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ABSTRACT: Salmonellosis in poultry is detrimental to the advancement of the breeding industry and poses hazards to human health. Approximately 2,600 Salmonella varieties exist, among which S. Enteritidis, S. Pullorum, S. Typhimurium, and S. Infantis are prevalent serotypes in the poultry industry in recent years. They can also infect humans by contaminating poultry eggs and meat. Therefore, identifying these serotypes is crucial for successful preventive and control interventions. The White–Kauffmann–Le Minor scheme is time-consuming and requires expensive reagents. Whole-genome sequencing (WGS) and other molecular biology techniques require skilled technical staff. In comparison, the polymerase chain reaction (PCR) is more accurate, rapid, and inexpensive, thus proving suitable for widespread application in the poultry industry. Here, we selected 4 specific primers: lygD, mdh, ipaJ, and SIN_02055, which correspond to detecting S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Infantis, respectively. They were integrated into a 1-step multiplex PCR method. We optimized the PCR method by utilizing specificity test results to determine the optimal annealing temperature (57°C). The PCR method exhibited excellent sensitivity for genomic DNA and bacterial cultures. We used the developed method to determine 157 clinical Salmonella isolates from various stages of the poultry production chain. The results aligned with serotype data generated via WGS analysis, demonstrating the method's excellent accuracy. In conclusion, this study developed a 1-step multiplex PCR method that simultaneously identifies S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Infantis, allowing routine mass detection in the grass-root poultry industry.

ABSTRACT: Salmonella, which is widely distributed in nature, is an important zoonotic pathogen affecting humans, livestock, and other animals. Salmonella infection not only hinders the development of livestock and poultry-related industries but also poses a great threat to human health. In this study, we collected 1,537 samples including weak chicks, dead embryos, fecal samples and environmental samples from 2020 to 2023 (for a period of 1 to 2 months per year) to keep a long-term monitor the prevalence of Salmonella in an intensive laying hen farm, 105 Salmonella strains were isolated with an isolation rate of 6.83% (105/1,537). It revealed a significant decrease in prevalence rates of Salmonella over time (P < 0.001). Before 2020, the predominant serotype was S. Enteritidis. S. Kentucky was first detected in November 2020 and its proportion was gradually found to exceed that of S. Enteritidis since then. S. Kentucky isolates were distributed in various links of the four regions in the poultry farm. A total of 55 S. Kentucky strains, were assigned to ST198 based on whole genome sequencing. Among them, 54 strains were resistant to 12 to 16 antibiotics, indicating that they were extensively drug-resistant (XDR). Seventeen antimicrobial resistance genes were detected in 55 S. Kentucky isolates. For most of these isolates, antibiotic resistance phenotypes were concordant with their genotypes. All S. Kentucky strains isolated from this farm in 2020 to 2023 showed a high similarity based on their core-genome SNP-based phylogeny. The traceability analysis revealed that S. Kentucky was introduced to the farm through newly purchased flocks. The long-term existence of XDR S. Kentucky ST198 poses a substantial risk because of the multiage management and circulation of workers in this poultry farm. Thus, this study is the first to report extensively drug-resistant S. Kentucky ST198 detected in this intensive poultry farm in China.

ABSTRACT: Bovine tuberculosis (bTB) caused by Mycobacterium bovis is an important zoonotic disease. This infection is difficult to control because of the limited ability of the tuberculin skin test (TST) and ancillary IFN-γ release assay to detect all infected animals. In this study, we aimed to develop an efficient assay based on the enzyme-linked immunospot (ELISpot) technique for the diagnosis of bTB, with IFN-γ monoclonal antibodies 3E9 and Bio-labeled 6F8 used as capture and detection antibodies, respectively. As expected, there were significantly more M. bovis-specific spot-forming units (SFU) in bTB-infected cattle than in healthy cattle when an M. bovis-specific antigen, CFP-10-ESAT-6 fusion protein (CE protein), was used. The M. bovis IFN-γ ELISpot assay demonstrated a high level of agreement (90.83%) with the BOVIGAM ELISA test (Thermo Fisher Scientific) for detecting bTB. Furthermore, 3 of 109 cattle tested negative by both the TST and the BOVIGAM ELISA tests, but positive by the ELISpot assay (TST− ELISA− ELISpot+). During subsequent long-term monitoring, these 3 cattle became TST+ ELISA+ ELISpot+. These results suggest that the M. bovis IFN-γ ELISpot assay we established could detect infected cattle earlier than the BOVIGAM ELISA test.

ABSTRACT: Salmonella is the most important foodborne pathogen in poultry production systems and can infect humans via consumption of contaminated food. Ducks, an important waterfowl widely raised in China, are also a vehicle that transmits Salmonella through the food supply chain. In this study, 701 samples were collected from each production stage of the duck production chain. Salmonella was isolated and identified, and the isolates were tested for drug sensitivity and molecular typing based on whole genome sequencing (WGS) to explore the prevalence of Salmonella in the duck production chain. Altogether, a total of 180 Salmonella isolates (25.7%) were obtained from the duck production chain, 82 (35.7%) isolates were from hatchery samples, followed by 64 (29.2%) from market samples, 17 (23.6%) from farm samples, and 17 (9.4%) from slaughterhouse samples. All isolates were divided into 9 serotypes, among which S. Typhimurium, S. Anatum, and S. Enteritidis were the dominant serotypes. The S. Typhimurium was distributed in various production stages in the duck production chain. Among the 16 antibiotics, selected 60 isolates were only resistant to NAL, indicating that resistance of Salmonella in the duck production chain was low. WGS phylogenetic relationship results based on core-genome SNPs showed that S. Typhimurium can spread across geographic regions and along between different stages of the duck production chain, eventually reaching the market where it is a potential threat to consumer health. This study explored the prevalence of Salmonella in the duck production chain which will provide data support for proposing some interventions to control Salmonella.

ABSTRACT: Salmonella enterica serovar Pullorum (S. Pullorum) causes pullorum disease (PD), which is an acute systemic disease, in chickens, and leads to serious economic losses in many developing countries because of its high morbidity and mortality rate in young chicks. The live-attenuated vaccine is considered to be an effective measure to control the Salmonella infection. In addition, the DIVA (differentiation of infected and vaccinated animals) feature without the interference of serological monitoring of Salmonella infection is an important consideration in the development of the Salmonella vaccine. In this study, we evaluated the immunogenicity and protective efficacy of a S. Pullorum rough mutant S06004ΔspiCΔrfaH as a live attenuated DIVA vaccine candidate in chickens. The S06004ΔspiCΔrfaH exhibited a significant rough lipopolysaccharides (LPS) phenotype which was agglutinated with the acriflavine, not with the O9 mono antibody. Compared to the wild-type, 50% lethal dose (LD50) of the rough mutant increased 100-fold confirmed its attenuation. The mutant strain also showed a decreased bacterial colonization in the spleen and liver. The immunization with the mutant strain had no effect on the body weight and no tissue lesions were observed in the liver and spleen. The high level of the S. Pullorum-specific IgG titers in the serum indicated that significant humoral immune responses were induced in the immunization group. The cellular immune responses were also elicited from the analysis of lymphocyte proliferation and expression of cytokines in the spleen. In addition, the S06004ΔspiCΔrfaH immunized group exhibited a negative response for the serological test, while the wild-type S06004 infection group was strongly positive for the serological test showing a DIVA capability. The survival rates in the vaccinated chickens were 87% after intramuscular challenge with wild-type S. Pullorum, while the survival rates were 20% in the control groups. Overall, these results have demonstrated that the rough mutant S06004ΔspiCΔrfaH strain can be developed as an efficient live attenuated DIVA vaccine candidate to control the systemic S. Pullorum infection without the interference of salmonellosis monitoring program in poultry.

Salmonella spp. are important zoonotic pathogens that are responsible for severe diseases in both animals and humans. Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) and biovar Pullorum (S. Pullorum) are typical infectious pathogens detected in the chicken industry that have caused great economic losses. To facilitate their detection and prevent contamination, we developed a rapid multiple PCR method, which can simultaneously detect Salmonella spp. and further identify the biovars S. Pullorum/Gallinarum. This PCR detection method is based on the cigR gene, which is conserved among Salmonella spp. but has a 42-bp deletion in S. Pullorum/Gallinarum. The specificity and sensitivity of the PCR assay was evaluated with 41 different strains: 34 Salmonella strains, including 5 S. Pullorum/Gallinarum strains, and 7 non-Salmonella strains. The lower limit of detection was 8.15 pg of S. Pullorum (S06004) genomic DNA and 20 cfu in PCR, which shows a great sensitivity. In addition, this method was applied to detect or identify Salmonella from processing chicken liver and egg samples, and the results corresponded to those obtained from serotype analysis using the conventional slide agglutination test. Overall, the new cigR-based PCR assay is efficient and practical for Salmonella detection and S. Pullorum/Gallinarum identification and will greatly reduce the workload of epidemiologic investigation.

This study investigated the prevalence of Salmonella and the molecular typing of all isolates in a goose production chain including hatchery, farm, slaughterhouse, and market. A total of 350 Salmonella isolates was detected from 1,030 samples, and 13 serotypes were recovered. The highest Salmonella contamination frequency was observed at the hatchery, which 51.8% (188/363) of samples were Salmonella positive. S. Potsdam and S. Typhimurium were the 2 most common serotypes. S. Potsdam was most frequently found in the hatchery, while S. Typhimurium was widely distributed in the goose production chain. In general, the antibiotic resistance of Salmonella isolates is low, which isolates from the market is comparatively higher than from other production links indicating a possibility of Salmonella cross-contamination in the market. By the multilocus sequence typing (MLST) analysis, 7 different ST types were identified. ST2039 was the most common ST type, which was mostly found from S. Potsdam isolates in hatchery indicating that S. Potsdam might have been long existed in hatchery. The pulsed-field gel electrophoresis (PFGE) analysis of S. Potsdam indicated that S. Potsdam could be transmitted along the production chain. The PFGE analysis of S. Typhimurium showed that PFGE pattern 29 (PF29) was distributed in hatchery, and also in farm and from humans indicating the risk of S. Typhimurium transmitting to humans by the food supply chain. Our study provided the evidence of Salmonella cross-contamination in the slaughterhouse and the retail market of goose production chain, and specific serotypes existed for a long time at a particular production link. The spread of Salmonella along the production chain, might cause harm to humans through cross-contamination. Further studies would be needed to control the Salmonella contamination in hatchery and prevent the transmission of the pathogen during the goose production.

Introduction: Early detection of vertical root fracture (VRF) is important for clinical endodontic practice. The purpose of this study was to measure the fracture width (distance between 2 sides of the fracture) of VRF teeth in vitro by using 2 micro-computed tomography (μ-CT) systems with different spatial resolution and voxel size., Methods: Thirty-seven endodontically treated teeth with VRF were scanned by 80-μm pixel size μ-CT. Fifteen teeth with no obvious fracture line, blurred image, or fracture space less than 100 μm were scanned by 9-μm pixel size μ-CT., Results: Presence of 2 VRF lines was more common in premolars (82%) than in molars (53%). In 7 premolars (32%) and 9 molars (60%), the VRF lines extended to within the apical 3 mm of the root. All fracture lines were detected by 9-μm pixel size μ-CT, but only 22 of 37 VRF teeth had vertical fracture identified by 80-μm μ-CT. From μ-CT examination, none of the fracture lines showed consistent and uniform fracture space. If 2 fracture lines were present, they were typically in opposite (not linear) directions. There was a significant correlation between 2 fracture lines or fracture lines extending within the 3 mm of the apex and fracture width greater than 100 μm., Conclusions: Application of 9-μm μ-CT can be accurately used for early detection of VRF. Fracture characteristics (eg, number of fracture lines, extension of fracture line) may affect the fracture width. Appropriate use of μ-CT technology can be helpful for early diagnosis of VRF., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)