Identification of a novel porcine Teschovirus 2 strain as causative agent of encephalomyelitis in suckling piglets with high mortality in China.

BACKGROUND: Porcine Teschovirus (PTV), also named Teschovirus A, is prevalent in pig populations, mainly causing neurological symptoms, diarrhea, pneumonia, and reproductive failure, however the morbidity and mortality are usually low in pig farms. CASE PRESENTATION: In this study, we reported a PTV outbreak investigation in one large-scale pig farm in China with severe symptoms including diarrhea, lethargy, locomotor ataxia, nystagmus, paralysis of the hind limbs, and coma in piglets. More importantly, the mortality reached 38% in suckling pigs, which is remarkably high in PTV history. A novel PTV strain, named HeNZ1, was isolated from cerebral samples of one suckling pig and the genome sequence was obtained by NGS sequencing. Phylogenetic and evolutionary divergence analyses revealed that HeNZ1 belongs to PTV genotype 2. Surprisingly, the VP1 coding region of HeNZ1 shares the highest sequence similarity with European PTV-2 strains, instead of China domestic PTV-2 strains, implying it may not derive from China local PTV-2 strains. Multiple sequence alignment and B cell epitope prediction of PTV VP1 and VP2 protein revealed 10 B cell epitopes, 5 mutant clusters and 36 unique mutation sites, of which 19 unique mutation sites are located in B cell epitopes and exposed on the surface of VP1 or VP2, implying significant antigenic drift potential of HeNZ1. CONCLUSION: These results indicate that HeNZ1 is a highly virulent PTV-2 strain, which capable of causing severe neurological symptoms and high mortality in piglets. Bioinformatic analysis suggest that HeNZ1 is genetically and antigenically different from other Chinese PTV-2 strains. Overall, current case expanded our understanding of PTV-2 clinical spectrum and revealed the emergence of a highly virulent PTV-2 strain with substantial genetic diversity and antigenic drift potential in VP1 and VP2.

Fecal metabolomic analysis of rabbits infected with Eimeria intestinalis and Eimeria magna based on LC-MS/MS technique.

Rabbit coccidiosis is a common parasitic disease leading to economic losses in the rabbit industry. The intestinal flora plays a key role in pathogenesis of coccidiosis, and fecal metabolome mediates host-microbiome interactions as a functional readout of the gut microbiome. In this study, the E. intestinalis-infected and E. magna-infected rabbit models were established to investigate metabolic alterations and metabolic pathways based on LC-MS/MS technique for the first time. Multivariate OPLS-DA analysis was performed to explore differential metabolites. In total, 288 metabolites were detected from infected and uninfected rabbits. The level of 33 metabolites increased and 4 decreased in rabbits infected with E. intestinalis. Eight pathways were significantly perturbed during E. intestinalis infection including biosynthesis of unsaturated fatty acids, fatty acid biosynthesis, etc. After rabbits infected with E. magna, 13 metabolites were altered and 7 metabolic pathways were dysregulated. These metabolites and metabolic pathways were mainly involved in tuberculosis, parathyroid hormone synthesis, etc. Besides, 25 metabolites differed in abundance between E. intestinalis infection group and E. magna infection group, the major perturbed metabolic pathways were lipid metabolism and endocrine system, respectively. In general, it is confirmed that E. intestinalis and E. magna infection destroyed the intestinal flora, which caused corresponding changes in metabolites, and provide novel insights into the molecular mechanisms of rabbit-parasite interactions.

Telocytes in ileum of the Chinese giant salamander: ultrastructural evidence.

Telocytes (TCs) and their telopodes (Tps) have been found in various organs of many mammals, including in lower animals. However, knowledge of TCs in lower animals is still very limited. This study identified TCs and their Tps in the ileum of the Chinese giant salamander, Andrias davidianus (Amphibia: Caudata), by transmission electron microscopy. The TCs/Tps were found near epithelial cells, glandular cells and unmyelinated nerve fibres. Moreover, exosomes were also found to be present in between TCs/Tps and these cells.

Telocytes in pancreas of the Chinese giant salamander (Andrias davidianus).

Telocytes (TCs), novel interstitial cells, have been identified in various organs of many mammals. However, information about TCs of lower animals remains rare. Herein, pancreatic TCs of the Chinese giant salamanders (Andrias davidianus) were identified by CD34 immunohistochemistry (IHC) and transmission electron microscopy (TEM). The IHC micrographs revealed CD34+ TCs with long telopodes (Tps) that were located in the interstitium of the pancreas. CD34+ TCs/Tps were frequently observed between exocrine acinar cells and were close to blood vessels. The TEM micrographs also showed the existence of TCs in the interstitium of the pancreas. TCs had distinctive ultrastructural features, such as one to three very long and thin Tps with podoms and podomers, caveolae, dichotomous branching, neighbouring exosomes and vesicles. The Tps and exosomes were found in close proximity to exocrine acinar cells and α cells. It is suggested that TCs may play a role in the regeneration of acinar cells and α cells. In conclusion, our results demonstrated the presence of TCs in the pancreas of the Chinese giant salamander. This finding will assist us in a better understanding of TCs functions in the amphibian pancreas.

Mask refinement network for tooth segmentation on panoramic radiographs.

OBJECTIVES: Instance-level tooth segmentation extracts abundant localization and shape information from panoramic radiographs (PRs). The aim of this study was to evaluate the performance of a mask refinement network that extracts precise tooth edges. METHODS: A public dataset which consists of 543 PRs and 16211 labelled teeth was utilized. The structure of a typical Mask Region-based Convolutional Neural Network (Mask RCNN) was used as the baseline. A novel loss function was designed focus on producing accurate mask edges. In addition to our proposed method, 3 existing tooth segmentation methods were also implemented on the dataset for comparative analysis. The average precisions (APs), mean intersection over union (mIoU), and mean Hausdorff distance (mHAU) were exploited to evaluate the performance of the network. RESULTS: A novel mask refinement region-based convolutional neural network was designed based on Mask RCNN architecture to extract refined masks for individual tooth on PRs. A total of 3311 teeth were correctly detected from 3382 tested teeth in 111 PRs. The AP, precision, and recall were 0.686, 0.979, and 0.952, respectively. Moreover, the mIoU and mHAU achieved 0.941 and 9.7, respectively, which are significantly better than the other existing segmentation methods. CONCLUSIONS: This study proposed an efficient deep learning algorithm for accurately extracting the mask of any individual tooth from PRs. Precise tooth masks can provide valuable reference for clinical diagnosis and treatment. This algorithm is a fundamental basis for further automated processing applications.

Alterations in the jejunal microbiota and fecal metabolite profiles of rabbits infected with Eimeria intestinalis.

BACKGROUND: Rabbit coccidiosis is a major disease caused by various Eimeria species and causes enormous economic losses to the rabbit industry. Coccidia infection has a wide impact on the gut microbiota and intestinal biochemical equilibrium. In the present study, we established a model of Eimeria intestinalis infection in rabbits to evaluate the jejunal microbiota and fecal metabolite profiles. METHODS: Rabbits in the infected group were orally inoculated with 3 × 103 E. intestinalis oocysts. On the eighth day of infection, jejunal contents and feces were collected for 16S rRNA gene sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, respectively. Jejunum tissues were harvested for hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and immunohistochemistry (IHC) staining. RESULTS: Histopathological analysis showed that the whole jejunum was parasitized by E. intestinalis in a range of life cycle stages, and PAS staining showed that E. intestinalis infection caused extensive loss of goblet cells. IHC staining revealed that TNF-α expression was higher in the E. intestinalis infection group. Moreover, both the jejunal microbiota and metabolites significantly altered after E. intestinalis infection. At the genus level, the abundances of Escherichia and Enterococcus significantly increased in the infected group compared with the control group, while those of Oscillospira, Ruminococcus, Bacteroides, Akkermansia, Coprococcus, and Sarcina significantly decreased. In addition, 20 metabolites and two metabolic pathways were altered after E. intestinalis infection, and the major disrupted metabolic pathway was lipid metabolism. CONCLUSIONS: Eimeria intestinalis infection induced intestinal inflammation and destroyed the intestinal homeostasis at the parasitized sites, leading to significant changes in the gut microbiota and subsequent corresponding changes in metabolites.

Effects of Continuous LPS Induction on Oxidative Stress and Liver Injury in Weaned Piglets.

Due to imperfections in their immune and digestive systems, weaned piglets are susceptible to invasions of the external environment and diseases, especially bacterial infections, which lead to slow growth, tissue damage, and even the death of piglets. Here, a model of weaned piglets induced by Escherichia coli lipopolysaccharide (LPS) was established to explore the effects of continuous low-dose LPS induction on the mechanism of liver injury. A total of forty-eight healthy 28-day-old weaned piglets (weight = 6.65 ± 1.19 kg) were randomly divided into two groups: the CON group and LPS group. During the experimental period of thirteen days, the LPS group was injected intraperitoneally with LPS (100 µg/kg) once per day, and the CON group was treated with the same volume of 0.9% NaCl solution. On the 1st, 5th, 9th, and 13th days, the serum and liver of the piglets were collected for the determination of serum biochemical indexes, an antioxidant capacity evaluation, and histopathological examinations. In addition, the mRNA expression levels of the TLR4 pathway and inflammatory cytokines were detected. The results showed that the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in the serum increased after LPS induction. The activities of total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) in the serum and liver homogenate of the LPS group were lower than those of the CON group, while the malondialdehyde (MDA) content in the serum and the activities of catalase (CAT) and superoxide dismutase (SOD) in the liver of the LPS group were higher than those in the CON group. At the same time, morphological impairment of the livers occurred, including hepatocyte caryolysis, hepatocyte vacuolization, karyopycnosis, and inflammatory cell infiltration, and the mRNA expression levels of TLR4, MyD88, NF-κB, TNF-α, IL-6, and IL-10 were upregulated in the livers after LPS induction. The above results were more obvious on the 1st and 5th days of LPS induction, while the trend during the later period was not significant. It was concluded that the oxidative stress and liver injury occurred at the early stage of LPS induction, while the liver damage weakened at the later stage. The weaned piglets probably gradually developed tolerance to the endotoxin after the continuous low-dose induction of LPS.

The influence on oxidative stress markers, inflammatory factors and intestinal injury-related molecules in Wahui pigeon induced by lipopolysaccharide.

INTRODUCTION: The intestinal structure is the foundation for various activities and functions in poultry. An important question concerns the changes in the intestinal status under endotoxin stimulation. This study aimed to investigate the mechanism of intestinal injury induced by lipopolysaccharide (LPS) in Wahui pigeons. METHODS: Thirty-six 28-day-old healthy Wahui pigeons were randomly divided into two groups. The experimental group was injected with LPS (100 µg/kg) once per day for five days, and the control group was treated with the same amount of sterile saline. Blood and the ileum were collected from pigeons on the first, third, and fifth days of the experiment and used for oxidative stress assessment, inflammatory factor detection, histopathological examination, and positive cell localization. In addition, intestinal injury indices and mRNA expression levels (tight junction proteins, inflammatory cytokines, and factors related to autophagy and apoptosis) were evaluated. RESULTS: Villi in the ileum were shorter in the LPS group than in the control group, and D-lactic acid levels in the serum were significantly increased. Glutathione and catalase levels significantly decreased, but the malondialdehyde content in the serum increased. TNF-α and IL-10 were detected at higher levels in the serum, with stronger positive signals and higher mRNA expression levels, in the LPS group than in the control group. In addition, the levels of TLR4, MyD88, NF-κB, and HMGB1 in the inflammatory signaling pathway were also upregulated. Finally, the mRNA expression of Claudin3, Occludin, and ZO-1 was significantly decreased; however, that of Beclin1 and Atg5 was increased in the LPS group. CONCLUSION: Ileal pathological changes and oxidative stress were caused by LPS challenge; it is proposed that this triggering regulates the inflammatory response, causing excessive autophagy and apoptosis, promoting intestinal permeability, and leading to intestinal injury in Wahui pigeons.

A novel human anti-BAFF neutralizing monoclonal antibody derived from in vitro immunization.

B-cell activating factor (BAFF) plays a key role in the normal regulation of B cell development and immune response. Its abnormal expression level is accompanied by the occurrence of various autoimmune diseases. Therefore, BAFF is an effective target for the treatment of such diseases. Here, we report a new anti-BAFF monoclonal antibody. Based on improved in vitro immunization method, we used a recombinant BAFF containing unnatural amino acid p-nitro-phenylalanine (pNO2Phe) as an antigen to trigger immune response in vitro. The plasma cells were sorted by flow cytometry (FACS), and the antibody library was constructed based on the sorted plasma cells. The high affinity antigen-binding fragments were panned by phage display technology, and finally the anti-BAFF human IgG was obtained. The antibody demonstrated its ability to neutralize BAFF effectively both in vitro and in vivo. We propose that this novel full-length human anti-BAFF monoclonal antibody is a promising therapeutic candidate for the treatment of autoimmune diseases.

The Spermatozoal Ultrastructure of the Chinese Mitten Crab (Eriocheir sinensis).

BACKGROUND: The Chinese mitten crab (Eriocheir sinensis) is an economically important aquatic species in China. The artificial breeding crabs are also increasing in number day by day. However, knowledge about spermatozoal organization of the crab is still very limited. AIMS AND OBJECTIVES: In the present study, the spermatozoal ultrastructure of the E. sinensis is illustrated for improving artificial breeding technique. MATERIALS AND METHODS: The spermatozoa are observed by light microscopy and transmission electron microscopy. RESULTS: Spermatozoa are located in the lumen of seminiferous tubules. The spermatocytes and spermatids are observed in the wall of seminiferous tubules. The spermatophores are both present in the lumen of vas deferens and seminal vesicles. A mature spermatozoon consists of a central electron dense acrosome and a peripheral electron lucent nucleus within structures-organelles complex. The acrosome is divided into three zones, including inner acrosome zone, outer acrosome zone and zonal texture. The centre of acrosome is the perforatorium within parallel arranged perforatorial tubules along vertical axis. The highest electron dense operculum surrounds the head side of perforatorium. CONCLUSION: The ultrastructure of spermatozoa of E. sinensis is illustrated. In particular, the outermost part of the acrosome appears as concentric circles and is described as zonal texture.

De novo generation of specific human IgGs by in vitro immunization using autologous proteins containing immunogenic p-nitrophenylalanine.

In vitro immunization can to used to produce monoclonal antibodies(mAbs), but this technology is limited by poor reproducibility and the requirement of pre-immunized lymphocytes. To improve this approach, we recently developed a method for rapid generation of antigen-specific B cells. Here, we report the application of this system to the production of human IgGs against tumor necrosis factor (TNF). We expressed mutant proteins with site-specific incorporated p-nitrophenylalanine (pNO2Phe), which stimulated an in vitro immune response in human immune cells. After constructing an antigen-specific antibody library from in vitro immunized B cells identified by fluorescence-activated cell sorting, we demonstrated that many point mutation events of the variable region occurred in our step-by-step co-cultivation system for affinity maturation in vitro. To mimic the class switching, we panned for high-affinity antigen-binding fragments by the phage display method, assembled them and identified hTNF-neutralizing human IgGs. This approach may provide a general method for raising high-affinity monoclonal antibodies against self-proteins. Furthermore, it supports mechanistic understanding in breaking human self-tolerance with pNO2Phe.

Telocytes in gastric lamina propria of the Chinese giant salamander, Andrias davidianus.

In this study, we attempt to identify gastric telocytes (TCs) of the Chinese giant salamander Andrias davidianus, by light microscopy, immunohistochemistry and transmission electron microscopy (TEM) methods. Toluidine blue staining showed TCs with one to two very thin and long telopodes (Tps) that were located in gastric lamina propria. Tps had characteristic structures, including podoms, podomers and dichotomous branching. Immunohistochemistry showed the existence of CD34(+)/PDGFRα(+) TCs with moniliform Tps in stroma and were close to gastric glands and blood vessels. TEM micrographs also demonstrated the presence of TCs in interstitium between gastric glands. TCs/Tps were located in close proximity to gastric glands, blood vessels, endocrine cells and stem cells. In particular, Tps frequently surrounded stem cells. TCs and Tps, Tps and stem cells established close contacts. Moreover, the exosomes were also found near TCs/Tps. Our data confirmed the presence of TCs in gastric lamina propria of the amphibian, and suggested that TCs cooperate with resident stem cells to regulate endocrine cells and gastric glands regeneration and homeostasis.

Micro-ultrastructure features of liver in Chinese Taihe black-bone silky fowl (Gallus gallus domesticus Brisson) / Características ultraestructurales de hígado en la gallina silky China Taihe de hueso negro (Gallus gallus domesticus Brisson)

In this work, the morphological features of liver in Chinese Taihe black-bone silky Fowl (BSF) were analyzed by light microscope and transmission electron microscopy. The results showed that two kinds of hepatocytes were present in Taihe BSF liver, i.e., the dark and the light hepatocyte. The dark hepatocyte was electron-dense and lager, with many organelles, mitochondria especially. The light hepatocytes were smaller than the dark. They had electron-lucent cytoplasm with a small number of organelles. Furthermore, there were lipolysosomes in the light hepatocyte. The numerous long and serried finger-like microvilli spread into bile canaliculus lumen. The glycogen granules intensely stained, spread in some hepatocytes. Numerous glycogen granules scattered in cytoplasm especially near bile canaliculi. However, lipid droplets were not observed in any hepatocytes. The natural apoptotic hepatocytes were observed in Taihe BSF liver. The hepatocytes which contain abundant uesicae-like endoplasmic reticulum closed the apoptotic hepatocytes and spread the process to approach the cell residual bodies. Besides, there was a macrophage with several phagosomes. In conclusion, the dark and the light hepatocyte were present in Chinese Taihe BSF liver. They were different from electron-dense and organelles. The hepatocytes of Taihe BSF could undergo natural apoptosis, regeneration and renew ability.

Fueron analizadas las características morfológicas ultraestructurales de hígado en la gallina sedosa china de hueso negro por microscopía óptica y microscopía electrónica de transmisión. Los resultados mostraron que se encontraron hepatocitos claros y oscuros en el hígado de la gallina china Taihe. El hepatocito oscuro era denso y de mayor tamaño. Tenía numerosos organelos, especialmente mitocondrias. Los hepatocitos claros eran más pequeños que los oscuros. El citoplasma presentó un pequeño número de organelos. Además, había lipolisosomas en los hepatocitos claros. Numerosas microvellosidades se extendían hacia los canalículos biliares. En algunos hepatocitos se observó una tinción marcada en los gránulos de glucógeno. Sin embargo, no se observaron gotas de lípidos en los hepatocitos. Se observaron los hepatocitos apoptóticos naturales en el hígado de la gallina Silky Taihe. Aquellos hepatocitos que contenían abundante retículo endoplásmico, cerraban los hepatocitos apoptóticos y extendían el proceso de acercamiento a cuerpos residuales celulares. También hubo un macrófago con varios fagosomas. En conclusión, los hepatocitos claros y oscuros estaban presentes en el hígado de la gallina Taihe china. Estos diferían de electrones de alta densidad y organelos. Los hepatocitos de la gallina Taihe presentaron una apoptosis natural y capacidad de regeneración.