The absence of luxS reduces the invasion of Avibacterium paragallinarum but is not essential for virulence.

The contagious respiratory pathogen, Avibacterium paragallinarum, contributes to infectious coryza in poultry. However, commercial vaccines have not shown perfect protection against infectious coryza. To search for an alternative approach, this research aimed to investigate whether the quorum-sensing system of pathogens plays a crucial role in their survival and pathogenicity. The LuxS/AI-2 quorum-sensing system in many Gram-negative and Gram-positive bacteria senses environmental changes to regulate physiological traits and virulent properties, and the role of the luxS gene in Av. paragallinarum remains unclear. To investigate the effect of the luxS gene in the quorum-sensing system of Av. paragallinarum, we constructed a luxS mutant. Bioluminescence analysis indicated that the luxS gene plays a vital role in the LuxS/AI-2 quorum-sensing system. The analysis of the LuxS/AI-2 system-related genes showed the level of pfs mRNA to be significantly increased in the mutant strain; however, lsrR, lsrK, and lsrB mRNA levels were not significantly different compared with the wild type. The ability of the luxS mutant strain to invade HD11 and DF-1 cells was significantly decreased compared with the wild-type strain. In addition, all chickens challenged with various doses of the luxS mutant strain developed infections and symptoms, and those challenged with the lowest dose exhibited only minor differences compared to chickens challenged with the wild-type strain. Thus, the deletion of the luxS gene reduces the invasion, but the luxS gene does not play an essential role in the pathogenesis of A. paragallinarum.

Assessment of probiotic Bacillus velezensis supplementation to reduce Campylobacter jejuni colonization in chickens.

Campylobacter jejuni continues to be a major public health issue worldwide. Poultry are recognized as the main reservoir for this foodborne pathogen. Implementing measures to decrease C. jejuni colonization on farms has been regarded as the most effective strategy to control the incidence of campylobacteriosis. The probiotics supplementation has been regarded as an attractive approach against C. jejuni in chickens. Here the inhibitory effects of one probiotic B. velezensis isolate CAU277 against C. jejuni was evaluated in vitro and in vivo. The in vitro antimicrobial activity showed that the supernatant of B. velezensis exhibited the most pronounced inhibitory effects on Campylobacter strains compared to other bacterial species. When co-cultured with B. velezensis, the growth of C. jejuni reduced significantly from 7.46 log10 CFU/mL (24 h) to 1.02 log10 CFU/mL (48 h). Further, the antimicrobial activity of B. velezensis against C. jejuni remained stable under a broad range of temperature, pH, and protease treatments. The in vivo experiments demonstrated that oral administration of B. velezensis significantly reduced the colonization of C. jejuni by 2.0 log10 CFU/g of feces in chicken cecum at 15 d postinoculation. In addition, the supplementary of B. velezensis significantly increased microbial species richness and diversity in chicken ileum, especially enhanced the bacterial population of Alistipes and Christensenellaceae, and decreased the existence of Lachnoclostridium. Our study presents that B. velezensis possesses antimicrobial activities against C. jejuni and promotes microbiota diversity in chicken intestines. These findings indicate a potential to develop an effective probiotic additive to control C. jejuni infection in chicken.

External validation of predictive models for new vertebral fractures following percutaneous vertebroplasty.

OBJECTIVE: To investigate the external validation and scalability of four predictive models regarding new vertebral fractures following percutaneous vertebroplasty. METHODS: Utilizing retrospective data acquired from two centers, compute the area under the curve (AUC), calibration curve, and Kaplan-Meier plot to assess the model's discrimination and calibration. RESULTS: In the external validation of Zhong et al.'s 2015 predictive model for the probability of new fractures post-vertebroplasty, the AUC for re-fracture at 1, 2, and 3 years postoperatively was 0.570, 0.617, and 0.664, respectively. The AUC for Zhong et al.'s 2016 predictive model for the probability of new fractures in neighboring vertebrae was 0.738. Kaplan-Meier plot results for both models indicated a significantly lower incidence of re-fracture in low-risk patients compared to high-risk patients. Li et al.'s 2021 model had an AUC of 0.518, and its calibration curve suggested an overestimation of the probability of new fractures. Li et al.'s 2022 model had an AUC of 0.556, and its calibration curve suggested an underestimation of the probability of new fractures. CONCLUSION: The external validation of four models demonstrated that the predictive model proposed by Zhong et al. in 2016 exhibited superior external generalization capabilities.

Chaperonin TRiC/CCT subunit CCT7 is involved in the replication of canine parvovirus in F81 cells.

Canine parvovirus (CPV) is one of the most common lethal viruses in canines. The virus disease is prevalent throughout the year, with high morbidity and mortality rate, causing serious harm to dogs and the dog industry. Previously, yeast two hybrid method was used to screen the protein chaperonin containing TCP-1 (CCT7) that interacts with VP2. However, the mechanism of interactions between CCT7 and VP2 on CPV replication remains unclear. In this study, we first verified the interaction between CCT7 and viral VP2 proteins using yeast one-to-one experiment and co-immunoprecipitation (CoIP) experiment. Laser confocal microscopy observation showed that CCT7 and VP2 were able to co-localize and were mostly localized in the cytoplasm. In addition, the study of VP2 truncated mutant found that the interaction region of VP2 with CCT7 was located between amino acids 231 and 320. Cycloheximide (CHX) chase experiments showed that CCT7 can improve the stability of VP2 protein. After further regulation of CCT7 expression in F81 cells, it was found that the expression level of VP2 protein was significantly reduced after knocking down CCT7 expression by RNA interference (RNAi) or HSF1A inhibitor, and increased after overexpressing host CCT7. The study reveals the role of VP2 interacting protein CCT7 in the replication process of CPV, which could provide a potential target for the prevention and control of CPV.

Transcriptional Differential Analysis of Nitazoxanide-Mediated Anticanine Parvovirus Effect in F81 Cells.

Canine parvovirus (CPV) is a single-stranded DNA virus that can cause typical hemorrhagic enteritis, and it is one of the common canine lethal viruses. In previous studies, we screened the Food and Drug Administration (FDA)'s drug library and identified nitazoxanide (NTZ), which has anti-CPV capabilities. To investigate the potential antiviral mechanisms, we first reconfirmed the inhibitory effect of NTZ on the CPV by inoculating with different doses and treating for different lengths of time. Then, the differences in the transcription levels between the 0.1%-DMSO-treated virus group and the NTZ-treated virus group were detected using RNA-seq, and a total of 758 differential expression genes (DEGs) were finally identified. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs revealed that these genes are involved in a variety of biological processes and/or signaling pathways, such as cell cycle, mitosis and cell proliferation and differentiation. A protein-protein interaction (PPI) analysis further identified hub genes associated with cell cycle and division among the DEGs. In addition, the expression levels of some of the enriched genes were detected, which were consistent with the high-throughput sequencing results. Moreover, when the cell cycle was regulated with cell cycle checkpoint kinase 1 (Chk1) inhibitor MK-8776 or Prexasertib HCl, both inhibitors inhibited the CPV. In summary, the transcriptome differential analysis results presented in this paper lay the foundation for further research on the molecular mechanism and potential targets of NTZ anti-CPV.

Characterization of the mechanism of bile salt hydrolase substrate specificity by experimental and computational analyses.

Bile salt hydrolases (BSHs) are currently being investigated as target enzymes for metabolic regulators in humans and as growth promoters in farm animals. Understanding structural features underlying substrate specificity is necessary for inhibitor design. Here, we used a multidisciplinary workflow including mass spectrometry, mutagenesis, molecular dynamic simulations, machine learning, and crystallography to demonstrate substrate specificity in Lactobacillus salivarius BSH, the most abundant enzyme in human and farm animal intestines. We show the preference of substrates with a taurine head and a dehydroxylated sterol ring for hydrolysis. A regression model that correlates the relative rates of hydrolysis of various substrates in various enzyme mutants with the residue-substrate interaction energies guided the identification of structural determinants of substrate binding and specificity. In addition, we found T208 from another BSH protomer regulating the hydrolysis. The designed workflow can be used for fast and comprehensive characterization of enzymes with a broad range of substrates.

Critical role of the RpoE stress response pathway in polymyxin resistance of Escherichia coli.

OBJECTIVES: Polymyxins, including colistin, are the drugs of last resort to treat MDR bacterial infections in humans. In-depth understanding of the molecular basis and regulation of polymyxin resistance would provide new therapeutic opportunities to combat increasing polymyxin resistance. Here we aimed to identify novel targets that are crucial for polymyxin resistance using Escherichia coli BL21(DE3), a unique colistin-resistant model strain. METHODS: BL21(DE3) was subjected to random transposon mutagenesis for screening colistin-susceptible mutants. The insertion sites of desired mutants were mapped; the key genes of interest were also inactivated in different strains to examine functional conservation. Specific genes in the known PmrAB and PhoPQ regulatory network were inactivated to examine crosstalk among different pathways. Lipid A species and membrane phospholipids were analysed by normal phase LC/MS. RESULTS: Among eight mutants with increased susceptibility to colistin, five mutants contained different mutations in three genes (rseP, degS and surA) that belong to the RpoE stress response pathway. Inactivation of rpoE, pmrB, eptA or pmrD led to significantly increased susceptibility to colistin; however, inactivation of phoQ or eptB did not change colistin MIC. RpoE mutation in different E. coli and Salmonella resistant strains all led to significant reduction in colistin MIC (16-32-fold). Inactivation of rpoE did not change the lipid A profile but significantly altered the phospholipid profile. CONCLUSIONS: Inactivation of the important members of the RpoE regulon in polymyxin-resistant strains led to a drastic reduction in polymyxin MIC and an increase of lysophospholipids with no change in lipid A modifications.

Campylobacter jejuni infection induces dynamic expression of avian host defense peptides in vitro and in vivo.

Campylobacter jejuni is considered as the leading cause of worldwide foodborne bacterial gastroenteritis. Chicken is the main reservoir of C. jejuni. Avian innate immune responses to C. jejuni remain poorly defined. Chicken host defense peptides (HDPs) are the major components of avian innate immune system. This study aimed to characterize the chicken HDPs responses to C. jejuni in vitro and in vivo. In the HD11 macrophage cell line, the HDPs, including AvBD1-2, CATH1-3, AvBD7, AvBD4, and AvBD6, were relatively higher expressed in untreated cells, whereas the expressions were suppressed after C. jejuni infection. In contrast, C. jejuni infection significantly increased the expression of the lower expressed HDPs, such as AvBD3, AvBD5, AvBD8-14, and CATHB1, in untreated cells. In the chicken challenge experiment, the immune tissues of spleens and cecal tonsils were collected from C. jejuni-infected and uninfected chickens at 1, 3 and 15 day post inoculation (DPI). In spleens of C. jejuni-infected chickens, only AvBD14 expression was elevated at 1 DPI. The majority of avian HDPs were significantly up-regulated at 3 DPI and dramatically decreased to the levels of uninfected controls at 15 DPI. In chicken cecal tonsils, only AvBD9 and AvBD14 were significantly up-regulated at 1 DPI with C. jejuni infection. Collectively, C. jejuni infection induced dynamic expression of chicken HDPs in both macrophage HD11 and immune tissues of chickens. Suppression of chicken HDPs expression may be an evasion strategy of C. jejuni for persistent colonization in chicken intestine by circumventing the chicken immune system.

Effects of OxyR regulator on oxidative stress, Apx toxin secretion and virulence of Actinobacillus pleuropneumoniae.

Introduction: Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, poses a significant threat to global swine populations due to its high prevalence, mortality rates, and substantial economic ramifications. Understanding the pathogen's defense mechanisms against host-produced reactive oxygen species is crucial for its survival, with OxyR, a conserved bacterial transcription factor, being pivotal in oxidative stress response. Methods: This study investigated the presence and role of OxyR in A. pleuropneumoniae serovar 1-12 reference strains. Transcriptomic analysis was conducted on an oxyR disruption mutant to delineate the biological activities influenced by OxyR. Additionally, specific assays were employed to assess urease activity, catalase expression, ApxI toxin secretion, as well as adhesion and invasion abilities of the oxyR disruption mutant on porcine 3D4/21 and PT cells. A mice challenge experiment was also conducted to evaluate the impact of oxyR inactivation on A. pleuropneumoniae virulence. Results: OxyR was identified as a conserved regulator present in A. pleuropneumoniae serovar 1-12 reference strains. Transcriptomic analysis revealed the involvement of OxyR in multiple biological activities. The oxyR disruption resulted in decreased urease activity, elevated catalase expression, enhanced ApxI toxin secretion-attributed to OxyR binding to the apxIBD promoter-and reduced adhesion and invasion abilities on porcine cells. Furthermore, inactivation of oxyR reduced the virulence of A. pleuropneumoniae in a mice challenge experiment. Discussion: The findings highlight the pivotal role of OxyR in influencing the virulence mechanisms of A. pleuropneumoniae. The observed effects on various biological activities underscore OxyR as an essential factor contributing to the pathogenicity of this bacterium.

Monoclonal antibody-based indirect competitive ELISA for quantitative detection of Enterobacteriaceae siderophore enterobactin.

Enterobactin (Ent) is a promising indicator to monitor intestinal level of Enterobacteriaceae for assessment of gut inflammation. In this study, we developed a monoclonal antibody (mAb)-based ELISA for Ent quantification. We immunized mice with an Ent conjugate vaccine. An mAb named 2E4, with the highest anti-Ent antibody titer, was selected for developing indirect competitive ELISA (ic-ELISA). The purified mAb 2E4 showed high affinity (3.1 × 10-10 M) and specificity to Ent. The limit of detection of ic-ELISA was 0.39 µg/mL. The intra- and inter-assay recovery rates of standard curve were up to 94.6% with the coefficients of variation between 4.0% and 12.3%, indicating high accuracy, repeatability, and reproducibility of the ic-ELISA. In addition, the ic-ELISA was able to quantitatively detect Ent produced in different bacterial cultures. Collectively, this study developed an ic-ELISA with excellent performance in Ent quantification, laying a solid foundation for Ent-based diagnostics of gut health.

First isolation and pathogenicity analysis of a genogroup U strain of infectious hematopoietic necrosis virus from rainbow trout in China.

Infectious hematopoietic necrosis virus (IHNV) is a major fish viral pathogen causing acute clinical disease and death in a variety of salmonids. IHNV isolates have been classified into five major genogroups according to the phylogenetic analysis of partial G gene fragments or the complete G gene sequence: U, M, E, L and J. Genogroup U strains have been reported in North America and Japan prior to 1982, and genogroup J is the only genogroup that has been reported in China. Here, one of IHNV strain (BjLL) was isolated from a local farm in China and were characterized in this study. The homogenate tissues of infected fry induced IHNV-positive cytopathic effects in epithelioma papulosum cyprinid (EPC) cells that were confirmed by RT-PCR and sequencing. The complete genome sequence of BjLL comprised 11,129 nucleotides, which had been submitted to GenBank (accession no. MF509592). By the sequence comparison and phylogenetic analysis for the G gene sequence of BjLL with 51 reference sequences in GenBank, we confirmed that this Chinese isolate belonged to genogroup U. Furthermore, virus exposure experiments with juvenile rainbow trout were conducted to assess the virulence and pathogenicity of BjLL. Compared with GS-2014 of genogroup J, BjLL was an obviously less virulent strain that could result in lower mortality. Besides, typical clinical symptoms and pathological damages could be seen in fish following infection of BjLL. The present study is the first report of genogroup U IHNV infection in China and will provide essential information for future studies on pathogenesis of IHNV BjLL and development of efficient control strategies.

Development of IMBs-qPCR detection method for Yersinia enterocolitica based on the foxA gene.

Yersinia enterocolitica is an important zoonotic pathogen, which seriously endangers food-safety risk. In this study, the recombinant outer membrane protein OmpF and its antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Y. enterocolitica in food samples, combining the quantitative PCR detection with primers of virulence factor gene foxA for Yersinia enterocolitica contamination. The results showed that the capture efficiency of approximately 80% using anti-OmpF antibody-immunomagnetic beads and linearly dependent capture under 101-105 CFU/mL Y. enterocolitica compared with less than 10% capture of other bacteria. The detection limit of 64 CFU/mL was obtained based on foxA gene PCR detection combined with capture of the anti-OmpF antibody-immunomagnetic beads to detect Yersinia enterocolitica in artificially contaminated milk and pork samples. Compared to the culture method, the developed IMBs-qPCR method has higher consistency, was less time consuming, which taken together provides an effective alternative method for rapid detection of Y. enterocolitica in food.

Critical Role of 3'-Downstream Region of pmrB in Polymyxin Resistance in Escherichia coli BL21(DE3).

Polymyxins, such as colistin and polymyxin B, are the drugs used as a last resort to treat multidrug-resistant Gram-negative bacterial infections in humans. Increasing colistin resistance has posed a serious threat to human health, warranting in-depth mechanistic research. In this study, using a functional cloning approach, we examined the molecular basis of colistin resistance in Escherichia coli BL21(DE3). Five transformants with inserts ranging from 3.8 to 10.7 kb displayed significantly increased colistin resistance, three of which containing pmrB locus and two containing pmrD locus. Stepwise subcloning indicated that both the pmrB with a single G361A mutation and at least a 103 bp downstream region of pmrB are essential for conferring colistin resistance. Analysis of the mRNA level and stability showed that the length of the downstream region drastically affected the pmrB mRNA level but not its half-life. Lipid A analysis, by mass spectrometry, revealed that the constructs containing pmrB with a longer downstream region (103 or 126 bp) have charge-altering l-4-aminoarabinose (Ara4N) and phosphoethanolamine (pEtN) modifications in lipid A, which were not observed in both vector control and the construct containing pmrB with an 86 bp downstream region. Together, the findings from this study indicate that the 3'-downstream region of pmrB is critical for the PmrB-mediated lipid A modifications and colistin resistance in E. coli BL21(DE3), suggesting a novel regulatory mechanism of PmrB-mediated colistin resistance in E. coli.

The Transcriptomic and Bioinformatic Characterizations of Iron Acquisition and Heme Utilization in Avibacterium paragallinarum in Response to Iron-Starvation.

Avibacterium paragallinarum is the pathogen of infectious coryza, which is a highly contagious respiratory disease of chickens that brings a potentially serious threat to poultry husbandry. Iron is an important nutrient for bacteria and can be obtained from surroundings such as siderophores and hemophores. To date, the mechanisms of iron acquisition and heme utilization as well as detailed regulation in A. paragallinarum have been poorly understood. In this study, we investigated the transcriptomic profiles in detail and the changes of transcriptomes induced by iron restriction in A. paragallinarum using RNA-seq. Compared with the iron-sufficiency control group, many more differentially expressed genes (DEGs) and cellular functions as well as signaling pathways were verified in the iron-restriction group. Among these DEGs, the majority of genes showed decreased expression and some were found to be uniquely present in the iron-restriction group. With an in-depth study of bioinformatic analyses, we demonstrated the crucial roles of the Hut protein and DUF domain-containing proteins, which were preferentially activated in bacteria following iron restriction and contributed to the iron acquisition and heme utilization. Consequently, RT-qPCR results further verified the iron-related DEGs and were consistent with the RNA-seq data. In addition, several novel sRNAs were present in A. paragallinarum and had potential regulatory roles in iron homeostasis, especially in the regulation of Fic protein to ensure stable expression. This is the first report of the molecular mechanism of iron acquisition and heme utilization in A. paragallinarum from the perspective of transcriptomic profiles. The study will contribute to a better understanding of the transcriptomic response of A. paragallinarum to iron starvation and also provide novel insight into the development of new antigens for potential vaccines against infectious coryza by focusing on these iron-related genes.

Exposure to Sublethal Ciprofloxacin Induces Resistance to Ciprofloxacin and Cross-Antibiotics, and Reduction of Fitness, Biofilm Formation, and Apx Toxin Secretion in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is increasingly resistant to antibiotics. However, little is known about the mechanisms of antibiotic resistance in this pathogen. In this study, we experimentally evolved the reference strain of both A. pleuropneumoniae serovar 1 and serovar 7, the most prevalent serovars worldwide, to quinolone resistance by sequential exposure to subinhibitory concentrations of ciprofloxacin. The adaptive ciprofloxacin-resistant mutants of A. pleuropneumoniae serovar 1 and serovar 7 had a minimum inhibitory concentration (MIC) increment from 0.004 to 1 or 2 µg/mL, respectively. Adaptation to ciprofloxacin was shown to confer quinolone resistance with a 32- to 512-fold increase (serovars 1 and 7, respectively) as well as cross-resistance to ampicillin with an increased MIC by 16,384- and 64-fold (serovars 1 and 7, respectively). The genetic analysis of quinolone resistance-determining region mutations showed that substitutions occurred in gyrA (S83A) and parC (D84N) of serovar 1, and gyrA (D87N) of serovar 7. The ciprofloxacin-resistant mutants showed significantly reduced bacterial fitness. The mutants also showed changes in efflux ability and biofilm formation. Notably, the transcription and secretion levels of Apx toxins were dramatically reduced in ciprofloxacin-resistant mutants compared with their wild-type strains. Altogether, these results demonstrated marked phenotypic changes in ciprofloxacin-resistant mutants of A. pleuropneumoniae. The results stress the need for further studies on the impact of both the genotypic and phenotypic characteristics of A. pleuropneumoniae following exposure to subinhibitory concentrations of antibiotics.

Ct value-based real time PCR serotyping of Glaesserella parasuis.

Glaesserella parasuis is the causative agent of Glässer's disease in swine. Serotyping plays an essential role in prevalence investigations and in the development of vaccination strategies for the prevention of this disease. Molecular serotyping based on variation within the capsule loci of the 15 serovars is more accurate and efficient than traditional serological serotyping. To reduce the running time and facilitate ease of data interpretation, we developed a simple and rapid cycle threshold (Ct) value-based real time PCR (qPCR) method for the identification and serotyping of G. parasuis. The qPCR method distinguished between all 15 serovar reference strains of G. parasuis with efficiency values ranging between 85.5 % and 110.4 % and, R2 values > 0.98. The qPCR serotyping was evaluated using 83 clinical isolates with 43 of the isolates having been previously assigned to a serovar by the gel immuno-diffusion (GID) assay and 40 non-typeable isolates. The qPCR results of 41/43 (95.3 %) isolates were concordant with the GID assay except two isolates of serovar 12 were assigned to serovar 5. In addition, the qPCR serotyping assigned a serovar to each of the 40 non-typeable isolates. Of the 83 isolates tested to assign a serovar, a concordance rate of 98.8 % (82/83) was determined between the qPCR and the previously reported multiplex PCR of Howell et al. (2015) (including those that were either serovars 5 or 12). Despite the inability to differentiate between serovars 5 and 12, the Ct value-based qPCR serotyping represents an attractive alternative to current molecular serotyping method for G. parasuis and could be used for both epidemiological monitoring and the guidance of vaccination programs.

Immunization of Chickens with the Enterobactin Conjugate Vaccine Reduced Campylobacter jejuni Colonization in the Intestine.

Campylobacter jejuni is the leading bacterial cause of human enteritis in developed countries. Chicken is the major animal reservoir of C. jejuni and a powerful infection model for human campylobacteriosis. No commercial vaccine against C. jejuni is available to date. The high affinity iron acquisition mediated through enterobactin (Ent), a small siderophore, plays a critical role in the colonization of C. jejuni in the intestine. Recently, an innovative Ent conjugate vaccine has been demonstrated to induce high-level of Ent-specific antibodies in rabbits; the Ent-specific antibodies displayed potent binding ability to Ent and inhibited Ent-dependent growth of C. jejuni. In this study, using specific-pathogen-free (SPF) chickens, we performed three trials to evaluate the immunogenicity of the Ent conjugate vaccine and its efficacy to control C. jejuni colonization in the intestine. The purified Ent was conjugated to the carrier keyhole limpet hemocyanin (KLH). Intramuscular immunization of chickens with the Ent-KLH conjugate for up to three times did not affect the body weight gain, the development of major immune organs and the gut microbiota. In the first two trials, immunizations of chickens with different regimens (two or three times of vaccination) consistently induced strong Ent-specific immune response when compared to control group. Consistent with the high-level of systemic anti-Ent IgG, C. jejuni colonization was significantly reduced by 3-4 log10 units in the cecum in two independent vaccination trials. The third trial demonstrated that single Ent-KLH vaccination is sufficient to elicit high level of systemic Ent-specific antibodies, which could persist for up to eight weeks in chickens. Taken together, the Ent-KLH conjugate vaccine could induce high-level of Ent-specific antibodies in chickens and confer host protection against C. jejuni colonization, which provides a novel strategy for Campylobacter control in poultry and humans.

Oral Immunization of Chickens with Lactococcus lactis Expressing cjaA Temporarily Reduces Campylobacter jejuni Colonization.

Campylobacter jejuni is the leading cause of human foodborne enteritis worldwide. Poultry products are regarded as the main source of human campylobacteriosis. Strategies are being developed to reduce colonization of poultry by Campylobacter. The membrane transport protein CjaA was reported to stimulate mucosal immune responses, which can reduce the C. jejuni load in chickens. In this study, oral immunization of broilers with food-grade Lactococcus lactis NZ3900/pNZ8149 carrying the C. jejuni cjaA gene was examined for the ability to reduce colonization of broilers by Campylobacter. The Usp45 signal peptide and the Escherichia coli heat-labile enterotoxin B subunit (LTB) gene fragments were inserted into the upstream and downstream of the cjaA gene for secretory expression and immune enhancement, respectively. The cjaA gene and the fusion cjaA-ltb gene were both expressed in recombinant L. lactis, and the single cjaA gene was secretory expressed in the recombinant strain. Oral administration of two recombinant L. lactis strains expressing the cjaA gene and the fusion cjaA-ltb gene both stimulated specific anti-CjaA serum IgY responses significantly. While the average intestinal sIgA responses in these groups were higher compared with the control groups, they were not significantly different. Chicken challenge experiments showed that the colonization levels of C. jejuni in the groups provided oral immunization with two recombinant L. lactis-delivered CjaA strains were significantly lower than that of the control group at 5 d postinoculation, but there was no significant difference in C. jejuni colonization among all groups at 9 d. These results indicated that recombinant L. lactis with secretory expression of CjaA is a promising live vector vaccine against C. jejuni colonization of chickens. The immunization regimen requires further optimization to ideally stimulate detectable levels of intestinal sIgA to enhance the level of inhibition of C. jejuni colonization.

Inhibitory Effects of Antiviral Drug Candidates on Canine Parvovirus in F81 cells.

Canine parvovirus (CPV) is a common etiological agent of acute enteritis, which occurs globally in domestic and wild carnivores. Despite the widespread use of inactivated or live attenuated vaccines, the emergence of antigenic variants and the influence of maternal antibodies have raised some concerns regarding the efficacy of commercial vaccines. While no specific antiviral therapy for CPV infection exists, the only treatment option for the infection is supportive therapy based on symptoms. Thus, there is an urgent medical need to develop antiviral therapeutic options to reduce the burden of CPV-related disease. In this study, a cytopathic effect (CPE)-based high-throughput screening assay was used to screen CPV inhibitors from a Food and Drug Administration (FDA)-approved drug library. After two rounds of screening, seven out of 1430 screened drugs were found to have >50% CPE inhibition. Three drugs-Nitazoxanide, Closantel Sodium, and Closantel-with higher anti-CPV effects were further evaluated in F81 cells by absolute PCR quantification and indirect immunofluorescence assay (IFA). The inhibitory effects of all three drugs were dose-dependent. Time of addition assay indicated that the drugs inhibited the early processes of the CPV replication cycle, and the inhibition effects were relatively high within 2 h postinfection. Western blot assay also showed that the three drugs had broad-spectrum antiviral activity against different subspecies of three CPV variants. In addition, antiapoptotic effects were observed within 12 h in Nitazoxanide-treated F81 cells regardless of CPV infection, while Closantel Sodium- or Closantel-treated cells had no pro- or antiapoptotic effects. In conclusion, Nitazoxanide, Closantel Sodium, and Closantel can effectively inhibit different subspecies of CPV. Since the safety profiles of FDA-approved drugs have already been extensively studied, these three drugs can potentially become specific and effective anti-CPV drugs.

The complex structure of bile salt hydrolase from Lactobacillus salivarius reveals the structural basis of substrate specificity.

The gut bacterial bile salt hydrolase (BSH) plays a critical role in host lipid metabolism and energy harvest. Therefore, BSH is a promising microbiome target to develop new therapies to regulate obesity in humans and novel non-antibiotic growth promoters for food animals. We previously reported the 1.90 Å apo crystal structure of BSH from Lactobacillus salivarius (lsBSH). In this study, we soaked the lsBSH crystal with glycocholic acid (GCA), a substrate, and obtained a 2.10 Å structure containing complex of lsBSH bound to GCA and cholic acid (CA), a product. The substrate/product sits in the water-exposed cavity molded by Loops 2 and 3. While the glycine moiety of GCA is exposed into a highly polar pocket, the sterane core of GCA is stabilized by aromatic and hydrophobic interactions. Comparison of product binding with BSH from Clostridium perfringenes reveals a distinct orientation of the sterane core in the binding site. The stability of the substrate-lsBSH complex and the putative catalytic mechanism were explored with molecular dynamics simulations. Site-directed mutagenesis of lsBSH demonstrated that Cys2 and Asn171 are critical for enzymatic activity, while Tyr24, Phe65 and Gln257 contribute to the substrate specificity. Together, this study provides structural insights into BSH-substrate interaction, the mechanism of catalysis and substrate specificity, which facilitate rational design of BSH inhibitors.