Shucheng Huang

Tibial dyschondroplasia is highly associated with suppression of tibial angiogenesis through regulating the HIF-1α/VEGF/VEGFR signaling pathway in chickens

Tibial dyschondroplasia (TD) is an intractable poultry problem that is characterized by the appearance of non-vascularized and non-mineralized cartilage masses in tibial growth plates (TGPs). However, the role of angiogenesis inhibition in the occurrence of TD remains unknown. In this study, we found that, compared to low-altitude Arbor Acres chickens (AACs), high-altitude Tibetan chickens showed higher tibial vascular distributions that were accompanied by up-regulation of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor A (VEGFA) and VEGF receptors. These observations provide insights into hypoxia-induced angiogenesis, which may be related to the absence of TD in high-altitude native Tibetan chickens. Importantly, hypoxia experiments also showed that during hypoxia, tibial angiogenesis was enhanced, which was due to pro-angiogenic factor up-regulation (including VEGFA, VEGFR1, VEGFR2, and IL-8), in AACs. Moreover, we observed that thiram-induced TD could strongly inhibit tibial angiogenesis in the hypertrophic zone through coordinated down-regulation of HIF-1α and pro-angiogenic factors, leading to a disruption in the blood supply to the TGP. Taken together, these findings reveal that the occurrence of TD is highly associated with inhibition of tibial angiogenesis through down-regulated expression of HIF-1α, VEGFA and VEGF receptors, which results in suppression of TGP development.

High altitude hypoxia as a factor that promotes tibial growth plate development in broiler chickens

Tibial dyschondroplasia (TD) is one of the most common problems in the poultry industry and leads to lameness by affecting the proximal growth plate of the tibia. However, due to the unique environmental and geographical conditions of Tibet, no case of TD has been reported in Tibetan chickens (TBCs). The present study was designed to investigate the effect of high altitude hypoxia on blood parameters and tibial growth plate development in chickens using the complete blood count, morphology, and histological examination. The results of this study showed an undesirable impact on the overall performance, body weight, and mortality of Arbor Acres chickens (AACs) exposed to a high altitude hypoxic environment. However, AACs raised under hypoxic conditions showed an elevated number of red blood cells (RBCs) and an increase in hemoglobin and hematocrit values on day 14 compared to the hypobaric normoxia group. Notably, the morphology and histology analyses showed that the size of tibial growth plates in AACs was enlarged and that the blood vessel density was also higher after exposure to the hypoxic environment for 14 days, while no such change was observed in TBCs. Altogether, our results revealed that the hypoxic environment has a potentially new role in increasing the blood vessel density of proximal tibial growth plates to strengthen and enhance the size of the growth plates, which may provide new insights for the therapeutic manipulation of hypoxia in poultry TD.

In Vitro Effect of Apigenin and Danshen in Tibial Dyschondroplasia Through Inhibition of Heat-Shock Protein 90 and Vascular Endothelial Growth Factor Expressions in Avian Growth Plate Cells

SUMMARY Tibial dyschondroplasia (TD) is one of the common skeletal abnormalities in fast-growing birds, and it is characterized by nonvascularized, unmineralized, and nonviable cartilage in the tibial growth plate that fails to form bone. The aim of this study was to check the in vitro effect of apigenin and danshen on heat-shock protein 90 (Hsp90) and vascular endothelial growth factor (VEGF) expressions in avian growth plate cells treated with sublethal concentration of thiram. Initially, chondrocytes from chicken growth plates were isolated on culturx ed medium with and without various concentration of thiram to determine the sublethal dose. Then, to check the effect of apigenin and danshen, the chondrocytes were treated first with a sublethal (2.5 μM) concentration of thiram and then with different doses (10, 20, 40, and 80 μM) of apigenin and danshen. The mRNA expression levels of Hsp90 and VEGF genes were evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The results showed that the expression levels of Hsp90 and VEGF mRNA transcripts were increased significantly (P < 0.05) in thiram-treated chondrocytes culture medium up to 1.5-fold, whereas apigenin and danshen therapy to chondrocytes in culture medium significantly (P < 0.05) reduced the Hsp90 and VEGF expression levels. In conclusion, up-regulation of both (Hsp90 and VEGF) genes and damage to chondrocytes in culture medium caused by thiram can be restored by using apigenin and danshen. Therefore, apigenin and danshen therapies are suggested and encouraged as a promising approach to control TD in broiler chickens.

First report of Metastrongylus pudendotectus by the genetic characterization of mitochondria genome of cox1 in pigs from Tibet, China

Lungworms, a world wild distributed parasites cause serious respiratory diseases to the pigs. A high infection rate of Metastrongylus lungworms has been found in Tibetan pigs being slaughtered in different slaughter houses of Tibet autonomous region. The main aim of our study was to detect and confirm the lungworm parasite by the genetic characterization of mitochondrial cox1genome isolated from the lungs of Tibetan pig. The adult lungworms were collected from the lungs of slaughtered pigs and identification was done through morphological examinations. Total genomic DNA of the extracted worms was performed and a fragment (∼450bp) of the cox1 of mitochondrial (mt) gene was amplified. Amplicons were cloned into PGEM(®)-T Easy vector and the positive clones were sequenced from a commercial company. Sequence and phylogenetic analysis were performed by software of DNAMAN and MEGA respectively. The results revealed that the lungworms infecting the Tibetan pigs were Metastrongylus pudendotectus (M. pudendotectus). To our knowledge, this is the first report for the isolation and identification for the genetic characterization of mitochondria (mt) genome of cox1 of M. pudendotectus derived from Tibetan pigs in Tibet, China.

Icariin Ameliorate Thiram-Induced Tibial Dyschondroplasia via Regulation of WNT4 and VEGF Expression in Broiler Chickens

Tibial dyschondroplasia (TD) is main bone problem in fast growing poultry birds that effect proximal growth plate (GP) of tibia bone. TD is broadly defined as non-vascularized and non-mineralized, and enlarged GP with tibia bone deformation and lameness. Icariin (Epimedium sagittatum) is a traditional Chinese medicine, which is commonly practiced in the treatment of various bone diseases. Recently, many researcher reports about the beneficial effects of icariin in relation to various types of bone conditions but no report is available about promoting effect of icariin against TD. Therefore, current study was conducted to explore the ameliorating effect of icariin in thiram-induced TD chickens. A total of 180 broiler chicks were equally distributed in three groups; control, TD induced by thiram (50 mg/kg), and icariin group (treated with icariin @10 mg/kg). All groups were administered with normal standard diet ad libitum regularly until the end of experiment. The wingless-type member 4 (WNT4) and vascular endothelial growth factor (VEGF) genes and proteins expression were analyzed by quantitative real-time polymerase chain reaction and western blot analysis respectively. Tibial bone parameters, physiological changes in serum, antioxidant enzymes, and chicken growth performance were determined to assess advantage and protective effect of the medicine in broiler chicken. The expression of WNT4 was decreased while VEGF increased significantly (P < 0.05) in TD affected chicks. TD enhanced the GP, lameness, and irregular chondrocytes, while reduced the liver function, antioxidant enzymes in liver, and performance of chickens. Icariin treatment up-regulated WNT4 and down-regulated VEGF gene and protein expressions significantly (P < 0.05), restored the GP width, increased growth performance, corrected liver functions and antioxidant enzymes levels in liver, and mitigated the lameness in broiler chickens. In conclusion, icariin administration recovered GP size, normalized performance and prevented lameness significantly. Therefore, icariin treatments are encouraged to reduce the incidence of TD in broiler chickens.

Role and regulation of growth plate vascularization during coupling with osteogenesis in tibial dyschondroplasia of chickens

Tibial dyschondroplasia (TD) is the most-prevalent leg disorder in fast-growing chickens; it is intractable and characterized by abnormal endochondral bone formation of proximal tibial growth-plates (TGPs). Previous studies have shown that bone is a highly vascularized tissue dependent on the coordinated coupling between angiogenesis and osteogenesis, but the underlying mechanisms of bone formation and bone remodeling are poorly defined in TD chickens. Here, we observed that inhibition of vasculogenesis and angiogenesis remarkably impaired vascular invasion in the hypertrophic chondrocyte zone of the TGPs, resulting in the massive death of chondrocytes due to a shortage of blood vessels and nutrients. Moreover, the balance of the OPG (osteoprotegerin)/RANKL (receptor activator of nuclear factor-kB ligand) system is also severely disrupted during the osteogenesis process while coupling with angiogenesis, both of which eventually lead to abnormal endochondral bone formation in TD chickens. Thus, the process of vascular formation in endochondral bone appears to initiate the pathological changes in TD, and improvement of this process during coupling with osteogenesis may be a potential therapeutic approach to treat this intractable disease.

Tibial dyschondroplasia is closely related to suppression of expression of hypoxia-inducible factors 1α, 2α, and 3α in chickens

Tibial dyschondroplasia (TD) cases has not been reported in Tibetan chickens (TBCs), but it is commonly seen in commercial broilers characterized by lameness. The underlying mechanism remains unclear. Hypoxia-inducible factors (HIFs) are important regulators of cellular adaptation to hypoxic conditions. In this study, we investigated the role of HIF-1α, -2α, and -3α in hypoxia and thiram-induced TD and their effect on tibial growth plate development in Arbor Acres chickens (AACs) and TBCs. RNA and protein expression levels of HIF-1α, -2α, and -3α were determined by using quantitative reverse transcriptase polymerase chain reaction and western blotting analyses, respectively. Interestingly, the results showed that HIF-1α, -2α, and -3α expressions in the tibial growth plate of TBCs were upregulated by hypoxia and the change was more significant in TBCs than in AACs. However, these factors were downregulated in thiram-induced TD. To further clarify the effect of thiram on tibial growth plate in commercial broilers, AACs were observed to exhibit more pronounced changes in their growth plate that that in TBCs. Taken together, these results demonstrate that HIF-1α, -2α, and -3α may be important in tibial growth plate development and in the prevention of TD. The present study contributes novel insights on a therapeutic target for poultry TD.

Epidemiological Survey and Phylogenetic Characterization of Cysticercus tenuicollis Isolated from Tibetan Pigs in Tibet, China

Cysticercus tenuicollis, commonly known as “water bell,” is a larva of Taenia hydatigena, which is the most significant parasite of pigs. However, until now very few information is available regarding the prevalence and genetic characterization of the Cysticercus tenuicollis in Tibetan pigs. Therefore, the aim of this study was to investigate the prevalence and phylogenetic analysis of Cysticercus tenuicollis in Tibetan pigs. For this purpose, the COX2 gene of Cysticercus tenuicollis was amplified and sequenced for the first time in Tibetan pigs. The overall prevalence of Cysticercus tenuicollis was 43.93% in Tibetan pigs, with further distribution of 42.86% in 2014 and 45.35% in 2015. In Tibetan male and female pigs, the prevalence of Cysticercus tenuicollis was 43.39% and 44.56%, respectively. The prevalence of Cysticercus tenuicollis in different growing stages (juveniles, subadults, and adults) varied from 30.20% to 63.79%. The phylogenetic analysis of the Cysticercus tenuicollis isolates showed very close resemblance to 16 reference strains, isolates from Gansu, Hunan, and Sichuan provinces of China. To the best of our knowledge, this is the first report on the prevalence and genetic characterization of Cysticercus tenuicollis derived from Tibetan pigs. The data of present study provides baseline information for controlling cysticerci infections in pigs in Tibetan Plateau, China.

Seroprevalence and risk factors associated with hepatitis E virus infections among people and pigs in Tibet, China

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important public health problem throughout the world. There is little information available on the autochthonous HEV infection in highland inhabitants (Tibet, average elevation >3000m) of China. Therefore, we conducted a cross-sectional survey to determine the sero-prevalence of Hepatitis E and association of different risk factors in local people and pigs in Tibet, China. For this purpose, serum samples were collected from 600 local volunteers and 453 Tibetan pigs from July 2014 to August 2015. All the samples were examined for the presence of anti-HEV IgG antibodies by using enzyme-linked immunosorbent assay (ELISA). While socio-demographic and behavioral characteristics were also obtained through questionnaire. The present survey found a close relationship among the HEV of Tibetan people and pigs. The prevalence of anti-HEV IgG was detected 39.33% (236/600) and 42.38% (192/453) in local people and pigs, respectively. There was a significance association of different age groups, ethnic groups, people having contact with pigs or consuming uncooked meat, and gender of Tibetan pigs. Our findings confirmed the presence of autochthonous HEV among people and pigs in Tibet, China. Therefore, effective measures should be taken to prevent and control HEV infection in Tibet, China.

Comparative analysis of gut microbial community in healthy and tibial dyschondroplasia affected chickens by high throughput sequencing

Tibial dyschondroplasia (TD) is a common skeletal problem of avian species. The cause and etiology of this disorder is still not clear. The intestinal bacterial community is a complex ecosystem and plays very important role in healthy life of human and animals. Many researchers have made the connection between diseases and alteration of gut microbiota. However, little is know about the role of gut microbiota in TD. The objective of this study was to explore the diversity and composition of small and large intestinal bacterial communities of TD chickens. The intestinal contests were collected from healthy and TD chickens at day 18 of the trail. The V3-V4 region of 16S rDNA was amplified and sequenced by high-throughput sequencing. Our results indicated that healthy chickens had higher abundance and diversity of gut microbiota than TD chickens but there was no significant difference (P > 0.05) among the gut microbiota of small and large intestine. However, the composition of bacterial communities were significantly different (P < 0.05) between healthy and TD chickens. The occurrence of gut pathogens associated with immunity and inflammation were much higher in the intestinal contests of TD chickens as compared to healthy ones. In summary, our results showed an interesting difference of gut microbiota between healthy and TD chickens, which provided a new thought about the pathogenesis of TD.