ClpP protease modulates bacterial growth, stress response, and bacterial virulence in Brucella abortus.

The process of intracellular proteolysis through ATP-dependent proteases is a biologically conserved phenomenon. The stress responses and bacterial virulence of various pathogenic bacteria are associated with the ATP-dependent Clp protease. In this study, a Brucella abortus 2308 strain, ΔclpP, was constructed to characterize the function of ClpP peptidase. The growth of the ΔclpP mutant strain was significantly impaired in the TSB medium. The results showed that the ΔclpP mutant was sensitive to acidic pH stress, oxidative stress, high temperature, detergents, high osmotic environment, and iron deficient environment. Additionally, the deletion of clpP significantly affected Brucella virulence in macrophage and mouse infection models. Integrated transcriptomic and proteomic analyses of the ΔclpP strain showed that 1965 genes were significantly affected at the mRNA and/or protein levels. The RNA-seq analysis indicated that the ΔclpP strain exhibited distinct gene expression patterns related to energy production and conversion, cell wall/membrane/envelope biogenesis, carbohydrate transport, and metabolism. The iTRAQ analysis revealed that the differentially expressed proteins primarily participated in amino acid transport and metabolism, energy production and conversion, and secondary metabolites biosynthesis, transport and catabolism. This study provided insights into the preliminary molecular mechanism between Clp protease to bacterial growth, stress response, and bacterial virulence in Brucella strains.

Molecular Characteristics of Bovine Viral Diarrhea Virus Strains Isolated from Persistently Infected Cattle.

In this study, we reported the isolation, identification, and molecular characteristics of nine BVDV strains that were isolated from the serum of persistently infected cattle. The new strains were designated as BVDV TJ2101, TJ2102, TJ2103, TJ2104, TJ2105, TJ2106, TJ2107, TJ2108 and TJ2109. The TJ2102 and TJ2104 strains were found to be cytopathic BVDV, and the other strains were non-cytopathic BVDV. An alignment and phylogenetic analysis showed that the new isolates share 92.2-96.3% homology with the CP7 strain and, thus, were classified as the BVDV-1b subgenotype. A recombination analysis of the genome sequences showed that the new strains could be recombined by the major parent BVDV-1a NADL strain and the minor parent BVDV-1m SD-15 strain. Some genome variations or unique amino acid mutations were found in 5'-UTR, E0 and E2 of these new isolates. In addition, a potential linear B cell epitopes prediction showed that the potential linear B cell epitope at positions 56-61 is highly variable in BVDV-1b. In conclusion, the present study has identified nine strains of BVDV from persistently infected cattle in China. Further studies on the virulence and pathogenesis of these new strains are recommended.

S1P promotes corneal trigeminal neuron differentiation and corneal nerve repair via upregulating nerve growth factor expression in a mouse model.

Corneal disease was the most critical cause of vision loss. This study aimed to research a new method and provide a theoretical basis for treating corneal injury. A mice corneal epithelial injury model was constructed by the method of mechanical curettage. Models were treated with sphingosine 1-phosphate (S1P) and si-Spns2. An immunofluorescence assay was used to detect ßIII-tubulin. The expressions of neurotrophic factor, S1P transporter, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway-related proteins were detected by western blot. Hematoxylin-eosin staining was processed to detect the effect of SIP on corneal repair in mice. si-Spns2 inhibited the effect of S1P. S1P significantly repaired the corneal injury, while si-Spns2 treatment made it more severe. Moreover, S1P could significantly increase the levels of NGF, BDNF, GDNF, Spns2, and p-ERK1/2. si-Spns2 inhibits the effect of S1P in the expression of these proteins. S1P significantly increased axonal differentiation of trigeminal ganglion neurons, which was inhibited after si-Spns2 treatment. S1P promoted corneal trigeminal neuron differentiation and corneal nerve repair via upregulating nerve growth factor expression in a mouse model. Treatment of corneal injury by S1P may be an effective approach.

Regulated necrosis, a proinflammatory cell death, potentially counteracts pathogenic infections.

Since the discovery of cell apoptosis, other gene-regulated cell deaths are gradually appreciated, including pyroptosis, ferroptosis, and necroptosis. Necroptosis is, so far, one of the best-characterized regulated necrosis. In response to diverse stimuli (death receptor or toll-like receptor stimulation, pathogenic infection, or other factors), necroptosis is initiated and precisely regulated by the receptor-interacting protein kinase 3 (RIPK3) with the involvement of its partners (RIPK1, TRIF, DAI, or others), ultimately leading to the activation of its downstream substrate, mixed lineage kinase domain-like (MLKL). Necroptosis plays a significant role in the host's defense against pathogenic infections. Although much has been recognized regarding modulatory mechanisms of necroptosis during pathogenic infection, the exact role of necroptosis at different stages of infectious diseases is still being unveiled, e.g., how and when pathogens utilize or evade necroptosis to facilitate their invasion and how hosts manipulate necroptosis to counteract these detrimental effects brought by pathogenic infections and further eliminate the encroaching pathogens. In this review, we summarize and discuss the recent progress in the role of necroptosis during a series of viral, bacterial, and parasitic infections with zoonotic potentials, aiming to provide references and directions for the prevention and control of infectious diseases of both human and animals.

Bacteriological and molecular typing of Clostridium perfringens strains isolated in retail beef in Beijing, China.

Clostridium perfringens is an important zoonotic pathogen. This study was designed to explore the prevalence and toxin types of C. perfringens in retail beef collected from Beijing, China. Among 221 beef samples collected, 53 samples were positive for C. perfringens, resulting in the average prevalence as 23.98%. By toxin gene-based typing, the most C. perfringens strains belong to type A (96.23%, 51/53), only 2 strains were identified as type D. By a multi-locus sequence typing (MLST)-based analysis, a total of 36 sequence types (STs) were detected, and the most STs (n=30) represented just a single strain. These finding suggested that the prevalence of C. perfringens in retail beef in Beijing was considerably high and these bacteria displayed extreme diversity in genetics.

Safety and Transcriptome Analysis of Live Attenuated Brucella Vaccine Strain S2 on Non-pregnant Cynomolgus Monkeys Without Abortive Effect on Pregnant Cynomolgus Monkeys.

Brucellosis, caused by Brucella spp., is an important zoonotic disease leading to enormous economic losses in livestock, posing a great threat to public health worldwide. The live attenuated Brucella suis (B. suis) strain S2, a safe and effective vaccine, is widely used in animals in China. However, S2 vaccination in animals may raise debates and concerns in terms of safety to primates, particularly humans. In this study, we used cynomolgus monkey as an animal model to evaluate the safety of the S2 vaccine strain on primates. In addition, we performed transcriptome analysis to determine gene expression profiling on cynomolgus monkeys immunized with the S2 vaccine. Our results suggested that the S2 vaccine was safe for cynomolgus monkeys. The transcriptome analysis identified 663 differentially expressed genes (DEGs), of which 348 were significantly upregulated and 315 were remarkably downregulated. The Gene Ontology (GO) classification and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that these DEGs were involved in various biological processes (BPs), including the chemokine signaling pathway, actin cytoskeleton regulation, the defense response, immune system processing, and the type-I interferon signaling pathway. The molecular functions of the DEGs were mainly comprised of 2'-5'-oligoadenylate synthetase activity, double-stranded RNA binding, and actin-binding. Moreover, the cellular components of these DEGs included integrin complex, myosin II complex, and blood microparticle. Our findings alleviate the concerns over the safety of the S2 vaccine on primates and provide a genetic basis for the response from a mammalian host following vaccination with the S2 vaccine.

Phylogenetic and genomic analysis reveals high genomic openness and genetic diversity of Clostridium perfringens.

Clostridium perfringens is associated with a variety of diseases in both humans and animals. Recent advances in genomic sequencing make it timely to re-visit this important pathogen. Although the genome sequence of C. perfringens was first determined in 2002, large-scale comparative genomics with isolates of different origins is still lacking. In this study, we used whole-genome sequencing of 45 C. perfringens isolates with isolation time spanning an 80-year period and performed comparative analysis of 173 genomes from worldwide strains. We also conducted phylogenetic lineage analysis and introduced an openness index (OI) to evaluate the openness of bacterial genomes. We classified all these genomes into five lineages and hypothesized that the origin of C. perfringens dates back to ~80 000 years ago. We showed that the pangenome of the 173 C. perfringens strains contained a total of 26 954 genes, while the core genome comprised 1020 genes, accounting for about a third of the genome of each isolate. We demonstrated that C. perfringens had the highest OI compared with 51 other bacterial species. Intact prophage sequences were found in nearly 70.0 % of C. perfringens genomes, while CRISPR sequences were found only in ~40.0 %. Plasmids were prevalent in C. perfringens isolates, and half of the virulence genes and antibiotic resistance genes (ARGs) identified in all the isolates could be found in plasmids. ARG-sharing network analysis showed that C. perfringens shared its 11 ARGs with 55 different bacterial species, and a high frequency of ARG transfer may have occurred between C. perfringens and species in the genera Streptococcus and Staphylococcus. Correlation analysis showed that the ARG number in C. perfringens strains increased with time, while the virulence gene number was relative stable. Our results, taken together with previous studies, revealed the high genome openness and genetic diversity of C. perfringens and provide a comprehensive view of the phylogeny, genomic features, virulence gene and ARG profiles of worldwide strains.

First identification of a Brucella abortus biovar 4 strain from yak in Tibet, China.

Brucellosis is one of the major zoonotic diseases in the world. In China, understanding on its causative agent Brucella is still limited. Recently, we isolated a Brucella strain XZ19-1 from yak in Lhasa, Tibet. Phenotypical characterization proved that it belongs to B. abortus biovar 4, a biotype that has never been reported in China. MLVA-16 genotyping revealed a novel profile (4-5-3-12-2-2-3-3-8-32-8-5-4-3-3-3) in this strain, while MLST sequence typing demonstrated that it belongs to ST 71. Furthermore, the whole genome of XZ19-1 strain was sequenced. Subsequent phylogenetic analysis demonstrated that XZ19-1was genetically more closely related to B. abortus strains originated from European countries rather than to those collected from China previously. Isolation and identification of XZ19-1 strain may thus indicate a unique Brucella lineage existing in Qing-Tibet plateau. These findings will help to improve the diagnosis and epidemiological studies of brucellosis in animals and human in this part of China.

Complete Genome Sequence of Mycoplasma bovis Strain XBY01, Isolated from Henan Province, China.

We report the complete genome sequence of Mycoplasma bovis strain XBY01, which was isolated from a severely diseased young calf in Henan Province, China, in 2019. The genome of XBY01 contains a single circular chromosome of 986,067 bp, with a GC content of 29.30%.

Mutations in PB2 and HA enhanced pathogenicity of H4N6 avian influenza virus in mice.

The H4 subtype avian influenza virus (AIV) continues to circulate in both wild birds and poultry, and occasionally infects mammals (e.g. pigs). H4-specific antibodies have also been detected in poultry farm workers, which suggests that H4 AIV poses a potential threat to public health. However, the molecular mechanism by which H4 AIVs could gain adaptation to mammals and whether this has occurred remain largely unknown. To better understand this mechanism, an avirulent H4N6 strain (A/mallard/Beijing/21/2011, BJ21) was serially passaged in mice and mutations were characterized after passaging. A virulent mouse-adapted strain was generated after 12 passages, which was tentatively designated BJ21-MA. The BJ21-MA strain replicated more efficiently than the parental BJ21, both in vivo and in vitro. Molecular analysis of BJ21-MA identified four mutations, located in proteins PB2 (E158K and E627K) and HA (L331I and G453R, H3 numbering). Further studies showed that the introduction of E158K and/or E627K substitutions into PB2 significantly increased polymerase activity, which led to the enhanced replication and virulence of BJ21-MA. Although individual L331I or G453R substitutions in HA did not change the pathogenicity of BJ21 in mice, both mutations significantly enhanced virulence. In conclusion, our data presented in this study demonstrate that avian H4 virus can adapt to mammals by point mutations in PB2 or HA, which consequently poses a potential threat to public health.

Complete Genome Sequence of Genotype Psittacine Beak and Feather Disease Virus, a Strain Identified from Budgerigars in China.

Psittacine beak and feather disease virus (PBFDV) has been reported in many countries, such as Australia, Poland, the United States, South Africa, etc. In this study, the complete genome of a PBFDV isolate was determined and characterized from budgerigars in China.

Complete Genome Sequence of an Avian Polyomavirus Strain First Isolated from a Pigeon in China.

Avian polyomavirus can infect multiple bird species and cause inflammatory disease with high mortality in young psittacine birds. In this study, we sequenced and analyzed an avian polyomavirus isolated from a pigeon in China, strain APV-P, which is closely related to a polyomavirus in psittacine birds.

Effects of Vaccination with the C-Strain Vaccine on Immune Cells and Cytokines of Pigs Against Classical Swine Fever Virus.

The attenuated C-strain vaccine against classical swine fever virus (CSFV) is one of the safest and most effective attenuated vaccines. However, little is known of the host immune response after vaccination with the C-strain vaccine. Blood samples from vaccinated pigs were collected to evaluate the number of immune cells, the level of specific CSFV antibody, and related cytokines induced by the vaccination of C-strain vaccine. The C-strain nucleic acid was gradually removed and specific antibody to vaccine kept increasing; the amount of the lymphocyte, Tc cell, and Th cell increased; some inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor-α mainly showed downregulated trends, but IL-6 and IL-8 were upregulated greatly; IL-2, IL-4, IL-5, IL-12p40, IL-13, interferon (IFN)-I, and Toll-like receptors (TLRs) kept high expression level after 28 days postvaccination (dpv); IFN-γ was upregulated slightly at 5 and 9 dpv, respectively. These results suggest that the C-strain vaccine induces a Th2 cell response to produce the specific antibody. The vaccine virus replicates at very low level. C-strain vaccine burden has close relationship with the expression of TLRs. The overexpression of TLRs initiates the innate immune system to clear up the vaccine. Meanwhile, ILs expressed by immune system induce the differentiation of B cells and produce specific antibody.

The role of heme oxygenase-1 (HO-1) in the regulation of inflammatory reaction, neuronal cell proliferation and apoptosis in rats after intracerebral hemorrhage (ICH).

OBJECTIVE: To investigate the role of heme oxygenase-1 (HO-1) in the regulation of inflammatory reaction, neuronal cell proliferation and apoptosis in rats after intracerebral hemorrhage (ICH). METHODS: Thirty-six adult Sprague Dawley (SD) male rats were randomly divided into sham operation, ICH and zinc protoporphyrin (ZPP) group. Rats (except for the sham operation group) were given 50 µL stereotactic injection of autologous blood from the femoral artery into the caudate nucleus, to establish an ICH model. In addition, rats in the ZPP group were given 10 mg/kg intraperitoneal injection of ZPP. At day 3 postoperative, neurobehavioral changes and brain water content were evaluated, brain tissue HO-1 expression was detected with immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR), brain tissue apoptosis was evaluated with TUNEL method, Caspase 3, Caspase 8 and Caspase 9 activity were detected with colorimetric method, level of TNF-α, IL-1ß, IL-6 and IL-8 were measured with the enzyme-linked immunosorbent assay (ELISA), while Bcl-2, Bax, p-NF-κB p65 and p-IκBα protein expression were detected with Western blot. RESULTS: ICH group compared to sham operation: HO-1 positive rate and mRNA expression were increased, neurological deficit score and cell apoptosis rate were increased, Caspase 3, Caspase 8 and Caspase 9 activity were increased, level of TNF-α, IL-1ß, IL-6 and IL-8 were increased, Bcl-2 expression was downregulated, Bax, p-NF-κB p65 and p-IκBα expression were upregulated. The differences were statistically significant (P<0.01). ZPP group compared to ICH: HO-1 positive rate and mRNA expression were decreased, neurological deficit score and cell apoptosis rate were decreased, Caspase 3, Caspase 8, Caspase 9 activity were decreased, level of TNF-α, IL-1ß, IL-6 and IL-8 were decreased, Bcl-2 expression was upregulated, Bax, p-NF-κB p65 and p-IκBα expression were downregulated, and the differences were statistically significant (P<0.01). CONCLUSION: HO-1 inhibitor, ZPP does have a protective effect on ICH rats. This might be due to its inhibition to the inflammatory reaction and neuronal cell apoptosis.

Antimicrobial Resistance Profile and Genotypic Characteristics of Streptococcus suis Capsular Type 2 Isolated from Clinical Carrier Sows and Diseased Pigs in China.

Streptococcus suis serotype 2 is an important zoonotic pathogen. Antimicrobial resistance phenotypes and genotypic characterizations of S. suis 2 from carrier sows and diseased pigs remain largely unknown. In this study, 96 swine S. suis type 2, 62 from healthy sows and 34 from diseased pigs, were analyzed. High frequency of tetracycline resistance was observed, followed by sulfonamides. The lowest resistance of S. suis 2 for ß-lactams supports their use as the primary antibiotics to treat the infection of serotype 2. In contrast, 35 of 37 S. suis 2 with MLSB phenotypes were isolated from healthy sows, mostly encoded by the ermB and/or the mefA genes. Significantly lower frequency of mrp+/epf+/sly+ was observed among serotype 2 from healthy sows compared to those from diseased pigs. Furthermore, isolates from diseased pigs showed more homogeneously genetic patterns, with most of them clustered in pulsotypes A and E. The data indicate the genetic complexity of S. suis 2 between herds and a close linkage among isolates from healthy sows and diseased pigs. Moreover, many factors, such as extensive use of tetracycline or diffusion of Tn916 with tetM, might have favored for the pathogenicity and widespread dissemination of S. suis serotype 2.

[Skull base reconstruction and peroperative treatment for cranio-orbital tumors].

OBJECTIVE: To investigate the peroperative treatment of cranio-orbital tumors and the method of the reconstruction of the skull base. METHODS: Between April 2008 and April 2011, 35 patients with cranio-orbital tumor were treated. There were 21 males and 14 females, aged 17-73 years (mean, 46.3 years). The first symptoms were orbital pain in 13 cases, hypopsia in 12 cases, exophthalmos or abnormal eye position in 5 cases, headache and dizziness in 2 cases, diplopia in 2 cases, and pulsating eyeball in 1 case. Some of the patients needed resecting the zygomatic arch, superciliary arch, and orbit roof. The autogenous bone, titanium net, frontal bone periosteum, biogel, and artificial meninges were used to reconstruct the skull base. RESULTS: Tumors were resected by one-stage operation, and the anterior skull bases were reconstructed. Postoperative MRI indicated that total removal of tumors was achieved in 30 cases, subtotal in 3 cases, and partial in 2 cases at 3 days. There was no operative death. Cerebrospinal rhinorrhea and infection occurred at 1 week in 1 and 2 cases respectively, and were cured after lumbar drainage and antibiotics. The patients were followed up 6 to 36 months (mean, 18 months). In patients having hypopsia, the visual function was improved in 9 cases at 1 month; in patients having orbital pain, pain relief was achieved at 2 weeks after operation; in patients having exophthalmos or abnormal eye position and pulsating eyeball, sympotoms disappeared after operation. In 27 patients with benign tumor, 24 were cured, without recurrence during follow-up; in 8 patients with malignant tumor, 6 had recurrence within 18 months and underwent second operation or radiotherapy, 2 relapsed cases died of cerebral hernia and respiratory circulating failure at 24 months after operation. No complication of enophthalmos, pulsating exophthalmos, or collapse of zygomatic region occurred. CONCLUSION: Using the autogenous bone, titanium net, frontal bone periosteum, biogel, and artificial meninges to reconstruct the skull base has reliable foundation, simple operation, and easy anatomical reconstruction, so it is an effective method after the removal of cranio-orbital tumors; better effectiveness would be obtained when combining with the peroperative nursing.

Long-term results of patients with head injuries treated in different hospitals after the Wenchuan, China, earthquake.

OBJECTIVE: To present an overview of mortality, disability, and depression among patients with head injuries admitted to multiple center hospitals after the 2008 Wenchuan earthquake in China. METHODS: A retrospective analysis was done of the medical records of earthquake victims with head traumas admitted to three different area hospitals; analysis included 18 months of follow-up. Demographic data, causes of injury, diagnosis, clinical classification, treatment, and prognosis were reviewed. RESULTS: This analysis included 1299 victims with craniocerebral injuries admitted to eight hospitals in or near the disaster zone. The victims were divided into three main groups. Group A comprised 68 victims treated in the local hospital; all 68 (100%) were hospitalized during the 24 hours after the earthquake, and 38 (55.88%) victims died 18 months later. Group B comprised 600 victims treated in regional hospitals; 264 (44%) were admitted within 72 hours after the earthquake, and 109 (18.17%) died. Group C comprised 631 victims transported to territory hospitals; 39 (6.18%) were dead at 18 months. Among survivors, 409 (31.48%) victims sustained a disability, and 879 (80.35%) have depression. CONCLUSIONS: In the first 72 hours after this major earthquake, the local and regional hospital neurosurgery departments were flooded with numerous victims with craniocerebral injuries. Most of these patients died or were disabled. The territory hospitals with expert neurosurgeons and advanced equipment did not effectively contribute to successful treatment of victims. The mobile hospital played an important role in initial triage and treatment.

Dynamic distribution and tissue tropism of classical swine fever virus in experimentally infected pigs.

BACKGROUND: Classical swine fever (CSF), caused by the Classical swine fever virus (CSFV), is an Office International des Epizooties (OIE) notifiable disease. However, we are far from fully understand the distribution, tissue tropism, pathogenesis, replication and excretion of CSFV in pigs. In this report, we investigated the dynamic distribution and tissue tropism of the virus in internal organs of the experimentally infected pigs using real-time RT-PCR and immunohistochemistry (IHC). RESULTS: A relative quantification real-time PCR was established and used to detect the virus load in internal organs of the experimentally infected pigs. The study revealed that the virus was detected in all 21 of the internal organs and blood collected from pigs at day 1 to day 8 post infections, and had an increasing virus load from day 1 to day 8 post infections. However, there was irregular distribution virus load in most internal organs over the first 2 days post infection. Blood, lymphoid tissue, pancreas and ileum usually contain the highest viral loads, while heart, duodenum and brain show relatively low viral loads. CONCLUSIONS: All the data suggest that CSFV had an increasing virus load from day 1 to day 8 post infections in experimentally infected pigs detected by real-time RT-PCR, which was in consistent with the result of the IHC staining. The data also show that CSFV was likely to reproduce in blood, lymphoid tissue, pancreas and the ileum, while unlikely to replicate in the heart, duodenum and brain. The results provide a foundation for further clarification of the pathogenic mechanism of CSFV in internal organs, and indicate that blood, lymphoid tissue, pancreas and ileum may be preferred sites of acute infection.

A novel RT-LAMP assay for rapid and simple detection of classical swine fever virus.

A simple and rapid assay for the detection of Classical swine fever virus (CSFV) was established using reverse transcription loop-mediated isothermal amplification (RT-LAMP). This study describes the amplification of the genomic RNA of CSFV under isothermal conditions (63 °C) within one hour, using a set of six primers (two outer primers, two inner primers and two loop primers). This RT-LAMP assay showed 100-fold higher sensitivity than the standard RT-PCR method and identified eighteen additional positive cases that were negative when tested by RT-PCR. This RT-LAMP was able to detect all the 13 strains of CSFV but not the BVDV. PRRSV. SIV. PRV-PCV, thus showed a good specificity. Products amplified by RT-LAMP can be visualized by agarose gel electrophoresis and in addition, either as a white precipitate at the bottom of the tube after a pulse spin or as a color change when dyed with SYBR Green I which are visible to the naked eye. Because RT-LAMP is low-cost and produces rapid results, it has the potential to be an excellent tool for CSFV surveillance in the field, especially in developing countries.

Investigation of integrons/cassettes in antimicrobial-resistant Escherichia coli isolated from food animals in China.

In this study, 326 Escherichia coli isolates from food animals collected during the last four decades in China were characterized using antimicrobial susceptibility testing and screening for integrons/cassettes. Minimum inhibitory concentration (MIC) testing indicated that the antimicrobial resistance of E. coli has increased since the 1970s. The findings of this study present a warning to veterinary practitioners about the excessive use of antimicrobials, and suggest the necessity for surveillance and control of antimicrobial resistance in veterinary clinical medicine in China.