Junbing Jiang

Diversity and abundance of the bacterial 16S rRNA gene sequences in forestomach of alpacas (Lama pacos) and sheep (Ovis aries)

Two bacterial 16S rRNA gene clone libraries were constructed from the forestomach of alpacas and sheep fed alfalfa. After the amplification using the universal 16S rRNA gene primers, equal quantities of PCR products from the same species were mixed and used to construct the two libraries. Sequence analysis showed that the 60 clones from alpacas were divided into 27 phylotypes with 25% clones affiliated with Eubacterium sp. F1. The 60 clones from sheep were divided into 21 phylotypes with 7 phylotypes affiliated with Prevotella ruminicola (40% clones). Clones closely related to Clostridium proteoclasticum, Eubacterium sp. F1, Clostridium cellobioparum, Mogibacterium neglectum, Eubacterium ventriosum, Clostridiaceae bacterium WN011, Clostridium coccoides, Clostridium orbiscindens, Eubacterium sp. F1, Cytophaga sp. Dex80-37, Treponema bryantii and Pelotomaculum sp. FP were only found in the forestomach of alpacas, and those to Anaerovorax odorimutans, Treponema zioleckii, Bifidobacterium indicum, Paludibacter propionicigenes, Paraprevotella clara, Eubacterium siraeum, Desulfotomaculum sp. CYP1, Clostridium bolteae, Clostridium termitidis and Clostridiaceae bacterium DJF_LS40 only in the rumen of sheep. Quantitative real-time PCR revealed that the forestomach of alpacas had significantly lower density of bacteria, with bacterial 16S rRNA gene copies (6.89 [Log10 (copies per gram of wet weight)]), than that of sheep (7.71, P<0.01). The two clone libraries also appeared different in Shannon index (library from alpacas 3.30 and from sheep 3.04). Our results showed that there were apparent differences in the bacterial diversity and abundance in the forestomach between alpacas and sheep.

Identification and characterization of microRNAs in white and brown alpaca skin

BackgroundMicroRNAs (miRNAs) are small, non-coding 21–25 nt RNA molecules that play an important role in regulating gene expression. Little is known about the expression profiles and functions of miRNAs in skin and their role in pigmentation. Alpacas have more than 22 natural coat colors, more than any other fiber producing species. To better understand the role of miRNAs in control of coat color we performed a comprehensive analysis of miRNA expression profiles in skin of white versus brown alpacas.ResultsTwo small RNA libraries from white alpaca (WA) and brown alpaca (BA) skin were sequenced with the aid of Illumina sequencing technology. 272 and 267 conserved miRNAs were obtained from the WA and BA skin libraries, respectively. Of these conserved miRNAs, 35 and 13 were more abundant in WA and BA skin, respectively. The targets of these miRNAs were predicted and grouped based on Gene Ontology and KEGG pathway analysis. Many predicted target genes for these miRNAs are involved in the melanogenesis pathway controlling pigmentation. In addition to the conserved miRNAs, we also obtained 22 potentially novel miRNAs from the WA and BA skin libraries.ConclusionThis study represents the first comprehensive survey of miRNAs expressed in skin of animals of different coat colors by deep sequencing analysis. We discovered a collection of miRNAs that are differentially expressed in WA and BA skin. The results suggest important potential functions of miRNAs in coat color regulation.

Microbial Community in the Forestomachs of Alpacas (Lama pacos) and Sheep (Ovis aries)

Abstract Four 2-yr old alpacas ((48±2.3) kg) and four 2-yr old sheep ((50±1.7) kg) were used to study the pH and microbial community of forestomach from alpacas (Lama pacos) and sheep (Ovis aries) fed fresh alfalfa as the sole forage at low altitude (793 m). The forestomach fluid was taken anaerobically via the esophagus. The electric pH meter and quantitative polymerase chain reaction systems were used to study the the pH and microbial community of forestomach. The results showed that the mean pH of forestomach fluid from alpacas was higher than that from sheep (P

In vitro antiviral activity and underlying molecular mechanisms of dipotassium glycyrrhetate against porcine reproductive and respiratory syndrome virus.

BACKGROUND Porcine reproductive and respiratory syndrome virus (PRRSV) has caused large economic losses in the swine industry. Currently, there is no effective way to prevent PRRSV infection. In this study, we investigated the inhibitory effect of dipotassium glycyrrhetate (DG), a derivative of glycyrrhetinic acid, on PRRSV infection ability. METHODS The cytotoxicity of DG was measured by MTT assay, and the effects of DG on PRRSV N gene/protein were investigated using real-time PCR, western blot and immunofluorescence assay. In addition, the effect of DG on cell apoptosis was analysed by fluorescence staining. RESULTS Our results indicated that DG could effectively inhibit virus replication and N gene expression in MARC-145 cells infected with PRRSV. When the infected cells received DG, the numbers of apoptotic cells were decreased, and the cleaved caspase-3 contents were decreased dramatically. CONCLUSIONS Our study demonstrates that DG could effectively inhibit the PRRS virus via multiple pathways including inhibition of virus replication and N gene expression and reduction of apoptotic cells. DG can serve as a potential chemical for PRRSV prevention and control.

Antiviral effects of the constituents derived from Chinese herb medicines on infectious bursal disease virus

Abstract Context: The prevalence of infectious bursal disease has brought about enormous financial losses to the world poultry industry. Chinese herb medicines can provide valuable materials for discovery and development of new drugs. Objective: To screen constituents derived from Chinese herb medicines for their antiviral activity against infectious bursal disease virus (IBDV) in vitro. Materials and methods: Twenty constituents derived from Chinese herb medicines and B87 strain of IBDV were used. The 50% cytotoxic concentration (CC50) and 50% effective concentration (EC50) were determined by visualization of cytopathologic effect (CPE) and 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) test on chicken embryo fibroblast. Selectivity index (SI) and inhibition ratio (%I) were calculated from the data obtained from the MTT test. Results: Antiviral assays showed dipotassium glycyrrhizinate and ligustrazine hydrochloride among the 20 constituents tested exhibited significant inhibitory activity against IBDV in a dose-dependent manner. EC50 of dipotassium glycyrrhizinate and ligustrazine hydrochloride were 663.2 ± 268.4 and 92.52 ± 21.13 µg/mL, and SI were >4.52 and >21.62, respectively. The time-of-addition and virucidal assay indicated that anti-IBDV activity of the two constituents could be due to their inhibiting virus replication and/or inactivating virus directly. The inhibition of virus attachment was not observed in the adsorption inhibition assay. Dipotassium glycyrrhizinate and ligustrazine hydrochloride exhibited more than 70% and 80% inhibition of IBDV, respectively, at the maximum safe concentration. Discussion and conclusion: We believe that dipotassium glycyrrhizinate and ligustrazine hydrochloride can be used to develop a new anti-IBDV compound, and it is worth applying the constituents in clinical practice.

Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors

Hepatocyte growth factor (HGF)/c-Met signaling has been considered as a key pathway in both melanocyte development and melanogenesis. To understand better the expression patterns and tissue distribution characterization of HGF and its receptor c-Met in skin of white versus brown alpaca (Vicugna pacos), we detected the tissue distribution of HGF and c-Met using immunohistochemistry and analyzed the expression patterns by using Western blot and quantitative real time PCR (qPCR). Immunohistochemistry analysis demonstrated that HGF staining robustly increased in the dermal papilla and mesenchymal cells of white alpaca skin compared with that of brown. However, c-Met staining showed strongly positive result, particularly inhair matrix and root sheath in brown alpaca skin. Western blot and qPCR results suggested that HGF and c-Met were expressed at significantly high levels in white and brown alpaca skins, respectively, and protein and transcripts possessed the same expression pattern in white and brown alpaca skins. The results suggested that HGF/c-Met signaling functions in alpaca coat color formation offer essential theoretical basis for further exploration of the role of HGF/c-Met signaling in pigment formation.

Sodium tanshinone IIA sulfonate inhibits the meq, ul49 and VP22 expression of Marek's disease virus.

BACKGROUND Our previous studies have demonstrated that sodium tanshinone IIA sulfonate (STS), a natural compound derived from Salviae Miltiorrhizae Radix et Rhizoma (Danshen), could effectively inhibit Mareks disease virus (MDV) infection both in vitro and in vivo, but the underlying mechanisms remain unclear. The main objective of the study was to explore the effect of STS on the meq, ul49 and VP22 expression of MDV in vitro. METHODS Quantitative real-time PCR (qRT-PCR) was used to analyse the effect of STS on meq and ul49 expression at both the DNA and messenger RNA (mRNA) level, and the effect of STS on VP22 was assessed by immunofluorescence assay and western blotting. RESULTS The DNA and mRNA copy numbers of meq and ul49 significantly decreased in the groups treated with STS compared with MDV control (P<0.05), which indicated that STS could inhibit the expression of meq and ul49 at both the DNA and mRNA level. Moreover, the expression of VP22 encoded by ul49 was also significantly inhibited (P<0.05). CONCLUSIONS STS possessed anti-MDV activity in chicken embryo fibroblasts. Its antiviral mechanisms may be ascribed to inactivating MDV directly, disturbing meq and ul49 replication and inhibiting the expression of VP22 encoded by ul49. These results suggested that STS is a promising natural compound to be further developed as an antiviral agent against MDV infection.

The cytological observation of immune adherence of porcine erythrocyte.

Abstract The immune adherence (IA) between the porcine erythrocytes and the opsonized Escherichia coli carried green fluorescent protein gene (GFP-E.coli) were detected by the fluorescence microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) with an attempt to verify the existence of IA between the porcine erythrocytes and complemented-opsonized microbes. Under fluorescence microscopy, GFP-E.coli opsonized by fresh rabbit serum complement adhered to the erythrocytes and could not be detached by PBS washing, and no IA was observed between the erythrocytes and nonopsonized GFP-E.coli after co-incubation. SEM and TEM also revealed the existence of IA between the serum complement-opsonized GFP-E.coli membrane and the erythrocyte membrane. The partial complement receptor type 1 (CR1)-like gene from porcine was generated by RT-PCR and rapid amplification of cDNA 3’ end (3’ RACE) (157bp and 578bp), both of which have high similarity with published mammals CR1 gene. The sequences were spliced based on homology comparison and submitted to GenBank (GenBank Accession No. JX033989). These results indicated that the porcine erythrocytes were able to bind to the opsonized microorganisms. Furthermore, the sequencing results confirmed that the CR1-like gene exists in porcine.

Antiviral activities of natural compounds derived from traditional chinese medicines against porcine circovirus type 2 (PCV2)

Twenty natural compounds isolated from traditional Chinese medicines were examined for their antiviral activities against PCV2 in vitro. Antiviral activity was analyzed by the assays of blocking of virus entry, inhibition of virus replication and inactivation of virus particles in the virus suspension. 50% cytotoxic concentration (CC50) and 50% effective concentration (EC50) for each compound were determined by using the MTT method and immunofluorescence assay (IFA), respectively. The results obtained from the inhibition of virus replication and inactivation of virus particles showed that the maximum inhibition rates for matrine and scutellarin were 57 and 72.69% and their SI were 29.87 and 18.66, respectively. However, the results from the assays of inhibition of virus entry and inhibition of virus replication demonstrated that both aesculetin and arecoline hydrochloride stimulated PCV2 replication, with the maximum stimulation rate of 387.3 and 219.4%. In summary, both matrine and scutellarin had antiviral activity against PCV2, while aesculetin and arecoline hydrochloride promoted PCV2 infection replication in vitro.

Screening compounds of Chinese medicinal herbs anti-Marek's disease virus.

Abstract Context: Marek’s disease (MD) seriously threatens the world poultry industry and has resulted in great economic losses. Chinese medicinal herbs are a rich source for lead compounds and drug candidates for antiviral treatments. Objective: To investigate the anti-MDV activity and mechanism of 20 compounds extracted from Chinese medicinal herbs. Materials and methods: Antiviral assay, time of addition experiments, and virucidal assay were performed on chicken embryo fibroblast cells. The 50% cytotoxic concentration and 50% effective concentration were determined and, accordingly, selectivity index and inhibition ratio were calculated. Results: Antiviral assay showed dipotassium glycyrrhizinate (DG) and sodium tanshinone IIA sulfonate (STS) exhibited significantly inhibitory activity against MDV in a dose-dependent manner. EC50 of DG and STS were 893.5 ± 36.99 µg/mL and 54.82 ± 2.99 µg/mL, and selective index (SI) were >3.36 and >9.12, respectively. Time of addition experiment and virucidal assay demonstrated DG inhibited viral replication in the full replication cycle and inactivated MDV particles in non-time-dependent manner, but STS interfered with the early stage of MDV replication and inactivated MDV particles in a time-dependent manner. Moreover, both DG and STS promoted apoptosis of cells infected by MDV. Discussion and conclusion: DG and STS have great potential for developing new anti-MDV drugs for clinic application.