Study on the genotypes of Echinococcus granulosus in yaks and sheep from Langkazi County in Tibet Autonomous Region of China based on mitochondrial cox1 and nad1.

To determine the genotypes of the epidemic strains of Echinococcus granulosus in livestock in Tibet, samples of E. granulosus cysts were collected from 11 yaks and 62 sheep. Genomic DNA was extracted from these samples, and gene fragments of mitochondrial cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit I (nad1) were amplified by PCR and sequenced. DNASTAR and MAGA7.0 were employed for homology analysis and phylogenetic tree construction. Echinococcus granulosus cysts were detected in 56.2% (41/73) of the samples screened. Of these, 63.4% (26/41) were identified as E. granulosus G1 genotype (common sheep strain), 24.4% (10 /41) as G3 genotype (buffalo strain), and 12.2% (5/41) were G6 genotype (camel strain). The study concludes that yaks and sheep in Langkazi county, Tibet, carry three E. granulosus genotypes (G1, G3, and G6), with the G1 genotype the predominant genotype in the region. This study clarifies the distribution of E. granulosus genotypes, providing genetic data and insight for the surveillance and prevention of echinococcosis.

BMP15 Modulates the H19/miR-26b/SMAD1 Axis Influences Yak Granulosa Cell Proliferation, Autophagy, and Apoptosis.

Bone morphogenetic protein 15 (BMP15) regulates the growth and development of follicles. In particular, the long non-coding RNA H19 plays an important role in mammalian reproduction. However, the function and regulatory mechanism of the interaction of BMP15 with H19 in yak granulosa cell (GC) proliferation, autophagy, and apoptosis are poorly understood. In our study, quantitative reverse-transcription-polymerase chain reaction analysis showed that H19 were highly expressed in yak healthy follicles. H19 was induced by BMP15 protein in yak GCs. In addition, we confirmed that overexpression of H19 promoted yak GC proliferation and autophagy and inhibited apoptosis. Bioinformatic analysis and luciferase reporter assays demonstrated that H19 directly binds to miR-26b, and SMAD1 was identified as a target of miR-26b. miR-26b overexpression inhibited GC proliferation and autophagy and promoted apoptosis through decreased SMAD1 expression, which was attenuated by H19 overexpression. RNA immunoprecipitation-quantitative polymerase chain reaction and dual-luciferase assays showed that miR-26b was sponged by H19 to preserve SMAD1 expression. Furthermore, SMAD1 mRNA expression was induced and miR-26b expression was reduced after yak GCs were treated with BMP15 protein. In conclusion, our results demonstrated that the H19/miR-26b/SMAD1 axis responds to BMP15 to regulate yack GC proliferation, autophagy, and apoptosis.

The Resistance Mechanism of Mycoplasma bovis From Yaks in Tibet to Fluoroquinolones and Aminoglycosides.

Mycoplasma bovis (M. bovis) is one of the important pathogens for yaks. Aminoglycosides and fluoroquinolones are frequently used medications for the treatment of M. bovis. Drug-resistant strains were inevitable with the abuse of antibiotics. The resistance of M. bovis to aminoglycosides was related to the base mutations in drug target genes. Amino acid mutations at the quinolone resistance-determining region (QRDR) in gyrA, gyrB, parC, and parE conferred resistance to fluoroquinolones. In order to investigate the resistance mechanism of M. bovis from yaks in Tibet to aminoglycosides and fluoroquinolones, six frequently used antibiotics and ten clinical M. bovis strains were administered for a drug sensitivity test for in vitro-induced highly resistant strains, a drug stable-resistance test, cross-resistance test, and analysis of target gene mutations. The results showed that the clinical strains of M. bovis from yaks in Tibet had varying degrees of resistance to fluoroquinolones and aminoglycosides. The mechanism of resistance to fluoroquinolones and aminoglycosides was identified preliminarily for M. bovis from yaks: the single-site base mutation mediated the resistance of M. bovis from yaks and both base mutations led to highly resistant strains (aminoglycosides: rrs3 and rrs4; fluoroquinolones: gyrA and parC). The active efflux system results of M. bovis showed that there was no active efflux system based on fluoroquinolones and aminoglycosides expressed in M. bovis from yaks. The research could provide a reference for clinical treatment of M. bovis.

Epidemiological Survey of Mycoplasma bovis in Yaks on the Qinghai Tibetan Plateau, China.

Mycoplasma bovis (M. bovis) is one of the most important pneumonia pathogens in yaks. It may result in more economic losses due to the cold and anoxia condition at Qinghai Tibetan plateau. However, to date, limited information on M. bovis infection in yaks is available in China. For this purpose, the seroprevalence of M. bovis was investigated in yaks living in the mentioned area through commercial ELISA kits. A total of 959 yaks were incorporated into this study. The prevalence of the disease in yaks was 48.70%. The serological results revealed a relatively high prevalence of M. bovis infection in yaks. The present study may greatly contribute to the prevention of this disease. More importance should be given to the potential threat caused by M. bovis in the special plateau.

Isolation, identification and biological characteristics of Mycoplasma bovis in yaks.

Mycoplasma bovis (M. bovis) is one of the important pathogens which may cause bovine respiratory disease syndrome (BRDS), and results in huge economic losses for yaks (Bos gaurus) breeding industry. However, there is limited information about M. bovis in yaks. In our study, 145 nasal mucus samples from yaks with pneumonia were collected to clarify. Bacteriological determination was carried out through biochemical identification and Polymerase Chain Reaction (PCR) detection. And ten strains of Mycoplasma bovis (M. bovis) were found from collected samples. Then, the growth curve of isolated strains was determined by the change of optical density (OD630), pH value and Color Change Cnit (CCU). K-B disk method was also used for antimicrobial susceptibility testing. Results of colony morphology and biochemical testing were consistent with the biological characters of M. bovis. The nucleotide sequences of uvrC specific gene and 16S rRNA gene among the 10 strains were highly homologous. The growth curve assay showed that the isolates cultured in PPLO medium were in lag phase for 24 h, entered stable period in 42 h, and entered decline phase after 78 h. The isolates were found resistant to macrolides, aminoglycosides and lincomycin at various degrees, but they were sensitive or moderately sensitive to doxycycline and kanamycin under antimicrobial susceptibility analysis. In conclusion, the results provided certain reference for the follow-up research and guiding for the treatment of M. bovis in yaks.

Probiotic potential of Lactobacillus on the intestinal microflora against Escherichia coli induced mice model through high-throughput sequencing.

The aim of this study was to evaluate the antibacterial potential of Lactobacillus screened from Tibetan yaks on clinical symptoms and intestinal microflora in enteroinvasive Escherichia coli (EIEC) induced mice model. In vitro study, Lactobacillus reuteri (LR1) exhibited stronger resistance to acid and bile and inhibited the growth of EIEC than Lactobacillus mucosae (LM1). The mice were randomly divided into four groups i.e. the LR1 group (LR1 1 × 109 CFU/day), LM1 group (LM1 1 × 109 CFU/day), blank control group and control group. Mice in control, LR1, and LM1 groups were challenged with EIEC on day 23. The body weight in the control and LM1 groups were significantly decreased after the infection with EIEC (P < 0.05), whereas the body weight of mice in the LR1 group did not change significantly (P > 0.05). The lowest diarrhea rate was recorded in the LR1 group after infection with EIEC. The results showed that the number of pathogens in the control group was higher than that in the experimental groups. The sequence analysis and OTU classification showed that the duodenum, ileum, and cecum of mice in the LR1 group had the highest number of OTUs compared with other groups. Whereas, the diversity analysis showed that in duodenum, ileum and cecum of mice in the LR1 group had the highest abundance and diversity. The composition of intestinal microbes indicated the presence of high proportions of Firmicutes, Proteobacteria and Bacteroidetes. Heat map analysis indicated high abundance of Bdello vibrio in the duodenum of mice in the LR1 group, while many pathogens were found in the different part of intestines in the control group, such as Streptococcus, Clostridium and Pseudomonas. In conclusion, pre-supplementation of LR1 alleviate the clinical symptoms caused by E. coli, and promote a healthy gut flora.

microRNA-125b Regulates Apoptosis by Targeting Bone Morphogenetic Protein Receptor 1B in Yak Granulosa Cells.

The intronic microRNA, miR-125b, plays a vital role in promyelocytic and hematopoietic stem cells, and in the development and apoptosis of cancer cells. In this study, we showed that miR-125b regulates granulosa cell (GC) apoptosis in the yak ovary. Bioinformatic analyses and luciferase reporter assays demonstrated that bone morphogenetic protein receptor type 1B (BMPR1B) is an miR-125b target. miR-125b overexpression induced apoptosis in yak GC, and affected the mRNA and protein expression of BMPR1B and the ratio of Bcl2/Bax. Silencing of miR-125b decreased the rate of yak GC apoptosis and increased the ratio of Bcl2/Bax. In addition, the effects of an miR-125b inhibitor were overturned by cotransfection with siRNA-BMPR1B2 (siRNA-299) in yak GC. Together, these results demonstrated that miR-125b regulates GC apoptosis in the yak ovary by targeting BMPR1B.

Negative Regulation of Interferon-ß Production by Alternative Splicing of Tumor Necrosis Factor Receptor-Associated Factor 3 in Ducks.

Tumor necrosis factor receptor-associated factor 3 (TRAF3), an intracellular signal transducer, is identified as an important component of Toll-like receptors and RIG-I-like receptors induced type I interferon (IFN) signaling pathways. Previous studies have clarified TRAF3 function in mammals, but little is known about the role of TRAF3 in ducks. Here, we cloned and characterized the full-length duck TRAF3 (duTRAF3) gene and an alternatively spliced isoform of duTRAF3 (duTRAF3-S) lacking the fragment encoding amino acids 217-319, from duck embryo fibroblasts (DEFs). We found that duTRAF3 and duTRAF3-S played different roles in regulating IFN-ß production in DEFs. duTRAF3 through its TRAF domain interacted with duMAVS or duTRIF, leading to the production of IFN-ß. However, duTRAF3-S, containing the TRAF domain, was unable to bind duMAVS or duTRIF due to the intramolecular binding between the N- and C-terminal of duTRAF3-S that blocked the function of its TRAF domain. Further analysis identified that duTRAF3-S competed with duTRAF3 itself for binding to duTRAF3, perturbing duTRAF3 self-association, which impaired the assembly of duTRAF3-duMAVS/duTRIF complex, ultimately resulted in a reduced production of IFN-ß. These findings suggest that duTRAF3 is an important regulator of duck innate immune signaling and reveal a novel mechanism for the negative regulation of IFN-ß production via changing the formation of the homo-oligomerization of wild molecules, implying a novel regulatory role of truncated proteins.

Characterization of Two Bovine viral diarrhea virus Strains Originating from Cattle in Tibet, China.

Here, we report two strains of Bovine viral diarrhea virus (BVDV), named XZ01 and XZ02, that were isolated from cattle in Tibet, China. They belong to subgenotype 1b. This report will help in understanding the molecular characteristics of BVDV in Tibetan cattle.

Sus scrofa miR-204 and miR-4331 Negatively Regulate Swine H1N1/2009 Influenza A Virus Replication by Targeting Viral HA and NS, Respectively.

The prevalence of swine pandemic H1N1/2009 influenza A virus (SIV-H1N1/2009) in pigs has the potential to generate novel reassortant viruses, posing a great threat to human health. Cellular microRNAs (miRNAs) have been proven as promising small molecules for regulating influenza A virus replication by directly targeting viral genomic RNA. In this study, we predicted potential Sus scrofa (ssc-, swine) miRNAs targeting the genomic RNA of SIV-H1N1/2009 by RegRNA 2.0, and identified ssc-miR-204 and ssc-miR-4331 to target viral HA and NS respectively through dual-luciferase reporter assays. The messenger RNA (mRNA) levels of viral HA and NS were significantly suppressed when newborn pig trachea (NPTr) cells respectively overexpressed ssc-miR-204 and ssc-miR-4331 and were infected with SIV-H1N1/2009, whereas the suppression effect could be restored when respectively decreasing endogenous ssc-miR-204 and ssc-miR-4331 with inhibitors. Because of the importance of viral HA and NS in the life cycle of influenza A virus, ssc-miR-204 and ssc-miR-4331 exhibited an inhibition effect on SIV-H1N1/2009 replication. The antiviral effect was sequence-specific of SIV-H1N1/2009, for the target sites in HA and NS of H5N1 or H9N2 influenza A virus were not conserved. Furthermore, SIV-H1N1/2009 infection reversely downregulated the expression of ssc-miR-204 and ssc-miR-4331, which might facilitate the virus replication in the host. In summary, this work will provide us some important clues for controlling the prevalence of SIV-H1N1/2009 in pig populations.

Identification and characterization of a protective antigen, PlpB of bovine Pasteurella multocida strain LZ-PM.

The Pasteurella multocida lipoprotein B (PlpB) was cloned from Pasteurella multocida (P. multocida) strain LZ-PM (serotype A) and expressed in Escherichia coli (E. coli). Sequence analysis showed that PlpB from different strains of P. multocida exhibited 80.8-99.4% sequence identity to each other, suggesting that PlpB might serve as a cross-protective antigen. The purified PCR product of PlpB gene consisting of 831 base pairs was inserted into the pET-32a (+) plasmid, and then transferred into E. coli. The protective immunity conferred by recombinant PlpB (rPlpB) on mice was evaluated. The results showed that mice immunized with 200 µg of purified rPlpB were protected (60% survival rate) against challenge infection with 1 MLD of P. multocida strain LZ-PM. In conclusion, our data indicated that the PlpB protein may be a potential target as a candidate subunit vaccine for P. multocida infection.

Seroprevalence investigation of bovine ephemeral fever in yaks in Tibetan Plateau of China from 2012 to 2015.

Bovine ephemeral fever (BEF) is caused by the arthropod-borne bovine ephemeral fever virus (BEFV), which is classified in family Rhabdoviridae and genus Ephemerovirus. However, it is still unclear whether yaks from the Tibetan plateau of China are exposed to BEFV. It is the first time that a survey was conducted to investigate the seroprevalence of BEFV infection in yaks (Bos grunniens) on the Tibetan Plateau of China. A total of 1123 serum samples were collected randomly from yaks from 2012 to 2015 and were assayed for BEFV antibodies by enzyme-linked immunosorbent assay (ELISA). The proportions of positive serum samples were assessed among the 1123 samples, as well as factors of geographical origin and years. The results showed that there were 454 serum samples that tested positive for BEFV, and the total positive rate is 40.4 %. The prevalence in 2012, 2013, 2014, and 2015 was 49.3, 36, 44.1, and 34.0 %, respectively, and the difference is statistically significant (P< 0.01). In different regions, the prevalence was ranged from 34.7 to 45.7 % with a significant difference among the different regions of (P < 0.05). Logistic regression analysis showed that yaks in Tibet (Xizang autonomous region) (45.7 %) had 1.6 times (OR = 1.589, 95 % CI = 1.141-2.215, P < 0.01) higher risk of being seropositive compared to yaks in Qinghai province, while no regional difference was found of Sichuan province compared to Qinghai (P > 0.05). The prevalence in 2012 (49.3 %) was more than 1.8 time (OR = 1.880, 95 % CI = 1.350-2.619, P < 0.001) at risk of acquiring the infection compared to the year of 2015. The prevalence of yaks in 2014 (44.1 %) had a 1.5 times (OR = 1.528, 95 % CI = 1.350-2.619, p < 0.001) at risk of being seropositive compared to yaks in 2015, while no year difference was found of 2013 compared to 2015 (P > 0.05). Our study suggests that the yaks from the high plateau are highly infected by BEFV, and geographical origin and years are main risk factors for BEF seroprevalence.

Prevalence of three Oesophagostomum spp. from Tibetan Pigs analyzed by Genetic Markers of nad1, cox3 and ITS1.

Parasitic nematodes of Oesophagostomum spp., commonly known as 'nodular worms' are one of the most widely distributed and prevalent emerging zoonotic nematodes. However, little is known about the prevalence and gene characteristics of those parasites in Tibetan pigs. Therefore, a study was carried out to investigate the prevalence, isolation and identification of Oesophagostomum spp from Tibetan pigs by genetic markers of nad1,cox3 and ITS1 for the first time. The results revealed that the infection rate of O. dentatum and O. quadrispinulatum by genetic markers of nad1 was 81.13%; 35 (66.04%); the O. dentatum and O. quadrispinulatum by genetic markers cox3 was 66.04%, and O. dentatum and O. stephanostomum by genetic markers ITS1 was found to be 77.36%. Interestingly, the O. stephanostomum specie was identified and isolated from 90.48% stomach and 69.23% colon samples by genetic markers of ITS1. The present study, for the first time has described the presence and genetic characterization of Oesophagostomun spp of O. dentatum, O. quadrispinulatum and especially O. stephanostomum in Tibetan pigs from the high and remote Tibetan plateau. A public concern should be raised in terms of economical losses and severe public health problem.

Prevalence, Associated Risk Factors, and Phylogenetic Analysis of Toxocara vitulorum Infection in Yaks on the Qinghai Tibetan Plateau, China.

Toxocara vitulorum has been rarely reported in yaks at high altitudes and remote areas of Sichuan Province of Tibetan Plateau of China. The current study was designed to investigate the prevalence, associated risk factors, and phylogenetic characteristics of T. vitulorum in yak calves on the Qinghai Tibetan plateau. Fecal samples were collected from 891 yak calves and were examined for the presence of T. vitulorum eggs by the McMaster technique. A multivariable logistic regression model was employed to explore variables potentially associated with exposure to T. vitulorum infection. T. vitulorum specimens were collected from the feces of yaks in Hongyuan of Sichuan Province, China. DNA was extracted from ascaris. After PCR amplification, the sequencing of ND1 gene was carried out and phylogenetic analyses was performed by MEGA 6.0 software. The results showed that 64 (20.1%; 95% CI 15.8-24.9%), 75 (17.2; 13.8-21.1), 29 (40.9; 29.3-53.2), and 5 (7.6; 2.5-16.8) yak calves were detected out to excrete T. vitulorum eggs in yak calve feces in Qinghai, Tibet, Sichuan, and Gansu, respectively. The present study revealed that high infection and mortality by T. vitulorum is wildly spread on the Qinghai Tibetan plateau, China by fecal examination. Geographical origin, ages, and fecal consistencies are the risk factors associated with T. vitulorum prevalence by logistic regression analysis. Molecular detection and phylogenetic analysis of ND1 gene of T. vitulorum indicated that T. vitulorum in the yak calves on the Qinghai Tibetan plateau are homologous to preveiously studies reported.

Molecular cloning and functional analysis of the duck TIR domain-containing adaptor inducing IFN-ß (TRIF) gene.

Toll-like receptors (TLRs) trigger the innate immune response by responding to specific components of microorganisms. The TIR domain-containing adaptor inducing IFN-ß (TRIF) plays an essential role in mammalian TLR-mediated signaling. The role of TRIF in ducks (duTRIF) remains poorly understood. In this study, we cloned and characterized the full-length coding sequence of duTRIF from duck embryo fibroblasts (DEFs). In healthy ducks, duTRIF transcripts were broadly expressed in different tissues, with higher expression levels in the spleen and liver. Using quantitative real-time PCR (qRT-PCR), we demonstrated the upregulation of duTRIF in DEFs infected with AIV or DTMUV, and DEFs treated with Poly I:C or LPS. Overexpression of duTRIF was able to induce the NF-κB and IFN-ß expression. Furthermore, the IFN induction function of duTRIF was impaired when Ala517 was mutated to Pro or His. Taken together, these results suggested that duTRIF regulated duck innate immune responses.

Genome sequencing of a virulent avian Pasteurella multocida strain GX-Pm reveals the candidate genes involved in the pathogenesis.

Pasteurella multocida (P. multocida) was first shown to be the causative agent of fowl cholera by Louis Pasteur in 1881. First genomic study was performed on an avirulent avian strain Pm70, and until 2013, two genomes of virulent avian strains X73 and P1059 were sequenced. Comparative genome study supplied important information for further study on the pathogenesis of fowl cholera. In the previous study, a capsular serotype A strain GX-Pm was isolated from the liver of a chicken, which died during an outbreak of fowl cholera in 2011. The strain showed multiple drug resistance and was highly virulent to chickens. Therefore, the present study performed the genome sequencing and a comparative genomic analysis to reveal the candidate genes involved in virulence of P. multocida. Sequenced draft genome sequence of GX-Pm was 2,292,886 bp, contained 2941 protein-coding genes, 5 genomic islands, 4 IS elements and 2 prophage regions. Notability, all the predicted drug-resistance genes were included in predicted genomic islands. A comparative genome study on virulent avian strains P1059, X73 and GX-Pm with the avirulent avian strain Pm 70 indicated that 475 unique genes were only identified in either of virulent strains but absent in the avirulent strain. Among these genes, 20 genes were contained within genomes of all three virulent strains, including a few of putative virulence genes. Further characterization of the pathogenic functions of these genes would benefit the understanding of pathogenesis of fowl cholera.

Seroprevalence of bovine tuberculosis infection in yaks (Bos grunniens) on the Qinghai-Tibetan Plateau of China.

Bovine tuberculosis (BTB), a chronic disease caused by Mycobacterium bovis, has been widely reported in bovines in developing countries, but there is little information on this infection in domestic yaks. Seroprevalence of antibodies to M. bovis in yaks from six and three regions of Tibet and Qinghai plateau, China, respectively, was investigated in 2011 by enzyme-linked immunosorbent assay kits. A total of 1,244 (Tibet 938, Qinghai 306) blood samples was collected and the results showed that 24 (2.6%) of Tibetan samples and 3 (1%) of Qinghai's samples were positive for BTB. The findings of the present study indicated that M. bovis infection is prevalent in Chinese yaks in Qinghai and Tibet. These observations should raise a serious public health concern considering the extent to which the herdsmen of the study areas are in contact with their animals and the levels at which they use untreated livestock products. This is the first study showing the infection of M. bovis in domestic yaks.

Seroprevalence of bovine viral diarrhea infection in Yaks (Bos grunniens) on the Qinghai-Tibetan Plateau of China.

The seroprevalence of bovine viral diarrhea virus (BVDV) infection in yaks was investigated in Qinghai and Tibet of China during the year 2011. A total of 549 (Tibet 287, Qinghai 262) serum samples was collected from Tibet and Qinghai and were examined for BVDV p80 antibody by ELISA. The results of the experiment showed that 145 (53.65 %) of Tibetan samples and 189 (72.14 %) of Qinghai's samples were positive for BVDV. The observations of the present study suggest that bovine viral diarrhea is common in yaks in Tibet and Qinghai, China.