Search

Background Species living at high altitude are subject to strong selective pressures due to inhospitable environments (e.g., hypoxia, low temperature, high solar radiation, and lack of biological production), making these species valuable models for comparative analyses of local adaptation. Studies that have examined high-altitude adaptation have identified a vast array of rapidly evolving genes that characterize the dramatic phenotypic changes in high-altitude animals. However, how high-altitude environment shapes gene expression programs remains largely unknown. Findings We generated a total of 910 Gb of high-quality RNA-seq data for 180 samples derived from 6 tissues of 5 agriculturally important high-altitude vertebrates (Tibetan chicken, Tibetan pig, Tibetan sheep, Tibetan goat, and yak) and their cross-fertile relatives living in geographically neighboring low-altitude regions. Of these, ∼75% reads could be aligned to their respective reference genomes, and on average ∼60% of annotated protein coding genes in each organism showed FPKM expression values greater than 0.5. We observed a general concordance in topological relationships between the nucleotide alignments and gene expression–based trees. Tissue and species accounted for markedly more variance than altitude based on either the expression or the alternative splicing patterns. Cross-species clustering analyses showed a tissue-dominated pattern of gene expression and a species-dominated pattern for alternative splicing. We also identified numerous differentially expressed genes that could potentially be involved in phenotypic divergence shaped by high-altitude adaptation. Conclusions These data serve as a valuable resource for examining the convergence and divergence of gene expression changes between species as they adapt or acclimatize to high-altitude environments.

Background Sleep bruxism (SB) patients show a higher incidence of leg movements than normal subjects. Study objectives The study aimed to characterize SB episodes and their relationships with limb movements (LMs). Materials and methods Polysomnographic (PSG) recordings were performed on eight SB patients. The intervals between the onsets of adjacent SB episodes and LMs were determined and linear correlation analyses were used to estimate the relationship between the SB index and SB episodes in clusters. The Pearson χ2 and partitions of χ2 tests were used to analyze the differences in incidence of SB episodes and clusters in different sleep stages. Results A majority of SB episodes (85.05%) were found to be accompanied by LMs and among them, 70.52% SB episodes occurred with movements of both upper and lower limbs and most of LMs (70.54%) occurred before the onset of SB episodes. Most of SB episodes especially those accompanied by LMs occurred with microarousals or awakenings. Linear correlation analysis showed a positive correlation between the SB index and SB episodes in clusters (r2 = 0.7027, P = 0.0093). In addition, the percentage of SB episodes in clusters accompanied by LMs was significantly smaller than that of SB episodes not accompanied by LMs (χ2 test, P Conclusions Most SB episodes might not be isolated events, but rather a part of a series of movements second to changes in arousal level.

Heterakis gallinarum is one of the common parasitic nematodes found in the caecum of poultry. To investigate the genetic diversity and genetic structure of the H. gallinarum population in Sichuan, we amplified and sequenced the complete mitochondrial (mt) cytochrome c oxidase subunit II (cox2) gene of 59 H. gallinarum isolates from seven different geographical regions, then analyzed their genetic polymorphisms. All cox2 genes of the 59 H. gallinarum isolates were 696 bp in length, with an average A + T content of 67.1%. Fifty-nine sequences contained 34 variable sites, and were classified into 23 haplotypes (HS1-HS23). The values of haplotype diversity (Hd) and nucleotide diversity (π) were 0.688 and 0.00288, respectively. Based on values of F

A total of 333 fecal specimens from horses in southwestern China were genotyped based on analysis of the small subunit rRNA (SSU rRNA) gene. Cryptosporidium hominis and Cryptosporidium andersoni were identified in 2 and 4 stool specimens, respectively. The identification of C. hominis was confirmed by sequence analysis of the 70‐kDa heat shock protein (HSP70) and oocyst wall protein (COWP) genes. Subtyping analysis of the 60‐kDa glycoprotein (GP60) gene sequence of C. hominis revealed a new rare subtype Id, named IdA15; only three Id isolates have been reported in humans to date. Multilocus sequence typing (MLST) analysis indicated that the C. andersoni subtype was A6, A5, A2, and A1 at the four minisatellite loci (MS1, MS2, MS3, and MS16, respectively). This is the first report to identify the presence of C. andersoni and C. hominis in horses in southwestern China and the first to identify a rare zoonotic subtype Id of C. hominis in horses. These findings suggest that infected horses may act as potential reservoirs of Cryptosporidium to transmit infections to humans.

Cladosporium cladosporioides causes asthma and superficial and deep infections, mostly in immunodeficient individuals and animals. This study aimed to investigate whether C. cladosporioides spores can enter the lungs through pulmonary circulation and influence pulmonary immune response. We intravenously injected mice with C. cladosporioides spore suspension and conducted several assays on the lungs. Pulmonary hemorrhage symptoms and congestion were most severe on days 1, 2, and 3 post-inoculation (PI). Extensive inflammatory cell infiltration occurred throughout the period of infection. More spores and hyphae colonizing the lungs were detected on days 1, 2, and 3 PI, and fewer spores and hyphae were observed within 21 d of infection. Numerous macrophages, dendritic cells, and neutrophils were observed on day 5 PI, along with upregulation of CD54, an intercellular adhesion molecule. Th1 and Th2 cells increased after infection; specifically, Th2 cells increased considerably on day 5 PI. These results suggest that days 2 and 5 PI represent the inflammatory peak in the lungs and that the Th2 and Th1 signaling pathways are potentially involved in pulmonary immune responses. In conclusion, the further adaptive immune responses played important roles in establishing effective pulmonary immunity against C. cladosporioides systemic infections based on innate immune responses.

The purpose of this study was to characterize the antimicrobial resistance produced by mobile genetic elements and integron gene cassettes in Escherichia coli isolated from the feces of captive giant pandas. We performed a standard disk diffusion antimicrobial susceptibility test with 84 E. coli isolates and further evaluated the mobile genetic elements and integron gene cassettes. The antimicrobial susceptibility test demonstrated that 43.37% (36/84) of the isolates showed multiple drug resistances. The E. coli isolates mainly showed resistance to aztreonam (86.90%, 73/84) and amoxicillin/clavulanic acid (80.95%, 68/84). The most frequently observed resistance patterns were ampicillin/amoxicillin-clavulanic acid (13.10%, n = 11), and doxycycline/amoxicillin-clavulanic acid (4.76%, n = 4). Further analyses detected 11 mobile genetic elements, of which merA (54/84, 64.30%) had the highest frequency. All isolates were negative for intI3, traA, tnpU, traF, tnp513, tnsA, ISkpn7, ISpa7, ISkpn6, and ISCR1. We further analyzed antimicrobial resistance-related integrons among 30 E. coli isolates (the 27 intI1-positive isolates and the 3 intI2-positive isolates); six gene cassette profiles (dfrA17+aadA5, aadA2, dfrA12+aadA2, dfrA1+aadA1, dfrA1, and aadA1) were identified in the 27 intI1-positive isolates, but not in the three intI2-positive ones. Our study sheds light on the prevalence of multiple drug resistances and the diversity of mobile genetic elements in E. coli isolates, and highlights the necessity to monitor antibiotic resistance in more E. coli strains from captive giant pandas.

Giardia duodenalis is a common enteric protozoan that infects a range of hosts including humans and other mammals. Multilocus genotyping of G. duodenalis in captive non-human primates (NHPs) from zoos in China is limited. In this study, we evaluated 302 NHP fecal samples collected from 32 different NHP species. The primates were from 12 zoos distributed across eight provinces and two municipalities (Chongqing and Beijing) of China. The overall infection rate was 8.3% (25/302). The six G. duodenalis-positive zoos and their infection rates were: Suzhou Zoo (40.0%, 4/10), Yangzhou Zoo (22.2%, 2/9), Dalian Zoo (16.7%, 4/24), Chengdu Zoo (12.8%, 6/47), Guiyang Forest Wildlife Zoo (12.1%, 7/58), and Changsha Zoo (4.7%, 2/43). Molecular analysis of three loci, beta-giardin (bg), triose phosphate isomerase (tpi), and glutamate dehydrogenase (gdh), showed high genetic heterogeneity, and seven novel subtypes (BIII-1, MB10-1, WB8-1, B14-1, MB9-1, DN7-1, and BIV-1) were detected within assemblage B. Additional analysis revealed 12 different assemblage B multilocus genotypes (MLGs), one known MLG and 11 novel MLGs. Based on phylogenetic analysis, 12 assemblage B MLGs formed two main clades, MLG-SW (10-12, 18) and MLG-SW (13, 14, 16, 17), the other four MLG-SW (15, 19, 20, 21) were scattered throughout the phylogenetic tree in this study. Using multilocus genotyping, this study expands our understanding of the occurrence of Giardia infection and genetic variation in Giardia in captive non-human primates from zoos in China.

Additional file 1: Table S1. Primer sequences, fragment lengths and annealing temperatures used in this study.

Additional file 2: Figure S1. Sequence variation in the ITS region of the rRNA gene of Enterocytozoon bieneusi isolates from pet rabbits. The ITS sequences of five known genotypes (SC02, I, N, J, and CHY1) and the six novel genotypes (SCR01, SCR02, SCR04 to SCR07), identified in this study, were aligned with each other.

Vibrio mimicus is a foodborne pathogen, which is widely distributed in the aquatic environment. Moreover, it is often involved in aquatic animal diseases. In recent years, V. mimicus is an emerging pathogen in some species of Siluriformes. The strain SCCF01 was isolated from yellow catfish (Pelteobagrus fulvidraco). In this study, we aimed to perform genomic analysis of V. mimicus strain SCCF01 to identify genetic features and evolutionary relationships. Information on gene function and classification was obtained by functional annotation, and circular graph of strain SCCF01 genome, which was created by Circos v0.64. Information on virulence genes (adhesion, flagellum system, exotoxin, and secretory system, etc.) was obtained by virulence genes annotation. Genome element prediction showed that most of the mobile elements were distributed in chromosome I. Therefore, chromosome I of SCCF01 genome has more plasticity than chromosome II and might be larger in size. Genomic linear relationship between the strain of V. mimicus and strain SCCF01 was analyzed by linear pairwise comparison but was unable to determine the relationship. Gene family analysis predicted that the evolutionary direction of strain SCCF01 was: clinical strain → environmental strain → SCCF01 strain. Phylogenetic analysis showed that the strain SCCF01 was more closely related to environmental strains. According to gene family analysis and phylogenetic analysis, we speculated that strain SCCF01 has probably diverged from environmental strains.

Giardia duodenalis is a zoonotic parasitic protist and poses a threat to human and animal health. This study investigated the occurrence of G. duodenalis infection in post-weaned calves from Sichuan province, China. Faecal samples were collected from a total of 306 post-weaned calves (3-12 months old) from 10 farms, including 4 intensive feeding farms and 6 free-ranging farms. The overall infection rate of G. duodenalis was 41.2% (126/306) based on the PCR results at any of the three genetic loci: beta-giardin (bg), triose-phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. Giardia duodenalis assemblages E (n = 115, 91.3%), A (n = 3, 2.4%), and A mixed with E (n = 8, 6.3%) were identified among the 126 positive specimens. Multilocus sequence typing of G. duodenalis revealed 34 assemblage E multilocus genotypes (MLGs), 1 assemblage A MLG and 7 mixed assemblage (A and E) MLGs. The eBURST data showed a high degree of genetic diversity within assemblage E MLGs. The phylogenetic tree revealed that MLG E3 was the primary MLG subtype in Sichuan province and also the most widely distributed in China.

Objective To investigate the anti-inflammatory activity of Ageratina adenophora essential oil (AAEO-CP) and its effects on the expression of Toll-like receptor 4 (TLR4) protein in lipopolysaccharide (LPS)-induced RAW264.7 cells. Methods RAW264.7 cells were divided into control group, LPS group, and LPS combined with AAEO-CP group. The cytotoxicity of AAEO-CP was detected by CCK-8 assay. The mRNA and protein expression of interleukin-6 (IL-6) and IL-10 were detected by real-time PCR and ELISA, respectively, and the protein expression of TLR4 in RAW264.7 cells was measured by Western blotting. Results AAEO-CP below 20 mg/mL was not cytotoxic to RAW264.7 cells. LPS increased the protein expression of TLR4, also increased the protein and mRNA expression of IL-6, but decrease the protein and mRNA expression of IL-10 in RAW264.7 cells. And all of the above results were reversed by AAEO-CP. Conclusion AAEO-CP can play the anti-inflammatory effects by increasing the expression of IL-10 protein and decreasing the expression of IL-6 protein, and inhibiting TLR4 protein in LPS-induced RAW264.7 cells.

Cryptosporidium spp. are opportunistic pathogens that cause diarrhea in a variety of animal hosts. Although they have been reported in many animals, no information has been published on the occurrence of Cryptosporidium spp. in red-bellied tree squirrels (Callosciurus erythraeus). A total of 287 fecal specimens were collected from Sichuan province in China; the prevalence of Cryptosporidium spp., measured by nested-PCR amplification of the partial small-subunit (SSU) rRNA gene, was 1.4% (4/287). Three different Cryptosporidium species or genotypes were identified: Cryptosporidium parvum (n = 1), Cryptosporidium wrairi (n = 1), and Cryptosporidium rat genotype II (n = 2). The present study is the first report of Cryptosporidium infection in red-bellied tree squirrels in China. Although there is a relatively low occurrence of Cryptosporidium, the presence of C. parvum and C. wrairi, which were previously reported in humans, indicates that red-bellied tree squirrels may be a source of zoonotic cryptosporidiosis in China.

Giardia duodenalis is a common protozoan parasite that can infect humans and animals. Although previous studies demonstrated that the assemblage E of G. duodenalis is prevalent in cattle, studies on its genetic diversity were mostly based on single loci and very few involved multilocus analysis. To better understand the genetic variability and structure of G. duodenalis assemblage E in Chinese dairy cattle, 651 multilocus sequences derived from nine provinces (Gansu, Guangdong, Henan, Jiangsu, Ningxia, Shaanxi, Shanghai, Sichuan and Xinjiang) of China were analyzed in this study. Results showed that a total of 220 haplotypes were identified in the G. duodenalis assemblage E, with a high haplotype diversity (Hd = 0.97225) and low nucleotide diversity (π = 0.00259). The genetic differentiation index (FST) and gene flow (Nm) results indicated low degree of genetic differentiation, implying frequent genetic communication. Combined with the analysis of molecular variance (AMOVA), genetic variation within populations (81.7%) was higher than that among populations (18.3%), indicating low degree of genetic differentiation between populations. Such low rates of gene differentiation supported no significant correlations with geographical divisions. Moreover, both negative Tajima's D and Fu's FS values of neutrality tests and unimodal curve of mismatch distribution analyses indicated that G. duodenalis assemblage E population in Chinese dairy cattle had experienced demographic expansion. Overall, these findings contribute to an improved understanding of the population genetics and evolutionary biology of G. duodenalis assemblage E and assist in its control in cattle.

Streptococcus agalactiae (group B streptococcus, GBS) infection poses a serious threat to fish farming, particularly in warm-water locations. Recently, an infectious disease of unknown etiology with accumulative mortality rates of 25% to 40% was observed in cyprinid fish, Schizopygopsis pylzovi , of the Sichuan Province, China during autumn 2015. The clinical and pathological features of the disease were similar to those caused by streptococcosis infection in other fish species. Two Gram-positive, chain-forming coccus strains (ZYW151011 and ZYW151012) isolated from dying S. pylzovi were considered as the main pathogenic agents according to the experimental infections, and the LD 50 of both isolates were 1.61 × 10 6 and 2.07 × 10 6 cfu per fish respectively. According to morphology, physiological and biochemical, 16S rRNA gene and cfb gene specific sequence analyses, the two isolates were identified as S. agalactiae . The results of serotypes and Multiple Locus Sequence Typing (MLST) indicated that the isolates were Ia. and a new sequence typing (ZST-1) was identified, which is a close relative to ST-261. The sensitivity results showed that both isolates were sensitive to ceftriaxone, enrofloxacin and ofloxacin but resistant to doxycycline, florfenicol and amoxicillin. To our knowledge, this is the first report on S. agalactiae natural infection in S. pylzovi , and the isolate ZST-1 is a new sequence type for the variation of glnA .

Anesthetics utilized for the immobilization of pregnant mammals are prone to crossing the placental barrier and cause adverse effects to the fetuses. In this study, we develop a facile method employing high performance liquid chromatography (HPLC) for the study of Telazol crossing the placental barrier of pregnant pigs. The method mainly relies on the efficient extraction strategy that includes the mobile phase composed of 10 mM ammonium acetate aqueous solution-acetonitrile (1:4, v/v). When the injected dose of Telazol is 10 mg/kg (5 mg/kg of each constituent drug, zolazepem and tiletamine), zolazepam can cross the placental barrier as it is detected in both uterus and umbilical cord with approximately the same content. Conversely, tiletamine is detected in neither uterus nor umbilical cord, indicating the absence of placental transfer of tiletamine. The different absorption rates of the two dosage-equal compounds by pigs are found to be the main cause of their different abilities to cross the placental barrier.

Cladosporium cladosporioides is a ubiquitous fungus, causing infections in plants, humans, and animals. Suppression subtractive hybridization (SSH) and quantitative real-time PCR (qRT-PCR) were used in this study to identify differences in gene expression between two C. cladosporioides strains, the highly virulent Z20 strain and the lowly virulent Zt strain. A total of 61 unigenes from the forward library and 42 from the reverse library were identified. Gene ontology (GO) analysis showed that these genes were involved in various biological processes, cellular components and molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the unigenes in the forward library corresponded to 5 different pathways and the reverse library unigenes were involved in 3 different pathways. The qRT-PCR results indicated that expressions of APL1, GUD1, CSE1, SPBC3E7.04c and MFS were significantly different between Z20 and Zt strains, while genes encoding the senescence-associated proteins, pse1, nup107, mip1, pex2, icl1 and α/β hydrolase exhibited no significant differences between the two strains. In addition, we found that 5 unigenes encoding mip1, chk1, icl1, α/β hydrolase and β-glucosidase may be associated with pathogenicity. One unigene (MFS) may be related to the resistance to 14 α-demethylase inhibitor fungicides, and 5 unigenes (PEX2, NUP107, PSE1, APL1, and SPBC3E7.04c) may be related to either low spore yield or earlier aging of the Zt strain. Our study may help better understand the molecular mechanism of C. cladosporioides infection, and therefore improve the treatment and prevention of C. cladosporioides induced diseases.

We evaluated the effects of selenium (Se) on antioxidant enzymes of piglet splenic lymphocytes exposed to deoxynivalenol (DON). We measured cell viability, the activities of several antioxidant enzymes, and lactate dehydrogenase (LDH), as well as total antioxidant capacity (T-AOC) and the levels of malonaldehyde (MDA) and hydrogen peroxide (H2O2). We found that DON exposure increased the concentrations of LDH, MDA, and H2O2 in all experimental groups in a dose-dependent manner, while the concentrations of other antioxidant enzymes were decreased. In Se-pretreated DON-exposed cells, damage to antioxidant enzymes was reduced, especially in the lower-dose DON groups over longer exposure times. These results may indicate that in piglet splenic lymphocytes, Se can alleviate DON-induced damage to antioxidant enzymes by improving glutathione peroxidase activity. Se may function as a potential antioxidative agent to alleviate DON-induced oxidative stress.

Glucosamine (GlcN) is a common drug used to treat osteoarthritis (OA). To prolong the action time of glucosamine on OA and improve its therapeutic effect, this research explored the potential application of GlcN-loaded thermosensitive hydrogels based on poloxamer 407 and poloxamer 188 for OA therapy by intra-articular injection. The thermosensitive hydrogels were prepared by cold method, and the effects of P407, P188, and GlcN on sol–gel transition temperature (Tsol–gel) were compared. After screening was performed, the optimized formulation showed good temperature sensitivity, and Tsol–gel was approximately 35 °C. In vitro release tests showed that GlcN was slowly released from the thermosensitive hydrogels. After the gels were intra-articularly administered to treat OA in rabbits, the degree of swelling and inflammatory factors were significantly decreased in the hydrogel group compared with those in the OA model group (P

Surfactants can improve the hydrophobicity of poorly water-soluble drugs and increase the stability of microparticles by reducing surface tension. This study describes that surfactant-engineered florfenicol instant microparticles (FIMs) increase bioavailability through a micellar solubilization mechanism. The FIMs were prepared by a modified emulsification method, and the optimal prescription was obtained by a combination of single factor investigation and response surface methodology. The microparticles prepared in this study reduce the polymer materials while increasing the drug content. FIM has a smaller particle size and modification of poloxamer, resulting in better solubility and higher bioavailability. The in vitro solubility of FIM is 1.43 times higher than that of the bulk drug, and the dissolution equilibrium can be achieved in 10 minutes. Compared with florfenicol, FIM showed a decrease in Tmax in the plasma concentration curve, with a peak concentration of 1.43 times and an area of 1.41 times. Considering the advantages of in vitro/in vivo performance and ease of preparation, FIMs may have great application prospects in pharmacy research.

Deoxynivalenol (DON) is a type of cytotoxic mycotoxin that targets the mitochondria in cells. However, effects of DON on mitochondrial dynamics have not yet been reported. In this study, we investigated the effects of DON on mitochondrial dynamics and mitochondrial autophagy. Spleen lymphocytes of primary pigs were treated with different concentrations of DON. 24 h later, the cells were collected and the indexes were measured as follow: contents of ROS and MDA; activities of SDH and SOD; total antioxidant capacity; expression of MFN1, MFN2 and OPA1; the mRNA expression of the genes encoding mitochondrial autophagy proteins LC3 and P62, as well as mitochondrial respiratory chain complex activities. The results showed that, compared with the control group, the contents of ROS and MDA in the DON groups increased, while the activities of T-AOC, SDH and SOD decreased in a dose-dependent manner. With increasing DON concentrations, the expression levels of MFN1/2, OPA1 and mitochondrial respiratory chain complex activities decreased, while LC3, P62 increased. We suspect that DON causes oxidative damage, which in turn leads to down-regulation of MFN1, MFN2, OPA1 and up-regulation of LC3 and P62 mRNA, thereby inhibiting mitochondrial fusion and promoting mitochondrial autophagy.

Pyroptosis is a different type of proinflammatory and lytic mode of cell death from apoptosis and necrosis, which play a killer and cleaner to the pathogenic microorganisms as an immune response when the host is infected by pathogenic microorganisms. Ketone musk (KM) is a component of the native musk, which is widely used to medicine and chemical engineering. In this research, we studied whether KM can suppress the pyroptosis in J774A.1 cells induced by lipopoysaccharide (LPS)/Adenosine Triphosphate (ATP) stimulation. The results showed that KM increased the viability of LPS/ATP-stimulated cells, decreased the production of interleukin (IL)-1β/18, and suppressed the activation of caspased-1 and NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome and gasdermin D (GSDMD). Our present study demonstrated that KM inhibited LPS/ATP-induced pyroptosis and the release of IL-1β/18 in J774A.1 cells by inhibiting the activation of GSDMD and caspase-1 and the assembly of NLRP3 inflammasome. Our finding may be of significance on investigating that KM has a positive potential application in the treatment of pyroptosis-mediated diseases.

Infection by the canine distemper virus (CDV) results in a fulminating infectious disease that causes serious harm to dogs. With breaking 'of the CDV into primates, some researchers wonder if the CDV will cause a serious infection in humans. To better understand the potential of the CDV to infect humans, the molecular characteristics of the CDV, how it infects target cells in the host, the key receptors involved in infection, and infection of human cells in vitro were assessed in this review. There is no direct evidence that CDV can colonize and grow in humans. Two key receptors, SLAM and nectin-4, in hunans and primates have high identity, and the CDV can infect human cells in vitro. Therefore, we must pay close attention to the potential threat of infection by the CDV in humans.

Figure S9. Pathological sections of tissue damaged by Trichosporon moniliiforme (JYZ932) infection. A: Hepatocyte necrosis (400×); B: Thickening of the cuticle (400×); C: Basically normal structure of skin(400×); D: Proliferation of hepatocytes in the liver (400×); E: Proliferation of lymphocytes in the skin (400×); F: Basically normal structure of skin (400×). (PDF 430 kb)

Figure S7. Pathological sections of tissue damaged by Trichosporon shinodae (JYZ1223) infection. A: Central venous congestion of the liver, local necrosis of hepatocytes, and mild lymphocyte infiltration (400×); B: Thickening of the cuticle and granular layer, local necrosis of cells in the reticular layer, and proliferation of lymphocytes (400×); C: Normal skin structure (400×); D: Central venous congestion of the liver, hemorrhage of the hepatic sinusoids, swelling of liver cells, and infiltration of a small number of lymphocytes (400×); E: Slight thickening of the cuticle of the skin, local necrosis of reticular cells, and infiltration of lymphocytes (400×); F: Normal skin structure (400×); G: Spore stained with PAS in the dermis of a mouse in group B (400×). (PDF 610 kb)

Figure S6. Pathological sections of tissue damaged by Trichosporon cutaneum (JYZ030202) infection. A: Central venous congestion of the liver, necrosis of liver cells around the veins, and infiltration of lymphocytes (400×); B1: Necrosis of cells in the papillary layer and infiltration of lymphocytes (400×); B2: Necrosis of reticular cells in the skin and infiltration of lymphocytes (400×); C: Normal skin structure (400×); D: Central venous congestion of the liver, hepatic sinusoidal congestion, swelling of liver cells, and disorder of the hepatic cord (400×); E: Necrosis of reticular cells in the skin and proliferation of lymphocytes (400×); F: Normal skin structure (400×); G: Spore stained with PAS in a lesion in the dermis of a mouse in group B (400×); H: Spore stained with PAS in a lesion in the dermis of a mouse in group E (400×). (PDF 721 kb)

Figure S8. Pathological sections of tissue damaged by Trichosporon middelhovenii (JYZ12922) infection. A: Diffuse congestion, venous congestion, hepatocyte swelling, unclear structure of the hepatic cord, and proliferation of lymphocytes (400×); B: Coagulative necrosis of reticular cells in the skin and proliferation of lymphocytes (400×); C: Thickening of the cuticle of the skin and proliferation of reticular lymphocytes (400×); D: Central venous congestion and interstitial widening (400×); E: Coagulative necrosis of skin cells, unclear structure of skin tissue, and proliferation of lymphocytes (400×); F: Thickening of the cuticle of the skin (400×); G: Spore stained with PAS in the dermis of a mouse in group C (400×); H: Spore stained with PAS in the dermis of a mouse in group B (400×). (PDF 679 kb)

Figure S1. Pathological sections of tissue damaged by Trichosporon gracile (JYZ1291) infection. A: Central venous congestion of the liver, interstitial widening, and a small amount of lymphocyte proliferation (400×); B: Thickening of the cuticle of the skin and a small amount of lymphocyte proliferation (400×); C: Mild congestion in the reticular layer (400×); D: Central venous congestion of the liver, hepatic sinusoidal congestion, swelling of hepatocytes and proliferation of lymphocytes (400×); E: Normal structure (400×); F: Thickening of the cuticle and granular layer (400×). (PDF 488 kb)

Figure S2. Pathological sections of tissue damaged by Trichosporon brassicae (JYZ1253) infection. A: Central venous congestion of the liver, hepatic sinusoidal congestion, infiltration of lymphocytes, and interstitial widening (400×); B1: Congestion in the dermal papillary layer and thickening of the cuticle of the skin (400×); B2: Massive cell necrosis of the reticular layer of the skin, local coagulation necrosis, and a large amount of lymphocyte infiltration (400×); C: Thickening of the cuticle of the skin and infiltration of a few lymphocytes (400×); D: Central venous congestion and interstitial widening of the liver (400×); E: Necrosis of the reticular cells of the skin, local coagulation necrosis, and infiltration of a large number of lymphocytes (400×); F: Normal structure (400×); G: Spore stained with PAS in a lesion of the dermis of a mouse in group B (400×). (PDF 674 kb)

Figure S5. Pathological sections of tissue damaged by Trichosporon jirovecii (JYZA10) infection. A: Central venous congestion of the liver, mild lymphocyte infiltration, and hepatocyte swelling (400×); B1: Thickening of the cuticle and necrosis of skin cells (200×); B2: Necrosis of reticular cell (400×); C: Normal skin structure (400×); D: Diffuse congestion of the liver and interstitial widening (400×); E: Thickening of the cuticle, infiltration of inflammatory cells, and local congestion of the reticular layer (400×); F: Normal skin structure (400×). (PDF 563 kb)

Figure S3. Pathological sections of tissue damaged by Trichosporon domesticum (JYZ983) infection. A: Local necrosis and swelling of hepatocytes and infiltration of a small number of lymphocytes (400×); B: Thickening of the cuticle of the skin (400×); C: Normal structure (400×); D: Mild interstitial widening and swelling of hepatocytes (400×); E: Small amount of lymphocyte proliferation (400×); F: Normal structure (400×). (PDF 451 kb)

The solubility of Mequindox(MEQ) in two kinds of binary mixtures(acetone+isopropanol; N-propanol+N-butanol) with different ratio was determined by shake flask method through UV–Vis spectrophotometer over the temperature from 293.15 to 323.15 K at atmospheric pressure(p = .1 MPa). The results showed that the solubility of mequindox in binary solvent mixtures was increased with temperature raised in the investigated temperature range. In the binary-solvent of acetone and isopropanol, the data was increased as the mole fraction of acetone increased. However, the solubility of MEQ was not significantly changed in the mixed solvents of N-propanol and N-butanol with different ratio. Furthermore, thermodynamic models including the modified Apelblat equation, λh equation, GSM equation and the Jouyban-Acree model were used to correlate the experimental data. In addition, thermodynamic properties, enthalpy (△H0sol), entropy (△S0sol) and Gibbs free energy (△G0sol), were calculated and analyzed in the dissolution process. This study provides a certain theoretical basis for the isolation purification and pharmaceutical formulation design of MEQ.

Brucella species are the causative agents of brucellosis, a worldwide zoonotic disease that affects a broad range of mammals and causes great economic losses. Small regulatory RNAs (sRNAs) are post-transcriptional regulatory molecules that participate in the stress adaptation and pathogenesis of Brucella. In this study, we characterized the role of a novel sRNA, BSR1141, in the intracellular survival and virulence of Brucella melitensis. The results show that BSR1141 was highly induced during host infections and under in vitro stress situations that simulated the conditions encountered within host phagocytes. In addition, a BSR1141 mutant showed reduced survival both under in vitro stress conditions and in mice, confirming the role of BSR1141 in Brucella intracellular survival. Bioinformatic and experimental approaches revealed that BSR1141 affects the expression of many target genes, including the Brucella virulence component virB2. These data indicate that BSR1141 could influence the expression of virB2, which is important for B. melitensis pathogenesis and intracellular survival. This work provides new insight into the mechanism of adaptation to environmental stress and into the pathogenesis of intracellular pathogens.

Enterocytozoon bieneusi is a common intestinal pathogen in a variety of animals. While E. bieneusi genotypes have become better-known, there are few reports on its prevalence in the Tibetan pig. This study investigated the prevalence, genetic diversity, and zoonotic potential of E. bieneusi in the Tibetan pig in southwestern China. Tibetan pig feces (266 samples) were collected from three sites in the southwest of China. Feces were subjected to PCR amplification of the internal transcribed spacer (ITS) region. Enterocytozoon bieneusi was detected in 83 (31.2%) of Tibetan pigs from the three different sites, with 25.4% in Kangding, 56% in Yaan, and 26.7% in Qionglai. Prevalence varies according to age group, from 24.4% (age 0–1 years) to 44.4% (age 1–2 years). Four genotypes of E. bieneusi were identified: two known genotypes EbpC (n = 58), Henan-IV (n = 24) and two novel genotypes, SCT01 and SCT02 (one of each). We compare our results with a compilation of published results on the host range and geographical distribution of E. bieneusi genotypes in China. Phylogenetic analysis showed these four genotypes clustered to group 1 with zoonotic potential. Multilocus sequence typing (MLST) analysis of three microsatellites (MS1, MS3, MS7) and one minisatellite (MS4) was successful in 47, 48, 23 and 47 positive specimens and identified 10, 10, 5 and 5 genotypes at four loci, respectively. This study indicates the potential danger of E. bieneusi to Tibetan pigs in southwestern China, and offers basic advice for preventing and controlling infections.

Increasing evidences have demonstrated that Ageratina adenophora (A. adenophora) can cause hepatotoxicity of animals. Liver is an important site in immune regulation and inflammatory responses. However, the information about hepatotoxicity induced by A. adenophora in relation to inflammation is still finite. To investigate the underlying mechanism, we conducted animal experiments with different dosage of A. adenophora. Mice were randomly divided into 4 groups and administrated with 0%, 10%, 20% and 30% levels of A. adenophora pallet diet in control, group A, B and C, respectively. The results showed that A. adenophora caused hepatotoxicity as revealed by increasing alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase. Then, the reactive oxygen species (ROS) levels were shown to be elicited by A. adenophora through flow cytometry assay in a dose-dependent manner. Furthermore, pyroptosis was activated by A. adenophora, which was characterized by increasing protein and mRNA levels of caspase-1, gasdermin D and interleukin-1β. Notably, ROS down-stream factors, including nod-like receptor inflammasome protein 3 and nuclear factor-κB, were also activated by A. adenophora. These data demonstrated that A. adenophora caused liver inflammatory injury and induced hepatocyte pyroptosis by activating NLRP3 inflammasome, which was triggered by elevating ROS production levels. This research might provide new insights into the mechanism of hepatotoxicity induced by A. adenophora.

Florfenicol (FF) is a novel animal-specific amidohydrin broad-spectrum antibiotic. However, its aqueous solubility is extremely poor, far below the effective dose required for veterinary clinic. Thus, FF is often used in large doses, which significantly limits its preparation and application. To overcome these shortcomings, the FF-hydroxypropyl-β-cyclodextrin (FF-HP-β-CD) inclusion complexes were developed using the solution-stirring method. The physical properties of FF-HP-β-CD were characterized. A comparison was conducted between FF and FF-HP-β-CD freeze-dried powder injection of their muscle irritation and the pharmacokinetics. The drug loading and saturated solubility of FF-HP-β-CD at 37 °C were 11.78% ± 0.04% and 78.93 ± 0.42 mg/mL, respectively (35.4-fold compared with FF). Results of scanning electron microscopy, differential scanning calorimetry, X-ray diffraction, and Fourier transform infrared showed that FF was entrapped in the inner cavity of HP-β-CD, and the inclusion complex formed in an amorphous state. In comparison with FF commercial injection, FF-HP-β-CD increased the elimination half-life (t1/2β), transport rate constant (K10, K12, K21), and maximum concentration (Cmax) after intramuscular injection in beagle dogs. Conversely, it decreased the distribution half-life (t1/2α), absorption rate constant (Ka), apparent volume of distribution (V1/F), and peak time (Tmax). These results suggest that FF-HP-β-CD freeze-dried powder injection is a promising formulation for clinical application.

MicroRNAs (miRNAs) are small, non-coding RNAs that have been proposed to control or fine-tune complex genetic pathways by post-transcriptional regulation of target genes. It was proved that numerous miRNAs have influence on the biology of adipocytes as well as on the function of adipose tissues. This study shows that miR-10b-5p expression was decreased in mice, rats, and human under obesity. In addition, the obtained results indicated that the expression level of miR-10b-5p was increased in 3T3-L1 pre-adipocytes without manifesting a significant role in 3T3-L1 cells proliferation. On the other hand, the downregulation of miR-10b-5p by the inhibitor played a role in 3T3-L1 cells differentiation and adipogenesis. Our results strongly suggest that Apol6 was the target gene of miR-10b-5p. The inhibition of miR-10b-5p promoted the differentiation of 3T3-L1 cells and adipogenesis by upregulating the Apol6 expression. Then, the upregulated Apol6 acted as an oncogene in certain obesity-related cancers. These results indicate that miR-10b-5p may have a therapeutic significance for obesity and obesity-related cancers.

Trichosporonis the dominant genus of epidermal fungi in giant pandas and causes local and deep infections. To provide the information needed for the diagnosis and treatment of trichosporosis in giant pandas, the sequence of ITS, D1/D2, and IGS1 loci in 29 isolates ofTrichosporonspp. which isolated from the body surface of giant pandas were combination to investigate interspecies identification and genotype. Morphological development was examined via slide culture. Additionally, mice were infected by skin inunction, intraperitoneal injection, and subcutaneous injection for evaluation of pathogenicity. The twenty-nine isolates ofTrichosporonspp. were identified as belonging to 11 species, andTrichosporon jiroveciiandT. asteroideswere the commonest species. Four strains ofT. laibachiiand one strain ofT. moniliiformewere found to be of novel genotypes, andT. jiroveciiwas identified to be genotype 1.T. asteroideshad the same genotype which involved in disseminated trichosporosis. The morphological development processes of theTrichosporonspp. were clearly different, especially in the processes of single-spore development. Pathogenicity studies showed that 7 species damaged the liver and skin in mice, and their pathogenicity was stronger than other 4 species.T. asteroideshad the strongest pathogenicity and might provoke invasive infection. The pathological characteristics of liver and skin infections caused by differentTrichosporonspp. were similar. So it is necessary to identify the species ofTrichosporonon the surface of giant panda. Combination of ITS, D1/D2, and IGS1 loci analysis, and morphological development process can effectively identify the genotype ofTrichosporonspp.

Adipokines can affect intrauterine development while calf birthweight (CBW) is a breeding standard of calves, which reflects the status of fetal intrauterine development. To explore the correlation between placental adipokines and CBW, 54 healthy Chinese Holstein cows were used in the present study. The cows were grouped according to the CBW of their calves. Placentas were collected immediately after delivery and enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction were used to detect the placental expression levels of adiponectin, leptin, visfatin and resistin. Our results show that the mRNA transcription and blood placental content of adiponectin, leptin, visfatin and resistin increased with increasing CBW. The analysis showed that the mRNA transcription levels of placental adiponectin, leptin and resistin were positively correlated with CBW. The mRNA and protein expression levels of adiponectin, leptin and visfatin between the three groups were significantly correlated. Placental resistin mRNA levels correlated positively with adiponectin mRNA, but not leptin or visfatin. The protein expression levels of resistin were significantly positively correlated with those of adiponectin, leptin and visfatin. These results suggest that placental adipokines play important roles in regulating calf intrauterine growth.

We investigated infection by canine parvovirus and genetic variation of the VP2 gene. We collected feces samples of 50 diarrheal dogs in Sichuan Province, China. Analyses polymerase chain reaction (PCRs), agarose gel electrophoresis, and amplification of the complete sequence of canine parvovirus were done. We observed 19PCR-positive samples. Sequencing analyses of 15PCR-positive samples based on amplification of the complete VP2 gene showed all to be CPV-2a,and to be polymerized with Sichuan isolates. These results suggest that the common epidemic strain in Sichuan Province is CPV-2a,and may originate from the same strain. Compared with reference strains, there were no significant variations in canine parvovirus in Sichuan Province, China.

Enterocytozoon bieneusi is a common intestinal pathogen and a major cause of diarrhea and enteric diseases in a variety of animals. While the E. bieneusi genotype has become better-known, there are few reports on its prevalence in the Tibetan pig. This study investigated the prevalence, genetic diversity, and zoonotic potential of E. bieneusi in the Tibetan pig in southwestern China. Tibetan pig feces (266 samples) were collected from three sites in the southwest of China. Feces were subjected to PCR amplification of the internal transcribed spacer (ITS) region. E. bieneusi was detected in 83 (31.2%) of Tibetan pigs from the three different sites, with 25.4% in Kangding, 56% in Yaan and 26.7% in Qionglai. Age group demonstrated the prevalence of E. bieneusi range from 24.4%(aged 0 to 1 years) to 44.4%(aged 1 to 2 years). Four genotypes of E. bieneusi were identified: two known genotypes EbpC (n=58), Henan-IV (n=24) and two novel genotypes, SCT01 and SCT02 (one of each). Phylogenetic analysis showed these four genotypes clustered to group 1 with zoonotic potential. Multilocus sequence typing (MLST) analysis three microsatellites (MS1, MS3, MS7) and one minisatellite (MS4) revealed 47, 48, 23 and 47 positive specimens were successfully sequenced, and identified ten, ten, five and five genotypes at four loci, respectively. This study indicates the potential danger of E. bieneusi to Tibetan pigs in southwestern China, and offers basic data for preventing and controlling infections.

This report is the first to describe Cryptosporidium infection in bamboo rats (Rhizomys sinensis). Ninety-two fresh fecal specimens were collected from a pet market in Ya'an City, China. One Cryptosporidium isolate from an asymptomatic host and two isolates from separate hosts with diarrhea were obtained by using Sheather's sucrose flotation technique and modified acid-fast staining. The Cryptosporidium spp. were genotyped by nested PCR and nucleotide sequencing of the small subunit rRNA (SSU rRNA), 70-kDa heat shock protein (HSP70), oocyst wall protein (COWP), and actin genes: isolates were identified as Cryptosporidium parvum with minor nucleotide differences at all four loci. Further subtyping was performed by PCR amplification and DNA sequence analysis of the 60-kDa glycoprotein (gp60) gene: two subtype families were detected, including a novel C. parvum subtype IIpA9 and a rare subtype IIoA13G1 (only reported in diarrheal patients of Sweden). Our results suggest that the bamboo rat is a reservoir host of C. parvum. Significantly, we discovered that the rare C. parvum subtype family IIo is also a zoonotic subtype and confirmed C. parvum subtype IIpA9 as a novel subtype family.

Giardia duodenalis and Cryptosporidium spp. are common gastrointestinal protozoa in mammals. Many studies have been conducted on the distribution of G. duodenalis and Cryptosporidium spp. genotypes in sheep and cattle. However, in China, information about molecular characterization and genetic analysis of G. duodenalis and Cryptosporidium spp. in goats is limited. In this study, 342 fecal samples from adult goats were collected from 12 farms in Sichuan Province, China. The occurrence of G. duodenalis and Cryptosporidium spp. in adult goats was 14.9% (51/342) and 4.7% (16/342), respectively. All G. duodenalis were identified as assemblage E, with two novel genotypes (assemblages E17 and E18) being detected at the beta-giardin (bg) locus. Based on three loci-beta-giardin (bg), triose phosphate isomerase (tpi), and glutamate dehydrogenase (gdh)-multilocus sequence typing revealed three novel multilocus genotypes (MLGs) of assemblage E (MLG-E1, E2, E3 (sc)). Small Subunit (SSU) rRNA-based PCR identified two Cryptosporidium species, namely C. xiaoi (11/16) and C. suis (5/16). This study is not only the first to report C. suis infection in adult goats in China but is also the first to use the MLG approach to identify G. duodenalis in adult goats.

Euptox A (9-oxo-10, 11-dehydroageraphorone), the main toxin isolated from Eupatorium adenophorum, is known to induce immunotoxicity in animals. However, the precise mechanism underlying the effects of Euptox A on splenocytes is unclear. Here, we aimed to investigate the molecular mechanisms underlying the effect of Euptox A in mouse spleens after its intragastric administration and found that Euptox A exhibits proautophagic effects in splenocytes. Euptox A markedly arrested the splenocytes in the G0/G1 phase, which was accompanied by inhibition of the expression of the positive regulators CDK4, CDK2, cyclin D1, PCNA, and E2F1, and promotion of the expression of the negative regulators p53, p21 Waf1/Cip1, p27 Kip1, and Chk1. We also found that Euptox A did not markedly induce splenocyte apoptosis, but induced autophagy while increasing the subcellular localization of punctate LC3, ratio of LC3-II/LC3-I, and Beclin 1 levels, and decreasing p62 levels. Euptox A also significantly inhibited p-PI3K, p-p38 MAPK, p-Akt, and p-mTOR expression, but increased PTEN and p-AMPK expression. These results indicated that Euptox A induced splenocyte autophagy by inhibiting the PI3K/Akt/mTOR pathway, suppressing p38 MAPK expression, and activating AMPK. These findings provide new insights into the mechanisms involved in spleen toxicity caused by Euptox A in mice.

Growth performance and meat quality are important traits for the pig industry and consumers. Adipose tissue is the main site at which fat storage and fatty acid synthesis occur. Therefore, we combined high-throughput transcriptomic sequencing in adipose and muscle tissues with the quantification of corresponding phenotypic features using seven Chinese indigenous pig breeds and one Western commercial breed (Yorkshire). We obtained data on 101 phenotypic traits, from which principal component analysis distinguished two groups: one associated with the Chinese breeds and one with Yorkshire. The numbers of differentially expressed genes between all Chinese breeds and Yorkshire were shown to be 673 and 1056 in adipose and muscle tissues, respectively. Functional enrichment analysis revealed that these genes are associated with biological functions and canonical pathways related to oxidoreductase activity, immune response, and metabolic process. Weighted gene coexpression network analysis found more coexpression modules significantly correlated with the measured phenotypic traits in adipose than in muscle, indicating that adipose regulates meat and carcass quality. Using the combination of differential expression, QTL information, gene significance, and module hub genes, we identified a large number of candidate genes potentially related to economically important traits in pig, which should help us improve meat production and quality.

Canine parvovirus 2 (CPV-2) was first identified in 1978, and is responsible for classic parvoviral enteritis. Despite the widespread vaccination of domestic carnivores, CPVs have remained important pathogens of domestic and wild carnivores. In this study, we isolated CPV-2 from Tibetan mastiffs and performed a global analysis of the complete VP2 gene sequences of CPV-2 strains in China. Six isolates were typed as new CPV-2a, according to key amino acid positions. On a phylogenetic tree, these six sequences formed a distinct clade. Five isolates occurred on the same branch as KF785794 from China and GQ379049 from Thailand; CPV-LS-ZA1 formed a separate subgroup with FJ435347 from China. One hundred ninety-eight sequences from various parts of China and the six sequences isolated here formed seven distinct clusters, indicating the high diversity of CPVs in China. Of 204 VP2 sequences, 183 (91.04%) encoded the mutation Ser297Ala, regardless of the antigenic type, implying that most Chinese CPV-2 strains contain the VP2 mutation Ser297Ala. However, the biological significance of this change from prototype CPV-2a/2b to new CPV-2a/2b types remains unclear. This study is the first to isolate new CPV-2a from the Tibetan mastiff. Our data show that new CPV-2a/2b variants are now circulating in China.

Fecal specimens from two Bactrian camels were collected in the Ya’an city zoo of China and were examined for Cryptosporidium by centrifugal flotation. One specimen was found to be parasitized by Cryptosporidium via microscopy, and the oocysts were measured to have an average size of 7.03 × 5.50 μm (n > 50). The isolate was genotyped by polymerase chain reaction (PCR) amplification and DNA sequence analysis of the partial 18S rRNA, COWP, and A135 genes, and was confirmed to be Cryptosporidium andersoni with minor nucleotide differences. Multilocus sequence typing (MLST) analysis indicated that the subtype of the camel-derived C. andersoni isolate was A4, A4, A4, and A1 at the four minisatellite loci (MS1, MS2, MS3, and MS16, respectively). Therefore, this isolate belongs to the most common MLST subtype reported in cattle in China and is distinct from two other known camel C. andersoni MLST subtypes (A6, A4, A2, A1 and A6, A5, A2, A1). Animal transmission experiments demonstrated that the C. andersoni isolate was not infectious to immunosuppressed or immunocompetent Kun-ming mice, Sprague-Dawley rats, and hamsters but was biologically similar to most bovine C. andersoni isolates characterized so far. Therefore, transmission of this camel-derived C. andersoni isolate is very likely to occur between camels and bovine.