Isolation and tracing of matrix-producing notochordal and chondrocyte cells using ACAN-2A-mScarlet reporter human iPSC lines.

The development of human induced pluripotent stem cell (iPSC)-based regenerative therapies is challenged by the lack of specific cell markers to isolate differentiated cell types and improve differentiation protocols. This issue is particularly critical for notochordal-like cells and chondrocytes, which are crucial in treating back pain and osteoarthritis, respectively. Both cell types produce abundant proteoglycan aggrecan (ACAN), crucial for the extracellular matrix. We generated two human iPSC lines containing an ACAN-2A-mScarlet reporter. The reporter cell lines were validated using CRISPR-mediated transactivation and functionally validated during notochord and cartilage differentiation. The ability to isolate differentiated cell populations producing ACAN enables their enrichment even in the absence of specific cell markers and allows for comprehensive studies and protocol refinement. ACAN's prevalence in various tissues (e.g., cardiac and cerebral) underscores the reporter's versatility as a valuable tool for tracking matrix protein production in diverse cell types, benefiting developmental biology, matrix pathophysiology, and regenerative medicine.

Identifying potential Q-markers for quality evaluation of Zhenyuan capsule by integrating chemical analysis, network pharmacology, molecular docking, and molecular dynamics simulations.

Quality markers (Q-markers) are of great significance for quality evaluation of herbal medicines. Zhenyuan Capsule (ZYC) is a kind of Chinese patent medicine used to treat cardiovascular diseases. However, reliable and effective Q-markers for ZYC are still lacking. Herein, a UHPLC-Q/Orbitrap-MS/MS was performed to characterise the preliminary chemical profile of ZYC. A total of 86 components were characterised among which 20 constituents were unambiguously identified by reference compounds. Based on network pharmacology, seven major ginsenosides with great importance in the network were identified as Q-markers among which ginsenoside Re with the highest betweenness was screened to inhibit the development of coronary heart disease (CHD) by binding with vascular endothelial growth factor A (VEGFA). Docking and molecular dynamics simulation studies suggested that ginsenoside Re stably bound to VEGFA. Quantitative determination and chemical fingerprinting analysis were performed using HPLC-DAD. The results showed that ginsenosides screened might function as potential Q-markers for ZYC.

Interaction of isoquinoline alkaloids with pyrimidine motif triplex DNA by mass spectrometry and spectroscopies reveals diverse mechanisms.

Isoquinoline alkaloids represent an important class of molecules due to their broad range of pharmacology and clinical utility. Prospective development and use of these alkaloids as effective anticancer agents have elicited great interest. In this study, in order to reveal structure-activity relationship, we present the characterization of bioactive isoquinoline alkaloid-DNA triplex interactions, with particular emphasis on the sequence selectivity and preference of binding to the two types of DNA triplexes, by electrospray ionization mass spectrometry (ESI-MS) and various spectroscopic techniques. The six alkaloids, including coptisine, columbamine, epiberberine, berberrubine, jateorhizine, and fangchinoline, were selected to explore their interactions with the TC and TTT triplex DNA structures. Berberrubine, fangchinoline, coptisine, columbamine, and epiberberine have preference for TC rich DNA sequences compared to TTT rich DNA triplex based on affinity values in MS. The experimental results from different fragmentation modes in tandem MS, subtractive and hyperchromic effects in UV absorption spectra, fluorescence quenching and enhancement in fluorescence spectra, and strong conformational changes in circular dichroism (CD) hinted that the interaction between isoquinoline alkaloid-TC/TTT DNA had diverse mechanisms including at least two different binding modes: the electrostatic binding and the intercalation binding. Interestingly, columbamine, berberrubine, and fangchinoline can stabilize TTT triplex as inferred from optical thermal melting profiles, while it was not the case in TC triplex. These results provide new insights into binding of isoquinoline alkaloids to pyrimidine motif triplex DNA.

LPS-induced inflammation disorders bone modeling and remodeling by inhibiting angiogenesis and disordering osteogenesis in chickens.

Inflammation plays a negative role in the growth and development of bone. However, the underlining mechanisms of inflammation caused abnormal bone development and even bone disease are still poorly understood, especially in chickens. In this study, we explored the influence of inflammation on bone formation in broilers for the first time by using lipopolysaccharide (LPS) to establish systemic inflammatory models in chickens with tibia as the research object. The measurements of production and tibial parameters showed an inefficient production performance and lower growth rate in LPS group. We also found a large amount of platelets, inflammatory cells in chickens' blood and higher levels of inflammatory factors in serum after LPS injection, meanwhile, increase in thrombus, chondrocyte nucleolysis, and osteoclasts and a reduction in blood vessels were observed in growth plate through histological observation. The qPCR analysis showed that the mRNA expression levels of NF-κB, TLR4, TF, TPO, and its receptor C-MPL enhanced, while VEGFA was inhibited in LPS group. In addition, in OPG/RANKL system, OPG was decreased while RANKL enhanced. It was also observed that the mRNA levels of MMP-9 and its inducing factor CD147 enhanced in LPS group. The western blot results were basically in consistent with mRNA test. Thus, we infer that inflammation can inhibit bone modeling and remodeling by affecting angiogenesis and osteogenesis, and result in negative effect on bone formation furtherly.

Epidemiological survey of fasciolosis in yaks and sheep living on the Qinghai-Tibet plateau, China.

Fasciolosis is one of the biggest threats to livestock and human population. For this purpose, the seroprevalence of Fasciola hepatica was investigated in yaks and sheep living on the Qinghai-Tibet plateau, China by piloting commercial ELISA kits. A total of 3276 yaks and 1092 sheep were incorporated in this study. The prevalence of the parasite in yaks and sheep was 38.3% and 26.4%, respectively. The serological results revealed a relatively high prevalence of F. hepatica infection in yaks and sheep, respectively. The present study may greatly contribute to the prevention of this parasitic zoonosis and great importance should be given to the potential threat caused by F. hepatica in this special plateau.

Tibial growth plate vascularization is inhibited by the dithiocarbamate pesticide thiram in chickens: potential relationship to peripheral platelet counts alteration.

The widespread use of thiram has raised concerns for health and its toxic effects, but the underlying toxicity mechanism on platelets and bones is poorly defined. Here, we found a significant increase in the number of platelets in chickens with the thiram intake, due to the increased expression of thrombopoietin mRNA in the dysfunction liver. Furthermore, the decreased vascular distribution and cell death of chondrocytes in the tibial growth plates (TGPs) were observed, resulting in bone growth inhibition, which is associated with the abnormal activation of platelets leading to the extraordinary decrease of vascular endothelial growth factor A (VEGFA) and angiopoietin-1 protein were released and their corresponding receptors VEGFR2 and Tie-2 expressions were also reduced in the TGPs. Taken together, these findings revealed that thiram has an adverse effect on bones and platelets, which may have a high risk of thrombosis and osteoarthritis.

Osthole: A Coumarin Derivative Assuage Thiram-Induced Tibial Dyschondroplasia by Regulating BMP-2 and RUNX-2 Expressions in Chickens.

Avian tibial dyschondroplasia affects fast growing broiler chickens accounting for almost 30% of leg ailments in broilers. The present project was designed to assess the efficacy of osthole against avian tibial dyschondroplasia (TD). Two hundred and forty chickens were equally allocated into control, TD and osthole groups (n = 80). The TD and osthole group chickens were challenged with tetramethylthiuram disulfide (thiram) at 50 mg/kg of feed from 4-7 days, followed by osthole administration at 20 mg/kg orally to the osthole group only from 8-18 days. Thiram feeding resulted in lameness, increased mortality, and decreased production parameters, alkaline phosphatase (ALP), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-PX) levels, along with significantly increased aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA) levels, and growth plate size. Moreover, the genes and protein expressions of BMP-2 and RUNX-2 were significantly down-regulated in TD affected chickens (p < 0.05). Osthole administration showed promising results by alleviating lameness; increased ALP, SOD, T-AOC, and GSH-Px levels; and decreased the AST, ALT, and MDA levels significantly. It restored the size of the growth plate and significantly up-regulated the BMP-2 and RUNX-2 expressions (p < 0.05). In conclusion, the oxidative stress and growth plate anomalies could be assuaged using osthole.

Recovery of chicken growth plate by Tanshinoneâ ¡A through wnt/ß-catenin pathway in thiram-induced Tibial Dyschondroplasia.

Tibial Dyschondroplasia (TD), a metabolic disease of fast growing poultry birds that effects the growth of bone and cartilage, is characterized by anorexia, mental depression and lameness. Wnt/ß-catenin pathway can mediate the occurrence of TD, and previous study showed the therapeutic effect of TanshinoneⅡA to TD Broilers. However there is no report about the effect of TanshinoneⅡA treating TD broiler chicken through wnt/ß-catenin pathway. The objective of this study was to explore the potential mechanism of how Tanshinone II A treats TD. Hematoxylin and eosin staining was used to study histologic pathology of growth plates. Key gene expressions were tested by western blot and reverse transcription quantitative real-time PCR. Results compared with control groups, showed the TD broilers' growth plate performed significantly better by treating with TanshinoneⅡA. After chickens treated by TanshinoneⅡA, the gene and protein expression of WNT5α and BMP-2 were increased (P < 0.05), but the ß-catenin were decreased (P < 0.05), which are all key genes expressed in wnt/ß-catenin pathway. Therefore, TanshinoneⅡA can potentially treat TD by affecting the expression of genes in wnt/ß-catenin pathway and it has availability to use as treatment for TD broilers.

Chlorogenic Acid Alleviates Thiram-Induced Tibial Dyschondroplasia by Modulating Caspases, BECN1 Expression and ECM Degradation.

Chlorogenic acid (CGA) is a widely applied traditional Chinese medicine ingredient which can be used for the treatment of osteoporosis. In this experiment, we investigated the potential therapeutic effect of chlorogenic acid on thiram-induced tibial dyschondroplasia (TD) and explored the underlying mechanisms that have been rarely mentioned by others yet. Performance indicator analysis and tibial parameter analysis showed that CGA exhibited a definite positive effect on thiram-induced TD chickens. In order to further explore the mechanisms underlying the positive actions of CGA, apoptotic, autophagic genes and MMPs involved in matrix mineralization of growth plate were evaluated in this study. The results showed that CGA decreased the expression of pro-apoptotic genes caspases-3 and caspases-9, leading to the reduction of apoptotic cells accumulated in growth plate. In addition, CGA also increased the level of BECN1, an important gene involved in autophagy, which benefits the survival of abnormal cells. Furthermore, CGA also increased the expression of MMP-9, MMP-10, and MMP-13, which can directly affect the ossification of bones. Altogether, these results demonstrate that CGA possesses a positive therapeutic effect on thiram-induced TD via modulating the expression of caspases and BECN1 and regulating the degradation of ECM (extracellular matrix).

Effect of Anacardic Acid against Thiram Induced Tibial Dyschondroplasia in Chickens via Regulation of Wnt4 Expression.

Tibial dyschondroplasia (TD) is a tibia bone problem in broilers. Anacardic acid (AA) is a traditional Chinese medicine, which is commonly used to treat arthritis in human. The purpose of the present study is to investigate the effect of AA against TD. A total of 300 day-old poultry birds were equally divided and distributed into three different groups: Control, TD and AA groups. The results showed that the feed conversion ratio was significantly lower in the TD group than control chickens. The tibia bone parameters including weight, length and width were of low quality in TD chickens, while the width of the tibial growth plate was enlarged remarkably. Whereas, in the AA treatment group, the tibia bone parameters showed improvement and tend to return to normal. The antioxidant parameters level of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), total and antioxidant capacity (T-AOC) was significantly decreased, while malondialdehyde (MDA) level was increased significantly in TD affected chickens. AA administration restored the antioxidant parameters significantly. The gene expression revealed a decrease in Wnt4 expression in TD chickens as compared to control chickens, while AA treatment up-regulated the Wnt4 expression. The present study demonstrates that the AA plays an important role to prevent the lameness and restore the size of tibial growth plate of chickens by regulating the expression of Wnt4.

Identification of differentially expressed MiRNAs profile in a thiram-induced tibial dyschondroplasia.

Tetramethyl thiuram disulfide (thiram) is a dithiocarbamate, which is widely used on seeds and storing food grains. The incorporation of thiram into the food chain could be a risk for both human beings and animals. Thiram-contaminated feed has been considered a common cause of tibial dyschondrolplasia (TD) in many avian species. The molecular mechanism of action of thiram on TD involving microRNA (miRNA) is not fully understood. For this purpose, the morbidity and pathologic changes were evaluated to understand the TD, and high-throughput RNA sequencing (RNA-Seq) was performed to explore the differentially expressed miRNAs (DEGs). RT-qPCR was used to confirm the validity as compared with sequencing data. The results showed that the marked alterations in the growth plate of the TD chickens were noticeable, with shrinking cells and irregular chondrocyte columns as compared with control group. In this study, we identified total 375 (p < 0.1), 340 (p < 0.05) and 266 (p < 0.01) significant DEGs between the TD and control groups. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs showed that the target miRNAs were significantly enriched in different treatment groups, such as apoptosis, mRNA surveillance pathway, mitophagy-animal, etc. This study provides theoretical basis for in-depth understanding the pathogenesis of thiram-induced TD and explore the new insights towards the proposed molecular mechanism of specific miRNA as biomarkers for effective gene diagnosis and treatment of TD in broilers.

Porcine epidemic diarrhea: an emerging disease in Tibetan pigs in Tibet, China.

Porcine epidemic diarrhea virus (PEDV) is a devastating cause of diarrhea in pigs worldwide, and has caused enormous economic losses in the pork industry. To date, however, it is still unclear whether Tibetan pigs from the Tibetan Plateau of China are exposed to PED. This study was to investigate the seroprevalence of PED in Tibetan pigs in Tibet, China. A total of 364 serum samples were collected from Tibetan pigs in Nyingchi in 2014 and 2015. Each sample was assayed for PED antibodies by commercial enzyme-linked immunosorbent assay (ELISA) kits. Our results showed that the seroprevalence of PED in Tibetan pigs was 39.56% with the further distribution of 26.37% and 52.75% in Nyingchi and Mainling (odds ratio was more commonly affected in Mainling than Nyingchi), respectively. Moreover, the seroprevalence of PED was 65.28%, 35.83%, and 28.57% in juveniles, sub adults, and adults, respectively. The prevalence was found as 40.63% in males and 38.37% in females. The present survey indicated that the infection of PED in Tibetan pigs was widely spread in Tibet, which should arouse public concern of the threat to the health of Tibetan pigs and the development of breeding industry in this unique region of the world.

Ligustrazine recovers thiram-induced tibial dyschondroplasia in chickens: Involvement of new molecules modulating integrin beta 3.

Tetramethyl thiuram disulfide (thiram) is a dithiocarbamate, which is extensively used in agriculture as pesticide and fungicide for treating grains intended for seed purposes and also for storing food grains. One of the most evident and detrimental effect produced by thiram is tibial dyschondroplasia (TD) in many avian species, by feeding diets containing thiram, a growth plate cartilage disease. TD is characterized by the lack of blood vessels and impaired vascular invasion of the hypertrophic chondrocyte resulting in the massive cell death. This study investigated the effects of ligustrazine on the treatment and control of thiram induced-TD. A total of 210 chicks were divided into three equal groups (n = 70): control group (received standard diet), TD group (feed on thiram containing diet from day 3-7), and ligustrazine group (feed on thiram containing diet from day 3-7 and after that ligustrazine @ 30 mg/kg from day 8 to day 18). During the experiment, the lameness, production parameters, tibia bone indicators, pathological index changes and integrin beta 3 (ITGB3) expressions were examined. The results reveal that ligustrazine plays an important role in improving angiogenesis and decreasing chondrocytes damage in TD chicks via a new molecule modulating ITGB3. So, the administration of ligustrazine can be an important way to cope with the losses and costs associated with TD in commercial poultry farming and animal welfare issue due to environmental contamination of thiram.

Molecular Detection of Indigenous Hepatitis E Virus (HEV) from Tibetan Pigs in Tibet, China.

Hepatitis E is an important public health concern throughout the world. Many molecular and serological surveys have reported the prevalence and genotypic characteristics of HEV in humans and animals worldwide. However, the genotypic characterization of this virus is very limited in Tibetan pigs. Hence, we aimed to explore the genotype of HEV, prevailing among Tibetan pigs in China. For this purpose, 253 bile samples of Tibetan pigs (free-range animals) were collected from different slaughterhouses during 2017-2018 and subsequently tested for HEV RNA by RT-nPCR. A total of 11 out of 253 (4.35%) samples tested were positive for HEV RNA. Based on the sequence alignment and phylogenetic analysis, all the isolated HEV strains belonged to genotype 4 and clustered into subtype 4b by sharing more than 84.8-95.2% identities with other reported strains. Our results concluded that HEV genotype 4 is prevailing among Tibetan pigs in Tibet, China.

Effect of Icariin on Tibial Dyschondroplasia Incidence and Tibial Characteristics by Regulating P2RX7 in Chickens.

Tibial dyschondroplasia (TD) is a disease of rapid growing chickens that occurs in many avian species; it is characterized by nonvascular and nonmineralized growth plates, along with tibia bone deformation and lameness. Icariin is widely used to treat bone diseases in humans, but no report is available regarding the effectiveness of icariin against avian TD. Therefore, this study was designed to determine its effect against TD. For this purpose, a total of 180 broiler chicks were distributed into three groups including control, TD, and icariin group. Control group was given a standard normal diet, while TD and icariin groups received normal standard diet containing 50 mg/kg thiram to induce TD from days 3 to 7 after hatch. After the induction of TD, the chicks of icariin group were fed with standard normal diet by adding 10 mg/kg icariin in water. Then morphological and production parameters analysis of tibial bone indicators, physiological index changes, and gene expression were examined. The results showed that icariin administration not only decreased the mortality but also mitigated the lameness and promoted the angiogenesis, which diminished the TD lesion and significantly increased the expression of P2RX7 (P < 0.05) in TD affected thiram induced chicks. In conclusion, present findings suggest that icariin has a significant role in promoting the recovery of chicken growth plates affected by TD via regulating the P2RX7. Our findings reveal a new target for clinical treatment and prevention of TD in broiler chickens.

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.

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.

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.

Revealing the parasitic infection in diarrheic yaks by piloting high-throughput sequencing.

Diarrhea a serious disease, reported to be related with changes in microbial diversity is widely epidemic in the yaks on the Qinghai Tibet plateau. Herein, we preformed high-throughput sequencing of the parasitic diversity in 3 groups of yaks i.e. healthy adult yaks (HA), diarrheic adult yaks (DA) and diarrheic yak calves (DC) in Gannan Tibetan Autonomous Prefecture. The results showed that 2 Phyla, 3 Classes, 5 Orders, 4 Families and 6 Genera were discovered in HA yaks; 2 Phyla, 3 Classes, 6 Orders, 6 Families and 8 Genera were found in DA yaks while 2 Phyla, 5 Classes, 5 Orders, 10 Families and 7 Genera were observed in DC yaks. At Phylum level, Nematoda and Apicomplexa were detected in all three yak groups; however, Apicomplexa was found to be conspicuously higher in the DC yaks as compared to DA yaks (p < 0.05). At Class level, Litostomatea, Chromadorea and Gregarinasina were found in all three yak groups, while Spirotrichea and Colpodea were only found in the DC yaks. At Order level, Vestibuliferida, Tritrichomonadida, Rhabditida and Eugregarinorida were observed in all three yak groups; and Neogregarinorida was noted HA and DC yaks while Trichomonadida and Hypotrichomonadida were only found in DA yaks. Tritrichomonadida was found to be relatively higher in the DA yaks when compared with HA yaks (p < 0.05) and DC yaks (p < 0.05). At Family level, Simplicimonadidae and Haemonchidae were found in all three yak groups. Trichostrongylidae was observed in both HA and DA yak groups. Syncystidae was established in both HA and DC yak groups except DA yaks. Trichomonadidae and Dictyocaulidae were only found in DA yaks. Plectidae, Strongylidae, Echinamoebidae, Lecudinidae, Pseudokeronopsidae and Panagrolaimidae were only discovered in DC yaks. Simplicimonadidae was found to be remarkably higher in DA yaks as compared to HA and DC yak groups (p < 0.05); and Haemonchidae was detected at higher levels in HA yaks as compared to DA and DC yaks (p < 0.05). At Genus level, Entamoeba, Buxtonella and Haemonchus were found in all three yak groups. Plectus and Echinamoeba were only found in DC yaks; while Trichostrongylus and Trepomonas were observed in HA and DA yak groups. The genus Gregarina was found in both diarrheic yak groups; while the genera of Tetratrichomonas and Dictyocaulus were observed in DA yaks only. The present study herein reported an insight of the change of parasitic diversity in diarrheic yaks at high altitude area, which shall make contribution towards the solid prevention of diarrhea in yaks.

Antimicrobial resistance and prevalence of diarrheagenic Escherichia coli (DEC), in diarrheic yaks of Tibetan Plateau, China.

This study was conducted to test the distribution of diarrheagenic Escherichia coli (DEC) associated genes in fecal isolates from diarrheic yaks of a high remote region of China. Briefly, we obtained 203 fecal samples from diarrheic adult yaks and E. coli strains were isolated and identified via standard methods The antibiotic sensitivity of isolates was determined via disk diffusion method and polymerase chain reaction (PCR) was used to detect the DEC virulence associated genes. Results of the current study showed a high rate of resistance to tetracycline (93.6%) and low rate of resistance to ofloxacin (16.7%) antibiotics. Meanwhile, five different diarrheagenic associated virulence traits were detected including; EAEC (11.80%), EHEC (25.62%), EIEC (17.18%), EPEC (36.92%) and ETEC (11.36%). Moreover, E. coli isolates were positive for all tested DEC associated virulence genes ranging from 1.48% to 33%. Additionally, four isolates were positive for more than one virulence genes. In conclusion, our investigation showed a relatively low number of E. coli virulence genes isolated from diarrheic Tibetan yaks, which could be attributed to the high altitude induced harsh environmental conditions that may not help in the growth and survival of pathogenic organisms. In addition, this study highlights the high level of antibiotic resistance in yaks, therefore; preventive measures should be taken to monitor the antibiotic usage in Tibet region of China.