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.

Cryptosporidium is an important zoonotic parasite that causes diarrhea in humans and animals worldwide. Previous studies suggested geographic differences in the distribution of Cryptosporidium species in sheep. However, molecular characterization studies of Cryptosporidium species in sheep have been carried out in only a few provinces in China, and the limited data available do not reflect the real situation. In this study, five districts, covering most areas where sheep are bred in China, were selected for examination of Cryptosporidium species, and Cryptosporidium infections were detected at all farms assessed, suggesting that Cryptosporidium is widespread in sheep in China. We also found geographic differences in the distribution of Cryptosporidium species but did not detect any differences between sheep age groups or seasons. Subtyping analyses showed that all of the subtypes identified in this study have been reported in humans, suggesting that sheep may be a potential source of zoonotic cryptosporidiosis.

Enterocytozoon bieneusi is a common species of microsporidia that not only influences human health but also threatens animal productive performance and value. However, there have been no systematic studies of the prevalence of E. bieneusi in sheep in China. A total of 953 fecal specimens were collected from sheep from 11 provinces across five regions of China and analyzed for E. bieneusi by nested PCR targeting the ribosomal internal transcribed spacer (ITS). Enterocytozoon bieneusi infections were detected in four regions, with an overall infection rate of 20.4% (194/953). The highest infection rate was detected in pre-weaned lambs (25.0%), followed by post-weaned lambs (22.2%) and adult sheep (14.6%). Enterocytozoon bieneusi was found in nine of the 11 tested provinces, with infection rates between 2.9–51.7%. Eleven genotypes were identified based on ITS analysis, including seven known genotypes (BEB6, CHG1, CHG3, CHS7, CHS8, COS-I and NESH5) and four novel genotypes (CHHLJS1, CHHLJS2, CHNXS1 and CHXJS1). All 11 genotypes were clustered into group 2, and the zoonotic genotype BEB6 was the dominant genotype (n = 129, 66.5%) in sheep. The prevalence of E. bieneusi was studied in five regions representing most areas where sheep are bred in China. This is the first report of E. bieneusi infection in sheep for seven Chinese provinces. Geographical differences were detected in the distribution of E. bieneusi genotypes, but no differences were found among sheep in different age groups. The zoonotic genotype BEB6 was the dominant genotype, indicating that sheep are a potential source of zoonotic microsporidiosis in China. These results improve our knowledge of the epidemiology of E. bieneusi in sheep in China.

The luxS gene is required for autoinducer-2 (AI-2) synthesis in many bacterial species. AI-2 is taken up by a specific receptor to regulate multiple bacterial activities. However, the lack of methods to identify AI-2 receptors has impeded investigations into the roles of AI-2. Here, a luxS mutant of Escherichia coli strain BL21 (DE3) was constructed (named BL21∆luxS), and the recombinant LsrB protein of Salmonella enterica was expressed in BL21∆luxS and BL21 cells, which were named LsrB (BL21∆luxS) and LsrB (BL21), respectively. The results of the activity of recombinant LsrB binding showed that LsrB (BL21) bound to endogenous AI-2 (produced from BL21 strain), while LsrB (BL21∆luxS) did not (as BL21∆luxS cannot produce AI-2). However, the results of recombinant LsrB binding showed that LsrB (BL21∆luxS) can bind exogenous AI-2, which was released from LsrB (BL21∆luxS) at 55 °C for 10 min, while LsrB (BL21) could not bind exogenous AI-2 (due to binding of endogenous AI-2 before). Furthermore, analysis of the thermal stability of AI-2 showed that that AI-2 activity was relatively high at incubation temperatures below 65 °C. These findings will be beneficial for screening of new AI-2 receptors in different bacterial species.

Hen eggs are one of the most popular foods worldwide, and their safety is critical. Employing 16S rRNA full-length sequencing is an effective way to identify microorganisms on or in eggs. Here, hen eggs collected from poultry farms over four seasons, as well as from markets in Shanghai, were analyzed with third-generation sequencing. Firmicutes (44.46%) and Proteobacteria (35.78%) were the two dominant phyla, and Staphylococcus, Acinetobacter, Aerococcus, Psychrobacter, and Lactobacillus were the dominant genera. The dominant genera on the eggshell surfaces from the farms varied with the seasons, and the highest contamination of Staphylococcus (32.93%) was seen in the eggs collected during the summer. For the market samples, Pseudomonas was the most abundant in content, with Staphylococcus being the most-often genera found on the eggshell surfaces. Moreover, several potential pathogenic bacteria including Riemerella anatipestifer (species), Klebsiella (genus), and Escherichia/shigella (genus) were detected in the samples. The results revealed the impacts of weather on the microbiota deposited on an eggshell’s surface, as well as the impacts due to the differences between the contents and the surface. The results can help disinfect eggs and guide antibiotic selection.