Campylobacter jejuni is one of the most important causes of food-borne infectious disease, and poses challenges to food safety and public health. Establishing a rapid, accurate, sensitive, and simple detection method for C. jejuni enables early diagnosis, early intervention, and prevention of pathogen transmission. In this study, an immunocapture magnetic bead (ICB)-enhanced loop-mediated isothermal amplification (LAMP) CRISPR/Cas12a method (ICB-LAMP-CRISPR/Cas12a) was developed for the rapid and visual detection of C. jejuni. Using the ICB-LAMP-CRISPR/Cas12a method, C. jejuni was first captured by ICB, and the bacterial genomic DNA was then released by heating and used in the LAMP reaction. After the LAMP reaction, LAMP products were mixed and detected by the CRISPR/Cas12a cleavage mixture. This ICB-LAMP-CRISPR/Cas12a method could detect a minimum of 8 CFU/mL of C. jejuni within 70 min. Additionally, the method was performed in a closed tube in addition to ICB capture, which eliminates the need to separate preamplification and transfer of amplified products to avoid aerosol pollution. The ICB-LAMP-CRISPR/Cas12a method was further validated by testing 31 C. jejuni-positive fecal samples from different layer farms. This method is an all-in-one, simple, rapid, ultrasensitive, ultraspecific, visual detection method for instrument-free diagnosis of C. jejuni, and has wide application potential in future work.

Avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is the causative agent of infectious bronchitis (IB) that has brought great threat and economic losses to the global poultry industry. Rapid and accurate diagnostic methods are very necessary for effective disease monitoring. At the present study, we screened a novel nanobody against IBV-N protein for development of a rapid, simple, sensitive, and specific competitive ELISA for IBV antibody detection in order to enable the assessment of inoculation effect and early warning of disease infection. Using the phage display technology and bio-panning, we obtained 7 specific nanobodies fused with horseradish peroxidase (HRP) which were expressed in culture supernatant of HEK293T cells. Out of which, the nanobody of IBV-N-Nb66-vHRP has highly binding with IBV-N protein and was easily blocked by the IBV positive serums, which was finally employed as an immunoprobe for development of the competitive ELISA (cELISA). In the newly developed cELISA, we reduce the use of enzyme-conjugated secondary antibody, and the time of whole operation process is approximately 1 h. Moreover, the IBV positive serums diluted at 1:1000 can still be detected by the developed cELISA, and it has no cross reactivity with others chicken disease serums including Newcastle disease virus, Fowl adenovirus, Avian Influenza Virus, Infectious bursal disease virus and Hepatitis E virus. The cut-off value of the established cELISA was 36%, and the coefficient of variation of intra- and inter-assay were 0.55–1.65% and 2.58–6.03%, respectively. Compared with the commercial ELISA (IDEXX kit), the agreement rate of two methods was defined as 98% and the kappa value was 0.96, indicating the developed cELISA has high consistency with the commercial ELISA. Taken together, the novel cELISA for IBV antibody detection is a simple, rapid, sensitive, and specific immunoassay, which has the potential to rapidly test IBV antibody contributing to the surveillance and control of the disease.

The CTX-M-55 type extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae is increasing in prevalence worldwide without the transmission mechanism being fully clarified, which threatens public and livestock health. Outer membrane vesicles (OMVs) have been shown to mediate the gene horizontal transmission in some species. However, whether bla_CTX-M-55 can be transmitted horizontally through OMVs in avian pathogenic Escherichia coli (APEC) has not been reported yet. To test this hypothesis, an ESBL-producing APEC was isolated and whole-genome sequencing (WGS) was performed to analyze the location of bla_CTX-M-55. Ultracentrifugation and size exclusion chromatography was used to isolate and purify OMVs, and the transfer experiment of bla_CTX-M-55 via OMVs was performed finally. Our results showed that the bla_CTX-M-55 was located on an IncI2 plasmid. The number and diameter of OMVs secreted by ESBL-producing APEC treated with different antibiotics were significantly varied. The transfer experiment showed that the OMVs could mediate the horizontal transfer of bla_CTX-M-55, and the frequency of gene transfer ranged from 10⁻⁵ to 10⁻⁶ CFU/mL with the highest frequency observed in the Enrofloxacin treatment group. These findings contribute to a better understanding of the antibiotics in promoting and disseminating resistance in the poultry industry and support the restrictions on the use of antibiotics in the poultry industry.

Carbapenems are atypical β-lactam antibiotics with a broade antibacterial spectrum and strong antibacterial activity; however, the emergence and spread of carbapenemases have led to a decline in their effectiveness. New Delhi metallo-β-lactamase (NDM) is an important carbapenemase that has attracted widespread attention and poses a major threat to public health. To investigate the epidemiological characteristics of bla_NDM in swine and chicken farms in southwestern China, we isolated 102 bla_NDM-positive Enterobacterales strains from 18 farms in Sichuan and Yunnan provinces in 2021, with Escherichia coli and Klebsiella spp. being the main reservoirs of bla_NDM, variant bla_NDM-5 being the most prevalent, and all strains being multi-drug resistant. Whole-genome sequencing analysis of 102 bla_NDM-positive Enterobacterales strains revealed that bla_NDM had spread primarily through its carriers on the same farm and among the 18 farms in this study. A high degree of genetic similarity between animal-derived bla_NDM-positive Escherichia coli strains and human-derived strains was also identified, suggesting a potential mutual transmission between them. Nanopore sequencing results indicated that bla_NDM is predominantly present on the IncX3 plasmid, that an insertion sequence might be important for recombination in the bla_NDM genetic environment, and that most of the plasmids carrying bla_NDM are transferable. Collectively, our results enrich the current epidemiological information regarding bla_NDM in pig and chicken farms in Southwest China, revealing its transmission pattern, as well as the potential risk of transmission to humans, which could help to better understand and control the spread of bla_NDM.

Avian coronavirus infectious bronchitis virus (IBV) causes severe economic losses in the poultry industry, but its control is hampered by the continuous emergence of new genotypes and the lack of cross-protection among different IBV genotypes. We designed a new immunogen based on a spike with the consensus nucleotide sequence (S_con) that may overcome the extraordinary genetic diversity of IBV. S_con was cloned into a pVAX1 vector to form a new IBV DNA vaccine, pV-S_con. pV-S_con could be correctly expressed in HD11 cells with corresponding post-translational modification, and induced a neutralizing antibody response to the Vero-cell-adapted IBV strain Beaudette (p65) in mice. To further evaluate its immunogenicity, specific-pathogen-free (SPF) chickens were immunized with the pV-S_con plasmid and compared with the control pVAX1 vector and the H120 vaccine. Detection of IBV-specific antibodies and cell cytokines (IL-4 and IFN-γ) indicated that vaccination with pV-S_con efficiently induced both humoral and cellular immune responses. After challenge with the heterologous strain M41, virus shedding and virus loading in tissues was significantly reduced both by pV-S_con and its homologous vaccine H120. Thus, pV-S_con is a promising vaccine candidate for IBV, and the consensus approach is an appealing method for vaccine design in viruses with high variability.