Porcine epidemic diarrhea virus (PEDV) causes devastating enteric disease that inflicts huge economic damage on the swine industry worldwide. A safe and highly effective PEDV vaccine that contains only the virus-neutralizing epitopes (not enhancing epitope), as well as a ready-to-use PEDV neutralizing antibody for the passive immunization of PEDV vulnerable piglets (during the first week of life) are needed, particularly for PEDV-endemic farms. In this study, we generated monoclonal antibodies (mAbs) to the recombinant S1 domain of PEDV spike (S) protein and tested their PEDV neutralizing activity by CPE-reduction assay. The mAb secreted by one hybrodoma clone (A3), that also bound to the native S1 counterpart from PEDV-infected cells (tested by combined co-immunoprecipitation and Western blotting), neutralized PEDV infectivity. Epitope of the neutralizing mAb (mAbA3) locates in the S1A subdomain of the spike protein, as identified by phage mimotope search and multiple sequence alignment, and peptide binding-ELISA. The newly identified epitope is shared by PEDV G1 and G2 strains and other alphacoronaviruses. In summary, mAbA3 may be useful as a ready-to-use antibody for passive immunization of PEDV-susceptible piglets, while the novel neutralizing epitope, together with other, previously known protective epitopes, have potential as an immunogenic cocktail for a safe, next-generation PEDV vaccine.
Coimplantation of endothelial cells (ECs) and mesenchymal stromal cells (MSCs) into the transplantation site could be a feasible option to achieve a sufficient level of graft-host vascularization. To find a suitable source of tissue that provides a large number of high-quality ECs and MSCs suited for future clinical application, we developed a simplified xeno-free strategy for isolation of human umbilical vein endothelial cells (HUVECs) and Wharton’s jelly-derived mesenchymal stromal cells (WJ-MSCs) from the same umbilical cord. We also assessed whether the coculture of HUVECs and WJ-MSCs derived from the same umbilical cord (autogenic cell source) or from different umbilical cords (allogenic cell sources) had an impact on in vitro angiogenic capacity. We found that HUVECs grown in 5 ng/ml epidermal growth factor (EGF) supplemented xeno-free condition showed higher proliferation potential compared to other conditions. HUVECs and WJ-MSCs obtained from this technic show an endothelial lineage (CD31 and von Willebrand factor) and MSC (CD73, CD90, and CD105) immunophenotype characteristic with high purity, respectively. It was also found that only the coculture of HUVEC/WJ-MSC, but not HUVEC or WJ-MSC mono-culture, provides a positive effect on vessel-like structure (VLS) formation, in vitro. Further investigations are needed to clarify the pros and cons of using autogenic or allogenic source of EC/MSC in tissue engineering applications. To the best of our knowledge, this study offers a simple, but reliable, xeno-free strategy to establish ECs and MSCs from the same umbilical cord, a new opportunity to facilitate the development of personal cell-based therapy.
Porcine epidemic diarrhea virus (PEDV) is the causative agent of a highly contagious enteric disease of pigs characterized by diarrhea, vomiting, and severe dehydration. PEDV infects pigs of all ages, but neonatal pigs during the first week of life are highly susceptible; the mortality rates among newborn piglets may reach 80–100%. Thus, PEDV is regarded as one of the most devastating pig viruses that cause huge economic damage to pig industries worldwide. Vaccination of sows and gilts at the pre-fertilization or pre-farrowing stage is a good strategy for the protection of suckling piglets against PEDV through the acquisition of the lactating immunity. However, vaccination of the mother pigs for inducing a high level of virus-neutralizing antibodies is complicated with unstandardized immunization protocol and unreliable outcomes. Besides, the vaccine may also induce enhancing antibodies that promote virus entry and replication, so-called antibody-dependent enhancement (ADE), which aggravates the disease upon new virus exposure. Recognition of the virus epitope that induces the production of the enhancing antibodies is an existential necessity for safe and effective PEDV vaccine design. In this study, the enhancing epitope of the PEDV spike (S) protein was revealed for the first time, by using phage display technology and mouse monoclonal antibody (mAbG3) that bound to the PEDV S1 subunit of the S protein and enhanced PEDV entry into permissive Vero cells that lack Fc receptor. The phages displaying mAbG3-bound peptides derived from the phage library by panning with the mAbG3 matched with several regions in the S1-0 sub-domain of the PEDV S1 subunit, indicating that the epitope is discontinuous (conformational). The mAbG3-bound phage sequence also matched with a linear sequence of the S1-BCD sub-domains. Immunological assays verified the phage mimotope results. Although the molecular mechanism of ADE caused by the mAbG3 via binding to the newly identified S1 enhancing epitope awaits investigation, the data obtained from this study are helpful and useful in designing a safe and effective PEDV protein subunit/DNA vaccine devoid of the enhancing epitope.
Background and Aim: The Sunda pangolin (Manis javanica) is on the International Union for Conservation of Nature Red List of Threatened Species (critically endangered) due to high levels of illegal trafficking for its products. Thailand is one of the habitats of this species, and it has become the main hub for its illegal trafficking. Rehabilitating these captive pangolins and reintroducing them back to the wild are challenging due to the limited knowledge on their diet, management, and diseases. Hemoparasites, including Babesia spp., can cause important protozoal infections in both domestic and wild animals, resulting in the failure of rehabilitation and conservation programs. However, Babesia spp. has not been reported in pangolins. The aim of the study was to determine the prevalence of Babesia spp. in the Sunda pangolin of Thailand. Materials and Methods: A total of 128 confiscated Sunda pangolins from across different regions in Thailand were investigated. These pangolins had been admitted to a regional Wildlife Quarantine Center for rehabilitation before release in the forest. Routine physical examinations were conducted on the animals. We collected blood samples from each pangolin for hematological analysis and to detect Babesia spp. using polymerase chain reaction (PCR) targeting the partial 18s rRNA gene. Results: Babesia-specific PCR detected 53 animals (41.4%) that were positive for Babesia spp. Blood smears were obtained from the positive samples and investigated under a light microscope to observe for trophozoites of Babesia spp. Examination of 40 PCR-positive and -negative samples found no significant differences between the hematological parameters of Babesia-positive and Babesia-negative samples. Eight PCR-positive samples were randomly selected and their DNA was sequenced. Seven and one of sequences match uncharacterized Babesia spp. with 100% and 99.2% similarity, respectively. Phylogenetic analysis demonstrated that our samples form a unique monophyletic clade along with other Babesia spp. detected in the wild. This clade is clearly separated from other Babesia spp. from small carnivores, ruminants, and rats. Conclusion: Our results provide evidence of infection of Sunda pangolins in Thailand by Babesia spp. These pangolins originated from different regions and had not lived together before blood collection. Thus, we suggest that the uncharacterized Babesia spp. found in this study constitute a new group of pangolin-specific Babesia spp. The prevalence of the uncharacterized Babesia spp. was not correlated to pangolin health. Further studies are required to characterize the genomes and phenotypes, including the morphology and pathogenicity of these protozoa. Such information will be helpful for the conservation and health management of the Sunda pangolin.
A 5-year-old female neutered domestic short-haired cat presented with abdominal enlargement. An abdominal ultrasound revealed that large multiple hepatic cysts with irregular walls, hypoechoic fluid, and internal septations occupied most of the liver parenchyma. Serum liver enzymes, bilirubin, and bile acids concentrations were within normal limits. A fecal examination using simple floatation and formalin-ether sedimentation techniques was negative for liver fluke (Platynosomum fastosum), intestinal protozoa, and other helminth eggs. Praziquantel was prescribed for two distinct courses one month apart without obvious improvement of the hepatic cysts. An abdominal laparotomy and histopathological examination finally enabled diagnosis of cyst-like lymphocytic cholangiohepatitis of the liver tissue. Twelve weeks of oral prednisolone resulted in marked ultrasonographic improvement of the hepatic cysts. The liver parenchyma was heterogeneous and filled with multiple small anechoic cavities. Twenty-three months after ceasing the prednisolone, there was no recurrence of hepatic cysts.
Background and Aim: The crocodile is a model for studying relevant sources of environmental contamination. They were determined an appropriate biomonitoring species for various toxins. The cytosolic and microsomal fraction of crocodiles plays a role in detoxifying xenobiotics. Cytochrome P450 1A2 (CYP1A2) metabolizes aflatoxin B1 (AFB1) to aflatoxin M1, while glutathione-S-transferase (GST) catalyzes carcinogenic agents. This study aimed to investigate the GST activity in various organs of Crocodylus siamensis. Further, the fate of microsomal and cytosolic fractions from various crocodile organs against AFB1-induced apoptosis in human hepatocarcinoma (HepG2) cells was investigated. Materials and Methods: The liver, lungs, intestines, and kidneys tissues from a 3-year-old crocodile (C. siamensis) (n=5) were collected. The cytosolic and microsomal fraction of all tissues was extracted, and protein concentrations were measured with a spectrophotometer. Subsequently, a comparison of GST activity from various organs was carried out by spectrophotometry, and the protective effects of CYP450 and GST activity from various crocodile organs were studied. In vitro AFB1-induced apoptosis in HepG2 cells was detected by reverse transcription-quantitative polymerase chain reaction. Comparisons between the metabolisms of the detoxification enzyme in organs were tested using the Kruskal–Wallis one-way analysis of variance and Dunn's multiple comparison tests. All kinetic parameters were analyzed using GraphPad Prism software version 5.01 (GraphPad Software Inc., San Diego, USA). Results: Total GST activity in the liver was significantly higher than in the kidneys, intestines, and lungs (p<0.05, respectively). The highest GST pi (GSTP) activity was found in the liver, while the highest GST alpha-isoform activity was in the crocodile lung. The kinetics of total GST and GST mu activity in the liver had the highest velocity compared to other organs. In contrast, the kinetics of GSTP enzyme activity was the highest in the intestine. The in vitro study of microsome and cytosol extract against apoptosis induced by AFB1 revealed that the level of messenger RNA expression of the Bax and Bad genes of HepG2 cells decreased in the treatment group in a combination of cytosolic and microsomal fractions of the crocodile liver but not for Bcl-2. Interestingly, the downregulated expression of Bax and Bad genes was also found in the microsome and cytosol of crocodile kidneys. Conclusion: The crocodile liver revealed very effective GST activity and expression of the highest kinetic velocity compared to other organs. The combination of liver microsomal and cytosolic fractions could be used to prevent cell apoptosis induced by AFB1. However, further study of the molecular approaches to enzyme activity and apoptosis prevention mechanisms should be carried out.
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