The function of Autoinducer-2 (AI-2) which acts as the signal molecule of LuxS-mediated quorum sensing, is regulated through the lsr operon (which includes eight genes: lsrK, lsrR, lsrA, lsrC, lsrD, lsrB, lsrF, and lsrG). However, the functions of the lsr operon remain unclear in avian pathogenic Escherichia coli (APEC), which causes severe respiratory and systemic diseases in poultry. In this study, the presence of the lsr operon in 60 APEC clinical strains (serotypes O1, O2, and O78) was investigated and found to be correlated with serotype and has the highest detection rate in O78. The AI-2 binding capacity of recombinant protein LsrB of APEC (APEC-LsrB) was verified and was found to bind to AI-2 in vitro. In addition, the lsr operon was mutated in an APEC strain (APEC94Δlsr(Cm)) and the mutant was found to be defective in motility and AI-2 uptake. Furthermore, deletion of the lsr operon attenuated the virulence of APEC, with the LD₅₀ of APEC94Δlsr(Cm) decreasing 294-fold compared with wild-type strain APEC94. The bacterial load in the blood, liver, spleen, and kidneys of ducks infected with APEC94Δlsr(Cm) decreased significantly (p < 0.0001). The results of transcriptional analysis showed that 62 genes were up-regulated and 415 genes were down-regulated in APEC94Δlsr(Cm) compared with the wild-type strain and some of the down-regulated genes were associated with the virulence of APEC. In conclusion, our study suggests that lsr operon plays a role in the pathogenesis of APEC.

Escherichia coli, Pasteurella multocida, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella spp. and Staphylococcus aureus are six bacterial pathogens of avian. However, these pathogens may cause many similar pathological changes, resulting in clinical isolates that are difficult to quickly and simultaneously detect and identify. Here, a multiplex polymerase chain reaction (m-PCR) assay is reported to rapidly identify targets genes (phoA, KMT1, ureR, toxA, invA, and nuc) of these six pathogens in clinical samples. Six pairs of specific primers were designed. The optimal reaction conditions, specificity, and sensitivity of the m-PCR assay were investigated. The results showed that betaine remarkably improved amplification of the target genes. Specific test results showed that all six pathogens were detected by the proposed m-PCR protocol without cross-amplification with viruses or parasites. Sensitivity test results showed that the m-PCR system could amplify the six target genes from bacterial genomes or cultures with template amounts of 500 pg or 2.8–8.6 × 10³ colony forming units, respectively. Furthermore, the six bacterial pathogens isolated from the infected tissue samples were successfully identified. The proposed m-PCR assay is a useful tool to monitor and diagnose bacterial infection in birds with high specificity, sensitivity and throughput.

Avian pathogenic Escherichia coli (APEC) is a causative agent of colibacillosis, one of the principal causes of morbidity and mortality in poultry worldwide. Nowadays, antibiotics are mainly used to prevent and control poultry colibacillosis, but the situation of drug resistance is serious. 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) is involved in methylation reactions, polyamine synthesis, vitamin synthesis, and quorum sensing (QS) pathways. In this study, compared with the APEC wild-type strain DE17, the pfs deletion strain DE17Δpfs was more susceptible to β-lactam antibiotics (amoxicillin, ceftazidime, cefuroxime) by drug sensitivity test and minimum inhibitory concentration (MIC), and the MIC of the DE17Δpfs was half that of the DE17. Quorum sensing signal molecule AI-2 is involved in antibiotic resistance. In the case of pfs inactivation, the DE17Δpfs cannot synthesize AI-2, so it is necessary to add AI-2 to study whether it affects APEC resistance. When the exogenous AI-2 was added, the MIC of all APEC did not change. Transcriptome sequencing indicated that the transcription levels of a lot of outer membrane protein genes and metabolic genes had changed due to the deletion of pfs. Moreover, the transcription levels of the efflux pump gene tolC and penicillin binding protein (fstI and mrcA) were significantly reduced (p < 0.05), while the transcription levels of the porin protein genes (ompF, ompC, and ompD) were significantly increased (p < 0.05). In addition, it was also found that the outer membrane permeability of the DE17Δpfs was significantly increased (p < 0.05). The results indicated that pfs does not affect APEC strain DE17 resistance to β-lactam antibiotics through AI-2, but pfs affects the sensitivity of APEC to β-lactam antibiotics by affecting antibiotic-related genes. This study can provide a reference for screening new drug targets.

Avian Pathogenic Escherichia coli (APEC) constitutes a major etiological agent of avian colibacillosis, which significantly hinders the development of the poultry industry. Conducting molecular epidemiological studies of APEC plays a crucial role in its prevention and control. This study aims to elucidate the molecular epidemiological characteristics of Avian Pathogenic Escherichia coli in Shanxi Province. In this study, 135 APEC strains were isolated and identified from 150 liver samples of diseased and deceased chickens exhibiting clinical symptoms, which were collected from farms in Shanxi Province between 2021 and 2023. The isolates were then analyzed for phylogenetic clustering, drug resistance, resistance genes, virulence genes, and biofilm formation capabilities. The results revealed that the proportions of the A, B1, B2, and D evolutionary subgroups were 26.67%, 32.59%, 17.78%, and 15.56%, respectively. The drug resistance testing results indicated that 92% of the isolates exhibited resistance to cotrimoxazole, kanamycin, chloramphenicol, amoxicillin, tetracycline, and other antibiotics. In contrast, 95% of the strains were sensitive to ofloxacin, amikacin, and ceftazidime. The most prevalent resistance genes included tetracycline-related (tetA) at 88.15%, followed by beta-lactam-related (bla-TEM) at 85.19%, and peptide-related (mcr1) at 12.59%. The virulence gene analysis revealed that ibeB, ompA, iucD, and mat were present in more than 90% of the isolates. The results revealed that 110 strains were biofilm-positive, corresponding to a detection rate of 81.48%. No significant correlation was found between the drug resistance genes, virulence genes, and the drug resistance phenotype. A moderate negative correlation was observed between the adhesion-related gene tsh and biofilm formation ability (r = −0.38). This study provides valuable insights into the prevention and control of avian colibacillosis in Shanxi Province.

Background: As a ubiquitous apicomplexan parasite, Toxoplasma gondii causes huge economic losses and poses a great threat to the health of animals, including humans, worldwide. In some kangaroo species, T. gondii can be fatal. To date, little information is available on T. gondii infection in the red kangaroos in east China. At a zoo in east China, thirteen red kangaroos consecutively developed clinical signs from July to November 2016, resulting in the deaths of seven, three of which were analyzed in this study. Methods: In the present study, ascitic fluid, blood and samples from the brain, heart, liver, spleen, lung, kidney, and mesenteric lymph nodes of three dead red kangaroos were collected. The pathogen was explored through microscopic observation, nested PCR, immunofluorescence antibody test (IFAT), hematoxylin–eosin (HE) staining, and immunohistochemistry (IHC) staining, respectively. Meanwhile, the potential source of the infection was also investigated by testing the blood of stray cats in the zoo for T. gondii using nested PCR. Results: Three dead red kangaroos were subjected to a necropsy, and organisms resembling T. gondii were detected in their ascitic fluids under microscope. This infection was further confirmed by a nested PCR assay, which resulted in a successful amplification and sequencing of the 433 bp fragment of the T. gondii 5.8S rRNA gene in all the dissected tissues, including heart, liver, spleen, lung, kidney, lymph nodes, cecum, and brain, as well as in body fluids (blood and ascitic fluid). Furthermore, the tachyzoites were observed in the heart, liver, spleen, lymph nodes, cecum, and brain through IFAT and HE staining. Administration of classic drugs (sulfadiazine and pyrimethamine) against T. gondii significantly alleviated the clinical signs of the sick kangaroos. The possible source of this infection was traced to a native stray cat, as T. gondii DNA was detected in its blood. Conclusions: In the present study, lethal T. gondii infection in red kangaroos has been described for the first time in east China, highlighting the necessity and urgency for close and long-term surveillance of this parasite infection in captive animals. The same strain of T. gondii detected in kangaroos as that found in stray cats wandering in the same area emphasizes the importance of controlling stray cat populations to mitigate the risk of Toxoplasma transmission to other animals.