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.

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.