Identification and Discrimination of Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum Based on a One-Step Multiplex PCR Assay

Salmonella enterica serovar Gallinarum biovars Pullorum (S. Pullorum) and Gallinarum (S. Gallinarum) can result in pullorum disease and fowl typhoid in avian species, respectively, and cause considerable economic losses in poultry in many developing countries. Conventional Salmonella serotyping is a time-consuming, labor-intensive and expensive process, and the two biovars cannot be distinguished using the traditional serological method. In this study, we developed a rapid and reliable one-step multiplex polymerase chain reaction (PCR) assay to simultaneously identify and discriminate the biovars Pullorum and Gallinarum. The multiplex PCR method focused on three specific genes, stn, I137_08605 and ratA. Based on bioinformatics analysis, we found that gene I137_08605 was present only in S. Pullorum and S. Gallinarum, and a region of difference in ratA was deleted only in S. Pullorum after comparison with that of S. Gallinarum and other Salmonella serovars. Three pairs of primers specific for the three genes were designed for the multiplex PCR system and their selectivity and sensitivity were determined. The multiplex PCR results showed that S. Pullorum and S. Gallinarum could be identified and discriminated accurately from all tested strains including 124 strains of various Salmonella serovars and 42 strains of different non-Salmonella pathogens. In addition, this multiplex PCR assay could detect a minimum genomic DNA concentration of 67.4 pg/μL, and 100 colony forming units. The efficiency of the multiplex PCR was evaluated by detecting natural-occurring Salmonella isolates from a chicken farm. The results demonstrated that the established multiplex PCR was able to identify S. Gallinarum and S. Pullorum individually, with results being consistent with traditional serotyping and biochemical testing. These results demonstrated that a highly accurate and simple biovar-specific multiplex PCR assay could be performed for the rapid identification and discrimination of Salmonella biovars Gallinarum and Pullorum, which will be useful, particularly under massive screening situations

Accurate identification and discrimination of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum by a multiplex PCR based on the new genes of torT and I137_14430

Most cases of chicken salmonellosis are caused by Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum, which lead to a significant morbidity and fatality rate. Although the conventional Kaufmann-White scheme is the reliable method for the serotyping of Salmonella, it does not distinguish between closely related biotypes like S. Pullorum and S. Gallinarum. Herein, we conducted a single one-step multiplex PCR assay that can identify and distinguish between S. Pullorum and S. Gallinarum in an accurate manner. This PCR method was based on three genes, including torT for S. Pullorum identification, I137_14430 for S. Gallinarum identification, and stn as the genus-level reference gene for Salmonella. By comparing S. Pullorum to S. Gallinarum and other serovars of Salmonella, in silico study revealed that only the former has a deletion of 126 bp-region in the carboxyl terminus of torT. The I137_14430 gene does not exist in S. Gallinarum. However, it is present in all other Salmonella serotypes. The multiplex PCR approach utilizes unique sets of primers that are intended to specifically target these three different genes. The established PCR method was capable of distinguishing between the biovars Pullorum and Gallinarum from the 29 distinct Salmonella serotypes as well as the 50 distinct pathogens that are not Salmonella, showing excellent specificity and exclusivity. The minimal amount of bacterial cells required for PCR detection was 100 CFU, while the lowest level of genomic DNA required was 27.5 pg/μL for both S. Pullorum and S. Gallinarum. After being implemented on the clinical Salmonella isolates collected from a poultry farm, the PCR test was capable of distinguishing the two biovars Pullorum and Gallinarum from the other Salmonella strains. The findings of the PCR assay were in line with those of the traditional serotyping and biochemical identification methods. This new multiplex PCR could be used as a novel tool to reinforce the clinical diagnosis and differentiation of S. Pullorum and S. Gallinarum, particularly in high-throughput screening situations, providing the opportunity for early screening of infections and, as a result, more effective management of the illness among flocks

Salmonella Enteritidis activates inflammatory storm via SPI-1 and SPI-2 to promote intracellular proliferation and bacterial virulence

Salmonella Enteritidis is an important intracellular pathogen, which can cause gastroenteritis in humans and animals and threaten life and health. S. Enteritidis proliferates in host macrophages to establish systemic infection. In this study, we evaluated the effects of Salmonella pathogenicity island-1 (SPI-1) and SPI-2 to S. Enteritidis virulence in vitro and in vivo, as well as the host inflammatory pathways affected by SPI-1 and SPI-2. Our results show that S. Enteritidis SPI-1 and SPI-2 contributed to bacterial invasion and proliferation in RAW264.7 macrophages, and induced cytotoxicity and cellular apoptosis of these cells. S. Enteritidis infection induced multiple inflammatory responses, including mitogen-activated protein kinase (ERK-mediated) and Janus kinase-signal transducer and activator of transcript (STAT) (STAT2-mediated) pathways. Both SPI-1 and SPI-2 were necessary to induce robust inflammatory responses and ERK/STAT2 phosphorylation in macrophages. In a mouse infection model, both SPIs, especially SPI-2, resulted in significant production of inflammatory cytokines and various interferon-stimulated genes in the liver and spleen. Activation of the ERK- and STAT2-mediated cytokine storm was largely affected by SPI-2. S. Enteritidis ΔSPI-1-infected mice displayed moderate histopathological damage and drastically reduced bacterial loads in tissues, whereas only slight damage and no bacteria were observed in ΔSPI-2- and ΔSPI-1/SPI-2-infected mice. A survival assay showed that ΔSPI-1 mutant mice maintained a medium level of virulence, while SPI-2 plays a decisive role in bacterial virulence. Collectively, our findings indicate that both SPIs, especially SPI-2, profoundly contributed to S. Enteritidis intracellular localization and virulence by activating multiple inflammatory pathways

Identification by PCR signature-tagged mutagenesis of attenuated Salmonella Pullorum mutants and corresponding genes in a chicken embryo model

A key feature of the fowl-specific pathogen Salmonella Pullorum is its vertical transmission to progeny via the egg. In this study, PCR signature-tagged mutagenesis identified nine genes of a strain of S. Pullorum that contributed to survival in the chicken embryo during incubation. The genes were involved in invasion, cell division, metabolism and bacterial defence. The competition index in vivo and in vitro together with a virulence evaluation for chicken embryos of all nine mutant strains confirmed their attenuation

Mucosal and Systemic Immune Responses to Influenza H7N9 Antigen HA1–2 Co-Delivered Intranasally with Flagellin or Polyethyleneimine in Mice and Chickens

Consecutive cases of human infection with H7N9 influenza viruses since 2013 in China have prompted efforts to develop an effective treatment. Subunit vaccines introduced by intranasal administration can block an infection at its primary site; flagellin (fliC) and polyethyleneimine (PEI) have been shown to be potent adjuvants. We previously generated the hemagglutinin (HA)1–2-fliC fusion protein consisting of the globular head domain (HA1–2; amino acids 62–284) of HA fused with Salmonella typhimurium fliC. In the present study, we investigated its effectiveness of both flagellin and PEI as mucosal adjuvants for the H7N9 influenza subunit vaccine. Mice immunized intranasally with HA1–2-fliC and HA1–2-PEI showed higher HA1–2-specific immunoglobulin (Ig)G and IgA titers in serum, nasal wash, and bronchial alveolar lavage fluid. Moreover, splenocyte activation and proliferation and the number of HA1–2-specific interferon (IFN)-γ- and interleukin (IL)-4-producing splenocytes were markedly increased in the fliC and PEI groups; in the latter, there were more cells secreting IL-4 than IFN-γ, suggesting that fliC induced T helper type (Th)1 and Th2 immune responses, and PEI induced Th2-biased responses, consistent with the serum antibody isotype pattern (IgG1/IgG2a ratio). Furthermore, virus challenge was performed in a chicken model. The results showed that chickens receiving fliC and PEI adjuvant vaccine exhibited robust immune responses leading to a significant reduction in viral loads of throat and cloaca compared to chickens receiving only HA1–2. These findings provide a basis for the development of H7N9 influenza HA1–2 mucosal subunit vaccines

The Impact of Industrial Green Transformation on Regional Economic Development in China: An Empirical Study Based on Spatial and Threshold Effects

At present, the high energy consumption, heavy pollution emissions, serious ecological damage and other problems of China’s industry are particularly prominent. Therefore, industrial governance is crucial and it is imperative to carry out industrial green transformation (IGT). Due to the differences in economic development, industrial structure and other aspects, the influence of IGT on different regions’ economic development has not been fully discussed. Using the provincial panel data of China from 2005 to 2019, this paper calculated each region’s IGT by constructing an index system and we discuss its impact on regional economic development, based on the spatial and threshold models. The results show that IGT in the eastern region is significantly higher than in the central and western regions. The IGT has a positive correlation in space and a significant positive spatial spillover effect on regional economic development. Moreover, there is a significant threshold effect of technological innovation in the influence of IGT on regional economic development. When technological innovation is low, the impact of IGT on regional economic development is not significant. As technological innovation rises and finally exceeds the critical point, the IGT will significantly promote regional economic development. This paper provides a scientific analysis and objective conclusions that may help to avoid radical measures in some regions in the process of “green development”

The Impact of Industrial Green Transformation on Regional Economic Development in China: An Empirical Study Based on Spatial and Threshold Effects

At present, the high energy consumption, heavy pollution emissions, serious ecological damage and other problems of China’s industry are particularly prominent. Therefore, industrial governance is crucial and it is imperative to carry out industrial green transformation (IGT). Due to the differences in economic development, industrial structure and other aspects, the influence of IGT on different regions’ economic development has not been fully discussed. Using the provincial panel data of China from 2005 to 2019, this paper calculated each region’s IGT by constructing an index system and we discuss its impact on regional economic development, based on the spatial and threshold models. The results show that IGT in the eastern region is significantly higher than in the central and western regions. The IGT has a positive correlation in space and a significant positive spatial spillover effect on regional economic development. Moreover, there is a significant threshold effect of technological innovation in the influence of IGT on regional economic development. When technological innovation is low, the impact of IGT on regional economic development is not significant. As technological innovation rises and finally exceeds the critical point, the IGT will significantly promote regional economic development. This paper provides a scientific analysis and objective conclusions that may help to avoid radical measures in some regions in the process of “green development”

Multi-objective global optimum design of collaborative robots

Optimum design is proven significant for improving task performances of robotic manipulators under certain constraints. However, when it is utilized for collaborative robots (Cobots), there are still many challenges such as complex smooth surface links, time-varying kinematic configurations, computational expensiveness, and nonstructural parameter optimization. Therefore, based on orthogonal design experiment (ODE) and finite element substructure method (FESM), a multi-objective optimum design method of Cobots is proposed with the structural dimensions and parameterized joint components as the optimization variables and the natural frequency, the Cartesian stiffness, and the mass of the robot as optimization objectives. Firstly, to obtain multiple global performance indexes (GPIs) of robots in real-time and efficiently, the FESM model of Cobots is established which can preserve the accuracy of the finite element method (FEM) while ensuring the computational efficiency. Then, the gray relational analysis method (GRAM) is used to construct the multi-objective optimization function which includes the global first-order natural frequency index (GFNFI), the global elastic deformation index (GEDI), and the mass of robots. The ODE is constructed, and the structural dimensions and parameterized joint components are taken as influencing factors. According to the orthogonal array (OA), the degree of gray incidence under different levels of influencing factors is solved. And the optimal combination of influencing factor levels is obtained by range analysis (RA), which is used to guide the design of Cobots. Finally, a Cobot SHIR5-I is taken as an illustrative example to perform optimum design in this paper