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.

Pseudomonas aeruginosa is one of the three most pathogenic bacteria that frequently cause life‑threatening opportunistic human infections, pneumonia, and lower respiratory tract infections in immunocompromised hosts, particularly in the burns ward. The present study aimed to establish a loop‑mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of P. aeruginosa‑specific gene hypothetical protein (GenBank ID: 882161). The gene was obtained through local and online BLAST, and specific primers were designed for this gene. Reaction conditions were optimized at 65˚C for 30 min and 80˚C for 2 min, whereas the reaction system contained 5.2 mM Mg2+, 8 U Bst 2.0 DNA polymerase, 1.4 mM deoxyribonucleotide and 0.2 and 1.6 µM of the outer and inner primers, respectively. The LAMP method was evaluated using 150 P. aeruginosa and 170 non‑P. aeruginosa strains. Positive reactions were observed on 150 P. aeruginosa strains, whereas all non‑P. aeruginosa strains exhibited negative results. Plasmids with the specific gene and mouse blood with P. aeruginosa were used for sensitivity assay. The detection limit of LAMP was 1 bacterium/reaction. Results indicated that the LAMP method targeted to hypothetical protein is a fast, specific, sensitive, inexpensive and suitable method for detection of P. aeruginosa.

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.