Molecular cloning, prokaryotic expression and its application potential evaluation of interferon (IFN)-ω of forest musk deer.

Forest musk deer (Moschus berezovskii) are currently a threatened species under conservation, and the development of captive populations is restricted by health problems. To evaluate the application potential of interferon (IFN)-ω in the prevention and control of forest musk deer disease, 5 forest musk deer IFN-ω (fmdIFNω) gene sequences were successfully obtained by homologous cloning method for the first time. FmdIFNω5 was selected and recombinant fmdIFNω protein (rIFNω) was successfully expressed by pGEX-6P-1 plasmid and E. coli expression system. The obtained protein was used to stimulate forest musk deer lung fibroblasts cells FMD-C1 to determine its regulatory effect on interferon-stimulated genes (ISGs). In addition, an indirect ELISA method based on anti-rIFNω serum was established to detect endogenous IFN-ω levels in 8 forest musk deer. The results showed that there were 18 amino acid differences among the 5 fmdIFNω subtypes, all of which had the basic structure to exert the activity of type I IFN and were close to Cervus elaphus IFN-ω in the phylogenetic tree. The protein expressed was 48 kDa, and the transcription levels of all ISGs were increased in FMD-C1 cells stimulated by rIFNω, and the amount of transcription accumulation was time-dependent. Meanwhile, Anti-rIFNω serum of mice could react with both rIFNω and forest musk deer serum, and the OD450nm value of forest musk deer serum with the most obvious symptoms was the highest, suggesting that the level of natural IFN-ω in different forest musk deer could be monitored by the rIFNω-based ELISA method. These results indicate that fmdIFNω has the potential as an antiviral drug and an early indication of innate immunity, which is of great significance for the prevention and control of forest musk deer diseases.

Virulence gene detection and antimicrobial resistance analysis of Enterococcus faecium in captive giant pandas (Ailuropoda melanoleuca) in China.

BACKGROUND: The emergence of multidrug resistance among enterococci makes effective treatment of enterococcal infections more challenging. Giant pandas (Ailuropoda melanoleuca) are vulnerable to oral trauma and lesions as they feast on bamboo. Enterococci may contaminate such oral lesions and cause infection necessitating treatment with antibiotics. However, few studies have focused on the virulence and drug resistance of oral-derived enterococci, including Enterococcus faecium, in giant pandas. In this study, we analyzed the prevalence of 8 virulence genes and 14 drug resistance genes in E. faecium isolates isolated from saliva samples of giant pandas held in captivity in China and examined the antimicrobial drug susceptibility patterns of the E. faecium isolates. RESULTS: Twenty-eight isolates of E. faecium were successfully isolated from the saliva samples. Four virulence genes were detected, with the acm gene showing the highest prevalence (89%). The cylA, cpd, esp, and hyl genes were not detected. The isolated E. faecium isolates possessed strong resistance to a variety of drugs; however, they were sensitive to high concentrations of aminoglycosides. The resistance rates to vancomycin, linezolid, and nitrofurantoin were higher than those previously revealed by similar studies in China and other countries. CONCLUSIONS: The findings of the present study indicate the drugs of choice for treatment of oral E. faecium infection in the giant panda.

Screening of candidate genes related to differences in growth and development between Chinese indigenous and Western pig breeds.

Neijiang (NJ) and Yacha (YC) are two indigenous pig breeds in the Sichuan basin of China, displaying higher resistance to diseases, lower lean ratio, and slower growth rate than the commercial Western pig breed Yorkshire (YS). The molecular mechanisms underlying the differences in growth and development between these pig breeds are still unknown. In the present study, five pigs from NJ, YC, and YS breeds were subjected to the whole genome resequencing, and then the differential single-nucleotide polymorphisms (SNPs) were screened using a 10-kb window sliding in 1-kb step using the Fst method. Finally, 48,924, 48,543, and 46,228 nonsynonymous single-nucleotide polymorphism loci (nsSNPs) were identified between NJ and YS, NJ and YC, and YC and YS, which highly or moderately affected 2,490, 800, and 444 genes, respectively. Moreover, three nsSNPs were detected in the genes of acetyl-CoA acetyltransferase 1 (ACAT1) insulin-like growth factor 2 receptor (IGF2R), insulin-like growth factor 2 and mRNA-binding protein 3 (IGF2BP3), which potentially affected the transformation of acetyl-CoA to acetoacetyl-CoA and the normal functions of the insulin signaling pathways. Moreover, serous determinations revealed significantly lower acetyl-CoA content in YC than in YS, supporting that ACAT1 might be a reason explaining the differences in growth and development between YC and YS breeds. Contents of phosphatidylcholine (PC) and phosphatidic acid (PA) significantly differed between the pig breeds, suggesting that glycerophospholipid metabolism might be another reason for the differences between Chinese and Western pig breeds. Overall, these results might contribute basic information to understand the genetic differences determining the phenotypical traits in pigs.

Antioxidant Capacity and Protective Effect of Cow Placenta Extract on D-Galactose-Induced Skin Aging in Mice.

Placental extract has been used for skin care and delaying skin aging. Cow placenta is an abundant resource with a large mass, which has not been harnessed effectively. Cow placenta extract (CPE) has the functions of antioxidation, anti-inflammatory, promoting growth and development, and promoting hair growth. However, little is known about the effect of oral administration of cow placenta extract on skin conditions. Therefore, the present study aimed to investigate the antioxidant capacity of CPE in vitro and in vivo and its protective effect on d-galactose (D-gal) induced skin aging in mice. The results showed that CPE had strong free radical scavenging, reducing and metal chelating activities. CPE can increase the activity of catalase (CAT), glutathione peroxidase (GSH-Px), peroxidase (POD), superoxide dismutase (SOD), and the content of glutathione (GSH), decrease the content of malondialdehyde (MDA). Moreover, CPE can decrease the gene and protein expression of matrix metalloproteinase 1a (MMP-1a) and matrix metalloproteinase 3 (MMP-3) and increase the expression of transforming growth factor-ß (TGF-ß) and tissue inhibitor of metalloproteinase 1 (TIMP-1) of mouse skin. Histopathological analysis showed CPE reduced the collagen damage caused by D-gal, increased collagen synthesis and reduced its degradation to delay skin aging.

High prevalence of blaCTX-M and blaSHV among ESBL producing E. coli isolates from beef cattle in China's Sichuan-Chongqing Circle.

Enterobacteria that produce extended-spectrum ß-lactamase (ESBL) such as Escherichia coli (E. coli) are common in our environment and known to cause serious health implications in humans and animals. ß-lactam antibiotics such as penicillins, cephalosporins and monobactams are the most commonly used anti-bacterials in both humans and animals, however, Gram negative bacteria (such as E. coli) that produces extended-spectrum ß-lactamases (ESBLs) have the ability to hydrolyze most ß-lactams therefore making them resistant to ß-lactam antibiotics. Recent extensive researches on the epidemiology and genetic characteristics of extended-spectrum ß-lactamase (ESBL)-producing E. coli reported the existence of ESBL-producing E. coli in humans, companion animals and poultry. Therefore, this experiment was performed to investigate the prevalence and genetic characteristics of ß-lactamase producing E. coli isolated from beef cattle farms in the Sichuan-Chongqing circle of China. Phenotypic confirmation of ESBL-producing E. coli was performed using the double disk synergy test. Polymerase Chain Reaction (PCR) was used to detect blaCTX-M, blaSHV and blaTEM gene codes, then after, isolates were divided into different phylogenetic groups and multi-locus sequence typing (MLST). The results showed that out of the 222 E. coli strains isolated from the beef cattle, 102 strains showed ESBL phenotypes. The PCR results showed that blaCTX-M was the predominant ESBL gene identified among the E. coli strains with 21 (9.5%) isolates having this gene, followed by blaSHV which was found in 18 (8.1%) isolates. The majority of these ESBL positive isolates were assigned to phylogroup A (19.8%) followed by phylogroup B1 (13.5%). In addition, from the MLST results on ESBL positive isolates (n = 30) we identified 19 STs, ST398 (ST398cplx) and ST7130 which were the prevalent population (20%). In conclusion, the high prevalence of CTX-M, and SHV in the study confirmed its association with E. coli infection; therefore, this calls for health concerns on ESBL-producing E. coli. As far as we know, this is the first comprehensive research report relating to ESBL-producing E. coli incidence in Chinese beef cattle.

Poloxamer modified florfenicol instant microparticles for improved oral bioavailability.

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.

Synthesis, Characterization, and Pharmacodynamics Study of Enrofloxacin Mesylate.

INTRODUCTION: Enrofloxacin is used in the treatment of a wide variety of bacterial infections in mammals. However, its poor solubility limits the clinical use. METHODS: In order to improve the solubility of enrofloxacin, the enrofloxacin mesylate (EM) were obtained by a chemical synthesis method. The characterization of EM was carried out using ultraviolet scan (UV), synchronous thermal analysis (SDT), fourier transform infrared spectrometer (FTIR) and mass spectrometry (MS), nuclear magnetic resonance (NMR) and X-ray powder diffraction analysis (XRPD). Acute toxicity of EM in Kunming mice was studied. Besides, pharmacokinetic studies were performed in New Zealand rabbits at a single oral dose of 10 mg/kg, and the antibacterial activity of EM was also evaluated. RESULTS: EM was successfully synthesized and purified. The stoichiometric ratio of mesylate to enrofloxacin was 1:1 and the aqueous solubility of EM was 483.01±4.06 mg/mL, the solubility of EM was about 2000 times higher than enrofloxacin. The oral lethal dose (LD50) of EM was 1168.364 mg/kg, and the pharmacokinetics indicated that the oral relative bioavailability of EM was about 1.79 times and 1.48 times higher than that of enrofloxacin and enrofloxacin hydrochloride, respectively. In addition, the in vitro antibacterial activity of EM was not significantly changed compared with enrofloxacin and enrofloxacin hydrochloride. CONCLUSION: EM has higher solubility, low toxicity for oral use, and increases the oral bioavailability in rabbit. This study may be of benefit for the development of new enrofloxacin drugs.

New genotypes and molecular characterization of Enterocytozoon bieneusi in pet birds in Southwestern China.

Enterocytozoon bieneusi, a unicellular enteric microsporidian parasite, can infect humans and a wide range of animals throughout the world. Although E. bieneusi has been identified in many animals, there is no information regarding the genotypes of E. bieneusi in pet birds in China. Birds are important sources of emerging infectious diseases that affect humans, and immunosuppressed individuals can be exposed to potential zoonotic agents shed by birds. The aim of the present study was to determine the prevalence and genotypic diversity of E. bieneusi in pet birds, as well as assessed its zoonotic potential. A total of 387 fecal samples were collected from Psittaciformes (n = 295), Passeriformes (n = 67), and Galliformes (n = 16) from four pet markets in Sichuan province, Southwestern China. The overall prevalence of E. bieneusi in pet birds was 25.1% based on nested polymerase chain reaction analysis of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene (Psittaciformes, 21.7%; Passeriformes, 37.3%; Galliformes, 50.0%). Eight genotypes of E. bieneusi were identified, including five known genotypes (D, SC02, BEB6, CHB1, and MJ5) and three novel genotypes (SCB-I, SCB-II, and SCB-III). In phylogenetic analysis, genotypes D and SC02 and one novel genotype SCB-II were clustered within group 1, genotype BEB6 was classified within group 2, and the remaining genotypes (CHB1, MJ5, SCB-I, and SCB-III) clustered with group 10. To the best of our knowledge, this is the first report of E. bieneusi infection in pet birds in China. Genotypes D, SC02, and BEB6 that have been previously identified in humans, were found in pet birds in this study, suggesting that these pet birds can be a potential source of human microsporidiosis in China.

First report of Blastocystis in giant pandas, red pandas, and various bird species in Sichuan province, southwestern China.

Blastocystis is a common enteric protist that colonizes humans and a wide range of animals. Although some studies have reported incidences of Blastocystis in humans and animals in China, there is no information available on the prevalence of Blastocystis in giant pandas, red pandas, or bird species. The aims of the present study were to determine the prevalence, subtype distribution, and genetic characterizations of Blastocystis in these animals in a captive situation in southwestern China, as well as assess the zoonotic potential of Blastocystis isolates. A total of 168 fecal specimens, including 81 from giant pandas, 23 from red pandas, 38 from black swans, 11 from ruddy shelducks, and 15 from green peafowl were collected at the Chengdu Research Base of Giant Panda Breeding in Sichuan province. The overall minimum prevalence of Blastocystis was 11.3% (19/168) based on PCR amplification of the barcode region of the SSU rRNA gene. The highest prevalence of Blastocystis was observed in ruddy shelduck (18.2%) and the lowest was found in green peafowl (6.7%). The prevalence of Blastocystis in giant pandas >5.5 years of age was higher than that in younger giant pandas. Two potentially zoonotic subtypes (ST1 and ST8) were identified, and ST1 (n = 12) was found to be more prevalent than ST8 (n = 7). To the best of our knowledge, this is the first report of the prevalence and subtypes of Blastocystis in giant pandas, red pandas, and bird species in China. The findings of this study will improve our understanding of the genetic diversity and public health potential of Blastocystis.

Genetic variation in mitochondrial cox2 of Heterakis gallinarum from poultry in Sichuan, China.

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 FST and Nm (FST = 0.01929, Nm = 12.71), there was a frequent gene flow but no significant genetic differentiation observed among the populations. The network map showed that the most prominent haplotype was HS1, and the other haplotypes (HS2-HS23) were centered on HS1 with a star-like topology, indicating that H. gallinarum had previously experienced a population expansion. To our knowledge, this is the first research on the population genetics of H. gallinarum based on mitochondrial cox2.

Epidemiology of Cryptosporidium infection in cattle in China: a review.

The present review discusses the findings of cryptosporidiosis research conducted in cattle in China and highlights the currently available information on Cryptosporidium epidemiology, genetic diversity, and distribution in China, which is critical to understanding the economic and public health importance of cryptosporidiosis transmission in cattle. To date, 10 Cryptosporidium species have been detected in cattle in China, with an overall infection rate of 11.9%. The highest rate of infection (19.5%) was observed in preweaned calves, followed by that in juveniles (10.69%), postweaned juveniles (9.0%), and adult cattle (4.94%). The dominant species were C. parvum in preweaned calves and C. andersoni in postweaned, juvenile, and adult cattle. Zoonotic Cryptosporidium species (C. parvum and C. hominis) were found in cattle, indicating the possibility of transmission between humans and cattle. Different cattle breeds had significant differences in the prevalence rate and species of Cryptosporidium. This review demonstrates an age-associated, breed-associated, and geographic-related occurrence of Cryptosporidium and provides references for further understanding of the epidemiological characteristics, and for preventing and controlling the disease.

Mitochondrial genomes of Heterakis gallinae and Heterakis beramporia support that they belong to the infraorder Ascaridomorpha.

Heterakis gallinae and Heterakis beramporia are the most prevalent nematode infecting native chicken breed, causing major economic losses. In the present study, the complete mitochondrial genomes (mt) of H. gallinae and H. beramporia were amplified by long-PCR and then sequenced. The complete mt genomes of H. gallinae and H. beramporia were 13,973bp and 14,012bp in size, respectively. Both mt genomes contain 12 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. All genes are transcribed in the same direction and the gene arrangement is identical to Ascaridia spp. Phylogenetic analysis based on the 12 protein-coding genes revealed that the family Heterakidae (represented by H. gallinae and H. beramporia) was more closely related to the infraorder Ascaridomorpha than it was to the infraorder Oxyuridomorpha. The present study determined the complete mt genome sequences for two Heterakis species, providing useful markers for studying the systematics, population genetics, and molecular epidemiology of these Heterakis parasites.

Absence of population genetic structure in Heterakis gallinarum of chicken from Sichuan, inferred from mitochondrial cytochrome c oxidase subunit I gene.

Population genetics information provides a foundation for understanding the transmission and epidemiology of parasite and, therefore, may be used to assist in the control of parasitosis. However, limited available sequence information in Heterakis gallinarum has greatly impeded the study in this area. In this study, we first investigated the genetic variability and genetic structure of H. gallinarum. The 1325 bp fragments of the mitochondrial COX1 gene were amplified in 56 isolates of H. gallinarum from seven different geographical regions in Sichuan province, China. The 56 sequences were classified into 22 haplotypes (H1-H22). The values of haplotype diversity (0.712) and nucleotide diversity (0.00158) in Sichuan population indicate a rapid expansion occurred from a relatively small, short-term effective population in the past. The haplotype network formed a distribution around H1 in a star-like topology, and the haplotypes did not cluster according to their geographical location. Similar conclusions could be made from MP phylogenetic tree. The Fst value (Fst<0.16965) and AMOVA analysis revealed that no significant genetic differentiation was observed among the seven different geographical populations. Neutrality tests (Tajima's D and Fu's Fs) and mismatch analysis indicated that H. gallinarum experienced a population expansion in the past. Our results indicated that H. gallinarum experienced a rapid population expansion in the past, and there was a low genetic diversity and an absence of population structure across the population.