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To reveal differences in virulence among strains of Salmonella enterica serovar Pullorum (S. Pullorum), we used 2 cell-based infection models and a chicken embryo infection model in this study. S. Pullorum strain S06004 was used to infect 4 different avian cell lines (HD-11, DF-1, LMH, DT-40), and the results showed that the infection of S06004 in both LMH and HD-11 cells was more stable than in DF-1 and DT-40 cells. Therefore, the HD-11 and LMH cell lines were used as the appropriate macrophage and epithelial cell models, respectively, to study the infection of S. Pullorum. Fifty strains isolated during the years 1962 to 2010 were then analyzed to compare their infection rates in HD-11 and LMH cells. The result showed that the infection rates of most strains were very similar to that of S06004, except for S9876 which displayed the highest infection rate among these strains. Based on the cell infection results, 10 strains were selected to be used in the chicken embryo infection model. Sixteen-day-old SPF chicken embryos were infected with the pathogen at a dose of 103 CFU/100 μL via allantoic cavity inoculation. The strains C79-13, 7101, and S06013 caused death of more than 80% embryos, whereas S09C12 and 6703 resulted less than 20% death. Thus, this study established cell-based infection models to screen S. Pullorum strains in vitro, and a chick embryo model to evaluate their in vivo virulence., (© 2018 Poultry Science Association Inc.)

Salmonella enterica subsp. enterica serovar Gallinarum biovar Pullorum (Salmonella Pullorum) is a host-specific serovar causing systemic infection with high mortality in young chicks. Pullorum disease is characterized by white diarrhea. However, arthritis has become increasingly frequent recently, particularly in southern China. The aim of the present study was to determine the pathogenesis and arthritis induction of new Salmonella Pullorum isolates. We isolated and identified five Salmonella Pullorum strains from broilers with bacterial arthritis and lameness in a commercial poultry farm. Four of five isolates were resistant to at least three classes of antibiotics and were defined as multidrug-resistant Salmonella Pullorum. All isolates had the same CRISPR sequence type and belonged to a single major cluster. The isolates exhibited high capability of biofilm formation, which may facilitate their dispersal and survival in hostile habitats, and showed high virulence based on embryo lethality and inoculation of newly hatched chicks. Tissue distribution analysis confirmed that SP1621 was more adapted to colonize the joint when compared to the white diarrhoea-causing Salmonella Pullorum reference strain S06004. Reproducible arthritis and typical joint lesions were observed in SP1621-infected chicks, and histopathological examination showed necrotic synovitis and cartilage tissue hyperplasia of the joint. Koch's postulates were confirmed when the novel Salmonella Pullorum strain was re-isolated from the joint tissues of experimentally inoculated chicks. These novel Salmonella Pullorum isolates have unique ability to induce arthritis in chickens, representing expanded pathogenic diversity in China. These results suggest the need for strict control strategies and new vaccines to prevent the disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this study, Salmonella prevalence and antimicrobial resistance were evaluated at various production stages in 2 geographically separated breeder farms (referred to as G and F). Day-old chicks for the breeder flock at farm F were purchased from farm G. A total of 219 Salmonella isolates, all identified as Salmonella enterica subsp. enterica serovar Enteritidis, were recovered from 1,430 samples (sick chicken carcasses and/or dead embryos). The isolation rates at breeder farms G and F were 10.53% (56/532) and 18.15% (163/898), respectively. Resistance to 4-6 antimicrobial agents was the most frequent phenotype during the laying stage at both farms, suggesting that chicks are exposed to higher risk of antimicrobial-resistant Salmonella infection during this stage of the breeding process. Using clustered regularly interspaced short palindromic repeat (CRISPR) typing, 5 CRISPR patterns were identified, out of which one pattern was shared by the 2 farms. In addition, pulsed-field gel electrophoresis (PFGE) typing result indicated that 2 clusters (PF-1 and PF-2) were shared among the 2 breeder farms, suggesting that strains were transmitted from breeder farm G to farm F via the trade of day-old chicks. Our findings suggested that the trade of day-old breeder chicks could be one of the potential Salmonella transmission routes, and antibiotics should be administered with caution during the laying stage.

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) and biovar Pullorum (S. Pullorum) are gram-negative bacteria, members of the most important infectious pathogens, and have caused common problems in the poultry industry, especially in the developing countries. O- and H-antigen specific anti-sera are commonly for slide and tube agglutination tests to identify Salmonella serovars. However, it is both labor intensive and time consuming, so there is an urgent need for a new technique for the rapid detection of the major Salmonella serovars. In this study, we developed a 1-step PCR assay to identify the serovar Gallinarum. This PCR-based assay was based on the SPUL_2693 gene, which was located in SPI-19 and found by comparing the genomes of the S. Pullorum and S. Gallinarum in the whole data of NCBI. The specificity of this gene was evaluated by bioinformatics analysis, and the results showed that the SPUL_2693 gene exists in all serovar Gallinarum. The specificity and sensitivity of this PCR assay were evaluated in our study. The developed PCR assay was able to distinguish the serovar Gallinarum from 27 different Salmonella serovars and 5 different non-Salmonella pathogens. The minimum limit of genomic DNA of S. Pullorum for PCR detection was 2.143 pg/μL, and the minimum limit number of cells was 6 CFU. This PCR assay was also applied to analyze Salmonella strains isolated from a chicken farm in this study. The PCR assay properly identified the serovar Gallinarum from other Salmonella serovars, and the results were in agreement with the results of a traditional serotyping assay. In general, the newly developed PCR-based assay can be used to accurately judge the presence of the serovar Gallinarum and can be combined with traditional serotyping assays, especially in the case of large quantities of samples.

Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is the pathogen of pullorum disease, which leads to severe economic losses in many developing countries. Traditional methods to identify S. enterica have relied on biochemical reactions and serotyping, which are time-consuming with accurate identification if properly carried out. In this study, we developed a rapid polymerase chain reaction (PCR) method targeting the specific gene ipaJ to detect S. Pullorum. Among the 650 S. Pullorum strains isolated from 1962 to 2016 all over China, 644 strains were identified to harbour ipaJ gene in the plasmid pSPI12, accounting for a detection rate of 99.08%. Six strains were ipaJ negative because pSPI12 was not found in these strains according to whole genome sequencing results. There was no cross-reaction with other Salmonella serotypes, including Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum), which show a close genetic relationship with S. Pullorum. This shows that the PCR method could distinguish S. Gallinarum from S. Pullorum in one-step PCR without complicated biochemical identification. The limit of detection of this PCR method was as low as 90 fg/μl or 10 2 CFU, which shows a high sensitivity. Moreover, this method was applied to identify Salmonella isolated from the chicken farm and the results were consistent with what we obtained from biochemical reactions and serotyping. Together, all the results demonstrated that this one-step PCR method is simple and feasible to efficiently identify S. Pullorum.