Establishment of an ELISA Based on a Recombinant Antigenic Protein Containing Multiple Prominent Epitopes for Detection of African Swine Fever Virus Antibodies.

African swine fever virus (ASFV) poses a significant threat to the global pig industry, necessitating accurate and efficient diagnostic methods for its infection. Previous studies have often focused on a limited number of epitopes from a few proteins for detecting antibodies against ASFV. Therefore, the current study aimed to use multiple B-cell epitopes in developing an indirect Enzyme-Linked Immunosorbent Assay (ELISA) for enhanced detection of ASFV antibodies. For the expression of recombinant protein, k3 derived from 27 multiple peptides of 11 ASFV proteins, such as p72, pA104R, pB602L, p12, p14.5, p49, pE248R, p30, p54, pp62, and pp220, was used. To confirm the expression of the recombinant protein, we used the Western blotting analysis. The purified recombinant K3 protein served as the antigen in our study, and we employed the indirect ELISA technique to detect anti-ASFV antibodies. The present finding showed that there was no cross-reactivity with antibodies targeting Foot-and-mouth disease virus (FMDV), Porcine circovirus type 2 (PCV2), Pseudorabies virus (PRV), Porcine reproductive and respiratory syndrome virus (PRRSV), and Classical swine fever virus (CSFV). Moreover, the current finding was sensitive enough to find anti-ASFV in serum samples that had been diluted up to 32 times. The test (k3-iELISA) showed diagnostic specificity and sensitivity of 98.41% and 97.40%, respectively. Moreover, during the present investigation, we compared the Ingenasa kit and the k3-iELISA to test clinical pig serum, and the results revealed that there was 99.00% agreement between the two tests, showing good detection capability of the k3-iELISA method. Hence, the current finding showed that the ELISA kit we developed can be used for the rapid detection of ASFV antibodies and used as an alternative during serological investigation of ASF in endemic areas.

Characteristics, pathogenic mechanism, zoonotic potential, drug resistance, and prevention of avian pathogenic Escherichia coli (APEC).

Although most Escherichia coli (E. coli) strains are commensal and abundant, certain pathogenic strains cause severe diseases from gastroenteritis to extraintestinal infections. Extraintestinal pathogenic E. coli (ExPEC) contains newborn meningitis E. coli (NMEC), uropathogenic E. coli (UPEC), avian pathogenic E. coli (APEC), and septicemic E. coli (SEPEC) based on their original host and clinical symptom. APEC is a heterogeneous group derived from human ExPEC. APEC causes severe respiratory and systemic diseases in a variety of avians, threatening the poultry industries, food security, and avian welfare worldwide. APEC has many serotypes, and it is a widespread pathogenic bacterium in poultry. In addition, ExPEC strains share significant genetic similarities and similar pathogenic mechanisms, indicating that APEC potentially serves as a reservoir of virulence and resistance genes for human ExPEC, and the virulence and resistance genes can be transferred to humans through food animals. Due to economic losses, drug resistance, and zoonotic potential, APEC has attracted heightened awareness. Various virulence factors and resistance genes involved in APEC pathogenesis and drug resistance have been identified. Here, we review the characteristics, epidemiology, pathogenic mechanism zoonotic potential, and drug resistance of APEC, and summarize the current status of diagnosis, alternative control measures, and vaccine development, which may help to have a better understanding of the pathogenesis and resistance of APEC, thereby reducing economic losses and preventing the spread of multidrug-resistant APEC to humans.

Isolation, Molecular Characterization, and Antibiotic Resistance of Avian Pathogenic Escherichia coli in Eastern China.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in avians, resulting in considerable losses in the poultry industry. APEC showed zoonotic potential initially related to the fact that APEC serves as the reservoir of virulence genes and antibiotic resistance genes for other E. coli. Thus, we determine the serotypes, phylogenetic groups, virulence genes distribution, and antibiotic resistance profiles of APEC isolates in eastern China. A total of 230 APEC were isolated from diseased chicken and duck with typical colibacillosis symptoms. Serotyping identified that O78 (44.78%) was the predominant serotype. The majority of APEC isolates were classified into B2 (29.57%), A (26.96%), D (20.00%), and B1 (18.26%), respectively. Among the 15 virulence genes, a high prevalence of ibeB (99.57%), fimC (91.74%), mat (91.30%), ompA (83.04%), and iss (80.43%) genes was observed. Except for low resistance rates for imipenem (1.7%) and polymyxin B (0.4%), most of the APEC isolates were resistant to erythromycin (98.7%), enrofloxacin (96.1%), tetracycline (95.2%), doxycycline (93.9%), lincomycin (90.0%), and streptomycin (90.0%). Moreover, all APEC exhibit multi-drug resistance. This study indicated that APEC isolates harbor a variety of virulence genes and showed multi-antibiotic resistance profiles, providing proof for understanding the epidemiological background and zoonotic potential of APEC in poultry farms.

Rapid Detection and Differentiating of the Predominant Salmonella Serovars in Chicken Farm by TaqMan Multiplex Real-Time PCR Assay.

Salmonella has been known as an important zoonotic pathogen that can cause a variety of diseases in both animals and humans. Poultry are the main reservoir for the Salmonella serovars Salmonella Pullorum (S. Pullorum), Salmonella Gallinarum (S. Gallinarum), Salmonella Enteritidis (S. Enteritidis), and Salmonella Typhimurium (S. Typhimurium). The conventional serotyping methods for differentiating Salmonella serovars are complicated, time-consuming, laborious, and expensive; therefore, rapid and accurate molecular diagnostic methods are needed for effective detection and prevention of contamination. This study developed and evaluated a TaqMan multiplex real-time PCR assay for simultaneous detection and differentiation of the S. Pullorum, S. Gallinarum, S. Enteritidis, and S. Typhimurium. In results, the optimized multiplex real-time PCR assay was highly specific and reliable for all four target genes. The analytical sensitivity corresponded to three colony-forming units (CFUs) for these four Salmonella serovars, respectively. The detection limit for the multiplex real-time PCR assay in artificially contaminated samples was 500 CFU/g without enrichment, while 10 CFU/g after pre-enrichment. Moreover, the multiplex real-time PCR was applied to the poultry clinical samples, which achieved comparable results to the traditional bacteriological examination. Taken together, these results indicated that the optimized TaqMan multiplex real-time PCR assay will be a promising tool for clinical diagnostics and epidemiologic study of Salmonella in chicken farm and poultry products.

DctR contributes to the virulence of avian pathogenic Escherichia coli through regulation of type III secretion system 2 expression.

Pathogens could precisely alter their gene expression to facilitate their survival and successful infection. The LuxR family transcriptional regulator DctR (also known as YhiF) was shown to participate in the regulation of acid fitness and adhesion of enterohemorrhagic E. coli (EHEC) O157:H7. Avian pathogenic Escherichia coli (APEC) causes significant economic losses to the poultry industries and also potentially threatens human health. However, the effects of DctR on the fitness and virulence of APEC have not been investigated yet. To assess the function of DctR in APEC, the dctR gene mutant and complemented strains were constructed and biologically characterized. Our results show that inactivation of the dctR gene led to decreased biofilm formation, diminished serum resistance, reduced adherence capacity, attenuated colonization and virulence of APEC in ducks. The altered capacities of the mutant strain were restored by genetic complementation. In addition, we found that DctR positively regulates the expression of E. coli type III secretion system 2 (ETT2) core genes in APEC. The expression of the inflammatory cytokines interleukin (IL)-1ß and IL-8 were decreased in HD-11 macrophages infected with the mutant strain compared with the wild-type strain. These observations indicate that regulator DctR contributes to the virulence of APEC through regulation of ETT2 expression.

Characterization of a Broad-Host-Range Lytic Phage SHWT1 Against Multidrug-Resistant Salmonella and Evaluation of Its Therapeutic Efficacy in vitro and in vivo.

Inappropriate use of antibiotics has accelerated to the emergence of multidrug-resistant bacteria, becoming a major health threat. Moreover, bacterial biofilms contribute to antibiotic resistance and prolonged infections. Bacteriophage (phage) therapy may provide an alternative strategy for controlling multidrug-resistant bacterial infections. In this study, a broad-host-range phage, SHWT1, with lytic activity against multidrug-resistant Salmonella was isolated, characterized and evaluated for the therapeutic efficacy in vitro and in vivo. Phage SHWT1 exhibited specific lytic activity against the prevalent Salmonella serovars, such as Salmonella Pullorum, Salmonella Gallinarum, Salmonella Enteritidis, and Salmonella Typhimurium. Morphological analysis showed that phage SHWT1 was a member of the family Siphoviridae and the order Caudovirales. Phage SHWT1 had a latent period of 5 min and burst size of ~150 plaque-forming units (PFUs)/cell. The phage was stable from pH 3-12 and 4-65°C. Phage SHWT1 also showed capacity to lyse Salmonella planktonic cells and inhibit the biofilm formation at optimal multiplicity of infection (MOI) of 0.001, 0.01, 0.1, and 100, respectively. In addition, phage SHWT1 was able to lyse intracellular Salmonella within macrophages. Genome sequencing and phylogenetic analyses revealed that SHWT1 was a lytic phage without toxin genes, virulence genes, antibiotic resistance genes, or significant genomic rearrangements. We found that phage SHWT1 could successfully protect mice against S. enteritidis and S. typhimurium infection. Elucidation of the characteristics and genome sequence of phage SHWT1 demonstrates that this phage is a potential therapeutic agent against the salmonellosis caused by multidrug-resistant Salmonella.