and cloacal samples were collected from each bird. Virus isolation and molecular methods did not detect AIV in any of the birds tested. A hemagglutination inhibition test was used to test for antibodies to high and low pathogenicity AIV subtypes. AIV subtype H9 antibodies were the only antibodies detected. The overall seroprevalence of AIV subtype H9 antibodies was 6.9%, and subtype H9 antibodies were found in UFPs at all eight sites. The overall geometric mean titer was 11.07 (range: 8-64). These results reveal that UFPs in Bangkok do not currently pose a risk of transmitting AIV to humans. However, monitoring of AIV in UFPs is necessary for disease control and to minimize the possibility of influenza outbreaks.

Assessing Coagulation Parameters in Healthy Asian Elephants (Elephas maximus) from European and Thai Populations.

The Asian elephant population is continuously declining due to several extrinsic reasons in their range countries, but also due to diseases in captive populations worldwide. One of these diseases, the elephant endotheliotropic herpesvirus (EEHV) hemorrhagic disease, is very impactful because it particularly affects Asian elephant calves. It is commonly fatal and presents as an acute and generalized hemorrhagic syndrome. Therefore, having reference values of coagulation parameters, and obtaining such values for diseased animals in a very short time, is of great importance. We analyzed prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen concentrations using a portable and fast point-of-care analyzer (VetScan Pro) in 127 Asian elephants from Thai camps and European captive herds. We found significantly different PT and aPTT coagulation times between elephants from the two regions, as well as clear differences in fibrinogen concentration. Nevertheless, these alterations were not expected to have biological or clinical implications. We have also sequenced the coagulation factor VII gene of 141 animals to assess the presence of a previously reported hereditary coagulation disorder in Asian elephants and to investigate the presence of other mutations. We did not find the previously reported mutation in our study population. Instead, we discovered the presence of several new single nucleotide polymorphisms, two of them being considered as deleterious by effect prediction software.

Coinfection of Chlamydia spp. and herpesvirus in juvenile farmed Siamese crocodiles (Crocodylus siamensis) in Thailand.

BACKGROUND AND AIM: For a decade, chlamydial and herpesvirus infections have caused significant morbidity and mortality in farmed crocodiles. In September 2017, a total of 160 juvenile freshwater Siamese crocodiles (Crocodylus siamensis) with conjunctivitis/pharyngitis lesions were admitted at the Veterinary Aquatic Animal Research Health Care Unit, Faculty of Veterinary Science, Mahidol University. All crocodiles did not respond well to antibiotics or supportive treatments and died. This study aimed to detect and identify the causative agents associated with conjunctivitis/pharyngitis and fatal outcomes in juvenile farmed Siamese crocodiles. MATERIALS AND METHODS: A total of 138 pharyngeal and conjunctival swabs and conjunctival scrapes were collected from live crocodiles. All swab and scrape samples were DNA-extracted and amplified by polymerase chain reaction (PCR) using Chlamydiaceae- and herpesvirus-specific primers. Tissue samples (brain, lung, liver, heart, spleen, and intestine) were collected from two representative postmortem animals. All tissue samples were processed for molecular and pathological analyses. RESULTS: PCR examinations identified chlamydial and herpesvirus DNA in 92% (126/138) and 100% (138/138), respectively, of the tested swab and scrape samples. Of those positive samples, 79% (26/33), 67% (4/6), and 98% (97/99) of the pharyngeal swabs, conjunctival swabs, and conjunctival scrapes, respectively, were positive for both chlamydial and herpesvirus DNA. Histopathological examination indicated necrosis and mononuclear cell infiltration in the liver, kidney, and intestine of the affected animals. The intracytoplasmic accumulation of Chlamydia was randomly observed in the examined tissue sample. Moreover, the presence of chlamydial and herpesvirus DNA was also detected in the tissue samples, including the heart, intestine, brain, lung, liver, and spleen, of the affected animals by PCR. Phylogenetic analyses revealed that Chlamydia spp. detected in the juvenile Siamese crocodiles was notably different from other known species in the Chlamydia genus, while the herpesvirus detected in the crocodiles was closely related to crocodyline herpesvirus 1. CONCLUSION: Based on histopathological and molecular examinations, this report provided the first evidence of coinfection of Chlamydia spp. and crocodyline herpesvirus 1 in juvenile Siamese crocodiles in Thailand.

Exogenous LIN28 Is Required for the Maintenance of Self-Renewal and Pluripotency in Presumptive Porcine-Induced Pluripotent Stem Cells.

Porcine species have been used in preclinical transplantation models for assessing the efficiency and safety of transplants before their application in human trials. Porcine-induced pluripotent stem cells (piPSCs) are traditionally established using four transcription factors (4TF): OCT4, SOX2, KLF4, and C-MYC. However, the inefficiencies in the reprogramming of piPSCs and the maintenance of their self-renewal and pluripotency remain challenges to be resolved. LIN28 was demonstrated to play a vital role in the induction of pluripotency in humans. To investigate whether this factor is similarly required by piPSCs, the effects of adding LIN28 to the 4TF induction method (5F approach) on the efficiency of piPSC reprogramming and maintenance of self-renewal and pluripotency were examined. Using a retroviral vector, porcine fetal fibroblasts were transfected with human OCT4, SOX2, KLF4, and C-MYC with or without LIN28. The colony morphology and chromosomal stability of these piPSC lines were examined and their pluripotency properties were characterized by investigating both their expression of pluripotency-associated genes and proteins and in vitro and in vivo differentiation capabilities. Alkaline phosphatase assay revealed the reprogramming efficiencies to be 0.33 and 0.17% for the 4TF and 5TF approaches, respectively, but the maintenance of self-renewal and pluripotency until passage 40 was 6.67 and 100%, respectively. Most of the 4TF-piPSC colonies were flat in shape, showed weak positivity for alkaline phosphatase, and expressed a significantly high level of SSEA-4 protein, except for one cell line (VSMUi001-A) whose properties were similar to those of the 5TF-piPSCs; that is, tightly packed and dome-like in shape, markedly positive for alkaline phosphatase, and expressing endogenous pluripotency genes (pOCT4, pSOX2, pNANOG, and pLIN28), significantly high levels of pluripotent proteins (OCT4, SOX2, NANOG, LIN28, and SSEA-1), and a significantly low level of SSEA-4 protein. VSMUi001-A and all 5F-piPSC lines formed embryoid bodies, underwent spontaneous cardiogenic differentiation with cardiac beating, expressed cardiomyocyte markers, and developed teratomas. In conclusion, in addition to the 4TF, LIN28 is required for the effective induction of piPSCs and the maintenance of their long-term self-renewal and pluripotency toward the development of all germ layers. These piPSCs have the potential applicability for veterinary science.

A new species of Chlamydia isolated from Siamese crocodiles (Crocodylus siamensis).

Chlamydia is a known pathogen in both saltwater and freshwater crocodiles. However, the exact species/strain has not been clearly identified. In this study, we successfully cultivated Siamese crocodile Chlamydia in McCoy cells at a temperature of 30°C. Electron microscopy; phylogeny based on nine conserved taxonomically informative markers, on ompA, or on seven housekeeping genes; and whole-genome sequencing and analysis of the isolate confirmed the identity of the isolate as a new member of the genus Chlamydia, a new species that we name Chlamydia crocodili.

Epigallocatechin-3-Gallate Protects Pro-Acinar Epithelia Against Salivary Gland Radiation Injury.

Antioxidant agents are promising pharmaceuticals to prevent salivary gland (SG) epithelial injury from radiotherapy and their associated irreversible dry mouth symptoms. Epigallocatechin-3-gallate (EGCG) is a well-known antioxidant that can exert growth or inhibitory biological effects in normal or pathological tissues leading to disease prevention. The effects of EGCG in the various SG epithelial compartments are poorly understood during homeostasis and upon radiation (IR) injury. This study aims to: (1) determine whether EGCG can support epithelial proliferation during homeostasis; and (2) investigate what epithelial cells are protected by EGCG from IR injury. Ex vivo mouse SG were treated with EGCG from 7.5-30 µg/mL for up to 72 h. Next, SG epithelial branching morphogenesis was evaluated by bright-field microscopy, immunofluorescence, and gene expression arrays. To establish IR injury models, linear accelerator (LINAC) technologies were utilized, and radiation doses optimized. EGCG epithelial effects in these injury models were assessed using light, confocal and electron microscopy, the Griess assay, immunohistochemistry, and gene arrays. SG pretreated with EGCG 7.5 µg/mL promoted epithelial proliferation and the development of pro-acinar buds and ducts in regular homeostasis. Furthermore, EGCG increased the populations of epithelial progenitors in buds and ducts and pro-acinar cells, most probably due to its observed antioxidant activity after IR injury, which prevented epithelial apoptosis. Future studies will assess the potential for nanocarriers to increase the oral bioavailability of EGCG.

Generation of Porcine Induced Neural Stem Cells Using the Sendai Virus.

The reprogramming of cells into induced neural stem cells (iNSCs), which are faster and safer to generate than induced pluripotent stem cells, holds tremendous promise for fundamental and frontier research, as well as personalized cell-based therapies for neurological diseases. However, reprogramming cells with viral vectors increases the risk of tumor development due to vector and transgene integration in the host cell genome. To circumvent this issue, the Sendai virus (SeV) provides an alternative integration-free reprogramming method that removes the danger of genetic alterations and enhances the prospects of iNSCs from bench to bedside. Since pigs are among the most successful large animal models in biomedical research, porcine iNSCs (piNSCs) may serve as a disease model for both veterinary and human medicine. Here, we report the successful generation of piNSC lines from pig fibroblasts by employing the SeV. These piNSCs can be expanded for up to 40 passages in a monolayer culture and produce neurospheres in a suspension culture. These piNSCs express high levels of NSC markers (PAX6, SOX2, NESTIN, and VIMENTIN) and proliferation markers (KI67) using quantitative immunostaining and western blot analysis. Furthermore, piNSCs are multipotent, as they are capable of producing neurons and glia, as demonstrated by their expressions of TUJ1, MAP2, TH, MBP, and GFAP proteins. During the reprogramming of piNSCs with the SeV, no induced pluripotent stem cells developed, and the established piNSCs did not express OCT4, NANOG, and SSEA1. Hence, the use of the SeV can reprogram porcine somatic cells without first going through an intermediate pluripotent state. Our research produced piNSCs using SeV methods in novel, easily accessible large animal cell culture models for evaluating the efficacy of iNSC-based clinical translation in human medicine. Additionally, our piNSCs are potentially applicable in disease modeling in pigs and regenerative therapies in veterinary medicine.

Satellite telemetry tracks flyways of Asian Openbill storks in relation to H5N1 avian influenza spread and ecological change.

BACKGROUND: Asian Openbills, Anastomus oscitans, have long been known to migrate from South to Southeast Asia for breeding and nesting. In Thailand, the first outbreak of H5N1 highly pathogenic avian influenza (HPAI) infection in the Openbills coincided with the outbreak in the poultry. Therefore, the flyways of Asian Openbills was determined to study their role in the spread of H5N1 HPAI virus to poultry and wild birds, and also within their flocks. RESULTS: Flyways of 5 Openbills from 3 colonies were monitored using Argos satellite transmitters with positioning by Google Earth Programme between 2007 and 2013. None of the Openbills tagged with satellite telemeters moved outside of Thailand. Their home ranges or movement areas varied from 1.6 to 23,608 km2 per month (95% utility distribution). There was no positive result of the viral infection from oral and cloacal swabs of the Openbills and wild birds living in the vicinity by viral isolation and genome detection during 2007 to 2010 whereas the specific antibody was not detected on both Openbills and wild birds by using microneutralization assay after 2008. The movement of these Openbills did not correlate with H5N1 HPAI outbreaks in domestic poultry but correlated with rice crop rotation and populations of the apple snails which are their preferred food. Viral spread within the flocks of Openbills was not detected. CONCLUSIONS: This study showed that Openbills played no role in the spread of H5N1 HPAI virus, which was probably due to the very low prevalence of the virus during the monitoring period. This study revealed the ecological factors that control the life cycle of Asian Openbills.

Generation of a pig induced pluripotent stem cell (piPSC) line from embryonic fibroblasts by incorporating LIN28 to the four transcriptional factor-mediated reprogramming: VSMUi001-D.

Pig induced pluripotent stem cell (piPSC) line was generated from embryonic fibroblast cells using retroviral transduction approaches carrying human transcriptional factors: OCT4, SOX2, KLF4, c-MYC and LIN28. The generated piPSC line, VSMUi001-D, was positive for alkaline phosphatase activity and expressed the pluripotency associated transcription factors including OCT4, SOX2, NANOG and surface markers SSEA-1, all iPSC hallmarks of authenticity. Furthermore, VSMUi001-D exhibited a normal karyotype and formed embryoid bodies in vitro and teratomas in vivo. Upon cardiac differentiation, VSMUi001-D displayed spontaneous beating and expressed cardiomyocyte markers, like cardiac Troponin T.