ISOLATION, IDENTIFICATION, AND GENOME ANALYSIS OF LUNG PATHOGENIC KLEBSIELLA PNEUMONIAE (LPKP) IN FOREST MUSK DEER.

Klebsiella pneumoniae is an important pathogen commonly associated with opportunistic infections. In this study, lung pathogenic K. pneumoniae (LPKP) was isolated and identified from suppurative pneumoniae in forest musk deer by conventional methods and by 16S ribosomal RNA sequence analysis. Median lethal dose and histopathologic analysis were used to demonstrate pathogenicity of the organism in mice. Furthermore, a draft genome of LPKP was sequenced, and its virulence genes were detected. One hundred and twenty-two virulence genes encoded determinant of capsule polysaccharide (CPS), lipopolysaccharide, fimbriae, outer membrane proteins, iron acquisition, and urease. In particular, 20 CPS-related genes were highly conserved in LPKP, K. pneumoniae U, K. pneumoniae NTUH-KP35, and K. pneumoniae KP-1. All of the strains were identified as capsular type K54. This is the first report of capsular type K54 K. pneumoniae causing suppurative pneumonia in an animal. The results of this study provided the basis for understanding the pathogenicity of LPKP and laid a foundation for the development of vaccines for the capsular type K54 K. pneumoniae disease.

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.

Isolation and bioinformatics analysis of the NADC30_Like CJS01 strain of the porcine reproductive and respiratory syndrome virus.

In this study, lung tissue was collected from nursery piglets suspected of being infected with porcine reproductive and respiratory syndrome virus (PRRSV) in a large­scale pig farm in Sichuan, China. Polymerase chain reaction (PCR) and reverse transcription quantitative­polymerase chain reaction (RT­qPCR) methods were used to ensure that no other pathogens were present. Virus isolation was also carried out where the presence of PRRSV was determined by indirect immunoinfluscent assay (IFA). Compared with the common PRRSV strain, the isolate did not produce evident Cytopathic effect (CPE) in the early stage of isolation. CPE was found in the late stage, and the titer was 104.17 TCID50/0.1 mL. The strain was named CJS01. Bioinformatics analysis showed that it was a NADC30­Like strain. The virus load was determined by measuring the nucleic acid load during the proliferation of the strain on Marc­145 cells. The strain showed good adaptability on cells, and the virus proliferated on cells for 84 hr when the highest nucleic acid load was achieved. By recombinant analysis of ORF3~7 genes and prediction of its epitope, it was found that CJS01 strain might interfere with the immunesystem of the infected animals.

Preliminary study on the role of novel LysR family gene kp05372 in Klebsiella pneumoniae of forest musk deer.

LysR-type transcriptional regulators are involved in the regulation of numerous cellular metabolic processes in Klebsiella pneumoniae, leading to severe infection. Earlier, we found a novel LysR family gene, named kp05372, in a strain of K. pneumoniae (designated GPKP) isolated from forest musk deer. To study the function of this gene in relation to the biological characteristics of GPKP, we used the suicide plasmid and conjugative transfer methods to construct deletion mutant strain GPKP-Δkp05372; moreover, we also constructed the GPKP-Δkp05372+ complemented strain. The role of this gene was determined by comparing the following characteristics of three strains: growth curves, biofilm formation, drug resistance, stress resistance, median lethal dose (LD50), organ colonization ability, and the histopathology of GPKP. Real-time polymerase chain reaction (RT-PCR) was used to test the expression level of seven genes upstream of kp05372. There was no significant difference in the growth rates when comparing the three bacterial strains, and no significant difference was recorded at different osmotic pressures, temperatures, salt contents, or hydrogen peroxide concentrations. The GPKP-Δkp05372 mutant formed a weak biofilm, and the other two strains formed medium biofilm. The drug resistance of the GPKP-Δkp05372 mutant toward cephalothin, cotrimoxazole, and polymyxin B was changed. The acid tolerance of the deletion strain was stronger than that of the other two strains. The LD50 values of the wild-type and complemented strains were 174-fold and 77-fold higher than that of the GPKP-Δkp05372 mutant, respectively. The colonization ability of the GPKP-Δkp05372 mutant in the heart, liver, spleen, kidney, and intestine was the weakest. The three strains caused different histopathological changes in the liver and lungs. In the GPKP-Δkp05372 mutant, the relative expression levels of kp05374 and kp05379 were increased to 1.32-fold and 1.42-fold, respectively, while the level of kp05378 was decreased by 42%. Overall, the deletion of kp05372 gene leads to changes in the following: drug resistance and acid tolerance; decreases in virulence, biofilm formation, and colonization ability of GPKP; and regulation of the upstream region of adjacent genes.

Affinity of Streptococcal G Protein to Forest Musk Deer (Moschus berezovskii) Serum Immunoglobulin G.

Forest musk deer (FMD; Moschus berezovskii) immunoglobulin G efficiently bound to streptococcal G protein (SPG) and weakly bound to staphylococcal A protein. The results suggested that horseradish peroxidase-conjugated SPG could be chosen as an enzyme-labeled antibody substitute and laid a foundation for immunologic research in FMD disease.

Elizabethkingia miricola infection in Chinese spiny frog (Quasipaa spinosa).

Elizabethkingia miricola is a Gram-negative rod which has been incriminated in severe infections in humans. Recently, a serious infectious disease was identified in Chinese spiny frogs (Quasipaa spinosa), in the Sichuan Province of China; the disease was characterized by corneal opacity, the presence of ascites and neurological symptoms. A Gram-negative bacillus was isolated from the liver, spleen and kidney of the diseased frogs. Experimental infection test revealed that the bacillus could infect the frogs Q. spinosa and the LD50 value was 1.19 × 106  cfu per frog. The isolated Gram-negative bacillus was identified as E. miricola according to phenotypic characteristics, 16S rRNA and gyrB gene sequence analysis. The isolated strain was only susceptible to florfenicol among all investigated chemotherapeutic agents. Histological examination revealed that E. miricola infection caused pathological lesions to multiple organs and tissues, especially in the liver, brain, kidney. These results confirmed that E. miricola is an emerging pathogen of Chinese spiny frogs.

Effects of compound probiotics on the weight, immunity performance and fecal microbiota of forest musk deer.

Probiotics are intended to provide health benefits when consumed, generally by improving or restoring the gut flora. The health problems of forest musk deer (FMD, Moschus berezovskii), a threatened species currently under conservation, restrict the development of captive musk deer. This study was conducted with the aim of analyzing the effects of forest musk deer compound probiotics (FMDPs) on weight, immunity performance and fecal microbiota in FMD by measuring average daily weight gain (ADG) and immune-related factors and by using high-throughput 16S rRNA sequencing to investigate differences in the fecal microbiota among the control group (4 samples), treatment group A (4 samples) and treatment group B (4 samples). The results showed that the ADG of treatment groups A and B was significantly higher than that of the control group (p = 0.032, p = 0.018). The increase in IgA and IgG levels in treatment group B was significantly higher than that in the control group (p = 0.02, p = 0.011). At the phylum and genus levels, the difference in bacterial community structure was significant between treatment group B and the control group. Both the alpha diversity and beta diversity results showed significant differences in the microbiota of FMD before and after FMDP feeding. In summary, the results indicated that FMDPs could promote the growth of growing FMD, improve immunity and balance the role of intestinal microbes.

A Novel Capillary Electrophoresis-Based High-Throughput Multiplex Polymerase Chain Reaction System for the Simultaneous Detection of Nine Pathogens in Swine.

Here we aimed to develop a capillary electrophoresis-based high-throughput multiplex polymerase chain reaction (PCR) system for the simultaneous detection of nine pathogens in swine. Nine pairs of specific primers and a set of universal primers were designed; the multiplex PCR was established. The specificity and cross-reactivity of this assay were examined, and the detection limit was determined using serial 10-fold dilutions of plasmids containing the target sequences. The assay was further tested using 144 clinical samples. We found that the nine specific amplification peaks were observed, and the assay had a high degree of specificity, without nonspecific amplification. The simultaneous detection limit for the nine viruses reached 10000 copies µL-1 when all of the premixed viral targets were present. Seventy-seven of the clinical samples tested positive for at least one of the viruses; the principal viral infections in the clinical samples were porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus. This approach has much potential for further development of high-throughput detection tools for the diagnosis of diseases in animals.