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Brazilian Journal of Microbiology :... Sep 2023Salmonella is present in the poultry production chain and is a major challenge in terms of food safety and animal health. The early Salmonella detection is one of the...
Salmonella is present in the poultry production chain and is a major challenge in terms of food safety and animal health. The early Salmonella detection is one of the main tools to control and prevent the transmission of this pathogen. Microbiological isolation and serotyping to identify and differentiate Salmonella serovars are laborious processes, time-consuming, and expensive. Therefore, molecular diagnostic methods can be rapid and efficient alternatives to the detection of this pathogen. Thus, the aim herein was to standardize and evaluate the use of loop-mediated isothermal amplification (LAMP) in comparison with real-time PCR (qPCR) for detection of Salmonella associated with a multiplex qPCR for simultaneous identification and differentiation of S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Gallinarum. The LAMP, qPCR, and multiplex qPCR assays were comparable in specificity. The three techniques were evaluated for specificity for 16 different serovars of Salmonella and for 37 strains of the serovars of interest. The limit of detection and the efficiency of the LAMP, qPCR, and multiplex qPCR reactions were determined. The techniques were applied to 33 samples of chicken carcasses and compared to the results of conventional microbiology for validation. As results, LAMP was specific in the detection of different Salmonella serovars but presented lower limit of detection ranging from 10 to 10 CFU/reaction. In comparison, qPCR could detect less cells (10 to 10 CFU/reaction), reaching equal specificity and better repeatability in the assays. The qPCR multiplexing for identification of the different serovars also showed good specificity, with the detection threshold between entre 10 and 10 CFU/reaction. The results obtained in the analyses on poultry carcasses suggested a correspondence between the results obtained in molecular methods and in conventional microbiology. Thus, the proposed assays are promising for the diagnosis of Salmonella in poultry carcasses, already proved to be faster and more efficient than conventional diagnostics techniques, being of great interest for poultry production, animal, and public health.
Topics: Animals; Poultry; Serogroup; Salmonella; Food Safety; Chickens; Sensitivity and Specificity
PubMed: 37582950
DOI: 10.1007/s42770-023-01095-y -
Frontiers in Veterinary Science 2023subspecies serovar Gallinarum biovar Gallinarum (SG) is associated with fowl typhoid fever, and the attenuated rough strain SG9R is widely used as a vaccine in many...
INTRODUCTION
subspecies serovar Gallinarum biovar Gallinarum (SG) is associated with fowl typhoid fever, and the attenuated rough strain SG9R is widely used as a vaccine in many regions. Reversion to virulence of vaccine strains was suspected as the cause during recent fowl typhoid fever outbreaks in poultry in South Africa and Eswatini.
METHODS
To compare nine field isolates with global wild-type SG9 strains and the two commercial SG9R vaccines in use, Nobilis SG9R and Cevac-SG, we used whole-genome comparison with single-nucleotide polymorphism (SNP) detection.
RESULTS
SNP phylogenic analysis showed that all the southern African field isolates were more closely related to the vaccine strains than wild-type SG9 strains. Furthermore, SNPs in the pyruvate dehydrogenase (E) and/or lipopolysaccharide 1,2-glucosyltransferase (J) genes, which are known markers of attenuation, were found in four of the field isolates along with intact , SPI-1, and SPI-2 gene clusters, providing conclusive evidence that these four isolates were originally vaccine strains that reverted to virulence. Five other field isolates lacked the SG9R attenuation markers, but variant analysis identified an SNP in the X gene, insertions in the X and H genes, and deletions in the K and A genes that were shared between the field isolates and vaccine strains but absent in wild-type SG9, indicating that these field isolates were also likely revertant vaccines.
DISCUSSION
Overall, this study highlights different mechanisms of reversion of two commercial vaccines, where virulence caused by field isolates closely related to the Nobilis SG9R vaccine was associated with the restoration of intact virulence gene clusters, and those derived from the Cevac-SG vaccine were characterized by point mutations resulting in restored E and J genes. A possible new marker of attenuation was identified as a point mutation in the X gene, as well as four new candidate genes that could potentially be used to distinguish current vaccine strains from wild-type strains using PCR assays.
PubMed: 37476827
DOI: 10.3389/fvets.2023.1191497 -
Frontiers in Veterinary Science 2023Most cases of chicken salmonellosis are caused by serovar Gallinarum biovars Gallinarum and Pullorum, which lead to a significant morbidity and fatality rate. Although...
Most cases of chicken salmonellosis are caused by 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 , it does not distinguish between closely related biotypes like . Pullorum and . Gallinarum. Herein, we conducted a single one-step multiplex PCR assay that can identify and distinguish between . Pullorum and . Gallinarum in an accurate manner. This PCR method was based on three genes, including for . Pullorum identification, for . Gallinarum identification, and as the genus-level reference gene for . By comparing . Pullorum to . Gallinarum and other serovars of study revealed that only the former has a deletion of 126 bp-region in the carboxyl terminus of . The gene does not exist in . Gallinarum. However, it is present in all other 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 serotypes as well as the 50 distinct pathogens that are not , 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 . Pullorum and . Gallinarum. After being implemented on the clinical isolates collected from a poultry farm, the PCR test was capable of distinguishing the two biovars Pullorum and Gallinarum from the other 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 . Pullorum and . 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.
PubMed: 37476820
DOI: 10.3389/fvets.2023.1220118 -
The Journal of Poultry Science 2023Benefits chitosan-fermented feed additives (CFFAs) particularly in the regulation of the immune system and antimicrobial activity. Therefore, we investigated the...
Benefits chitosan-fermented feed additives (CFFAs) particularly in the regulation of the immune system and antimicrobial activity. Therefore, we investigated the immune-enhancing and bacterial clearance effects of CFFA (fermented by ) on broiler chickens Gallinarum challenge. We administered 2% or 4% CFFA evaluated its immune-enhancing effects using several immunological experiments, including examination of lysozyme activity, lymphocyte proliferation, and expression of cytokines. We also evaluated the bacterial clearance effects of CFFA against Gallinarum. CFFA administration markedly enhanced lysozyme activity, lymphocyte proliferation, and the expression of interleukin (IL)-2, IL-12, tumor necrosis factor alpha, and interferon gamma in the spleen. In broilers challenged with Gallinarum, the clinical signs of Gallinarum infection and the number of viable bacterial colonies in the feces and tissues decreased in both CFFA groups. Therefore, CFFAs could be good candidates for feed additive to improve nonspecific immune responses and bacterial clearance.
PubMed: 37426541
DOI: 10.2141/jpsa.2023016 -
Microbial Pathogenesis Sep 2023Salmonella enterica serovar Gallinarum causes Fowl Typhoid in poultry, and it is host specific to avian species. The reasons why S. Gallinarum is restricted to avians,...
Salmonella enterica serovar Gallinarum causes Fowl Typhoid in poultry, and it is host specific to avian species. The reasons why S. Gallinarum is restricted to avians, and at the same time predominately cause systemic infections in these hosts, are unknown. In the current study, we developed a surgical approach to study gene expression inside the peritoneal cavity of hens to shed light on this. Strains of the host specific S. Gallinarum, the cattle-adapted S. Dublin and the broad host range serovar, S. Enteritidis, were enclosed in semi-permeable tubes and surgically placed for 4 h in the peritoneal cavity of hens and for control in a minimal medium at 41.2 °C. Global gene-expression under these conditions was compared between serovars using tiled-micro arrays with probes representing the genome of S. Typhimurium, S. Dublin and S. Gallinarum. Among other genes, genes of SPI-13, SPI-14 and the macrophage survival gene mig-14 were specifically up-regulated in the host specific serovar, S. Gallinarum, and further studies into the role of these genes in host specific infection are highly indicated. Analysis of pathways and GO-terms, which were enriched in the host specific S. Gallinarum without being enriched in the two other serovars indicated that host specificity was characterized by a metabolic fine-tuning as well as unique expression of virulence associated pathways. The cattle adapted serovar S. Dublin differed from the two other serovars by a lack of up-regulation of genes encoded in the virulence associated pathogenicity island 2, and this may explain the inability of this serovar to cause disease in poultry.
Topics: Animals; Female; Cattle; Serogroup; Chickens; Transcriptome; Salmonella enterica; Salmonella Infections, Animal; Salmonella enteritidis
PubMed: 37419218
DOI: 10.1016/j.micpath.2023.106236 -
Archives of Razi Institute Apr 2023Pullorum disease (PD) is one of the most common diseases in the world, with devastating consequences. In the chicken sector, there have been financial losses. It is...
Pullorum disease (PD) is one of the most common diseases in the world, with devastating consequences. In the chicken sector, there have been financial losses. It is brought on by ; definitive detection requires culture followed by biochemistry analysis and serotyping. This study aimed to verify the presence of bacteria by culture, biochemical characterization, PCR assay, and sequencing. One hundred samples were collected from 12 broiler chicken flocks of different ages for 8districts of Baghdad province, including cloacal swabs (65), visceral organs (15), and dropping (20). Salmonella colonies were identified by selective culture broth and agar with biochemical description for 75% of the total samples, with a higher incidence in visceral organs than dropping and cloacal swabs. ،The Sequencing and phylogenetic tree analysis of 16S rRNA gene for representative Salmonella isolates. The presence of Salmonella isolates in global genetic strains; was revealed a matching NCBI isolates similarity of 99.02% with (MF445124.1) and 98% with (MH352164.1), respectively. In the current state of molecular and genetic research, phlyogentic research announced the real presence of in Baghdadprovince's broiler chicken, also showing the phylogentic characteristics and links to some global isolates. The detection of in broiler flocks of the current study extent of health risks to other uninfected birds present in the free range.
Topics: Animals; Chickens; Phylogeny; Iraq; RNA, Ribosomal, 16S; Poultry Diseases; Salmonella Infections, Animal; Salmonella; Salmonella enterica
PubMed: 37396728
DOI: 10.22092/ARI.2022.359468.2424 -
Poultry Science Aug 2023Significant differences in pathogenicity between Salmonella Enteritidis and Salmonella Gallinarum exist despite the fact that S. Gallinarum is a direct descendant of S....
Significant differences in pathogenicity between Salmonella Enteritidis and Salmonella Gallinarum exist despite the fact that S. Gallinarum is a direct descendant of S. Enteritidis. It was hypothesized that such various properties may be in part the result of differences in structure and functions of type 1 fimbriae (T1Fs). In S. Enteritidis, T1Fs bind to oligomannosidic structures carried by host cell glycoproteins and are called mannose-sensitive T1Fs (MST1F). In S. Gallinarum, T1Fs lost ability to bind such carbohydrate chains, and were named mannose-resistant MRT1Fs (MRT1F). Therefore, the present study was undertaken to evaluate the role of MST1Fs and MRT1Fs in the adhesion, invasion, intracellular survival and cytotoxicity of S. Enteritidis and S. Gallinarum toward chicken intestinal CHIC8-E11cells and macrophage-like HD11 cells. Using mutant strains: S. Enteritidis fimH::kan and S. Gallinarum fimH::kan devoid of T1Fs and in vitro assays the following observations were made. MST1Fs have a significant impact on the chicken cell invasion by S. Enteritidis as MST1F-mediated adhesion facilitates direct and stable contact of bacteria with host cells, in contrast to MRT1Fs expressed by S. Gallinarum. MST1Fs as well as MRT1Fs did not affected intracellular viability of S. Enteritidis and S. Gallinarum. However, absolute numbers of intracellular viable wild-type S. Enteritidis were significantly higher than S. Enteritidis fimH::kan mutant and wild-type S. Gallinarum and S. Gallinarum fimH::kan mutant. These differences, reflecting the numbers of adherent and invading bacteria, underline the importance of MST1Fs in the pathogenicity of S. Enteritidis infections. The cytotoxicity of wild-type S. Enteritidis and its mutant devoid of MST1Fs to HD11 cells was essentially the same, despite the fact that the number of viable intracellular bacteria was significantly lower in the mutated strain. Using HD11 cells with similar number of intracellular wild-type S. Enteritidis and S. Enteritidis fimH::kan mutant, it was found that the lack of MST1Fs did not affect directly the cytotoxicity, suggesting that the increase in cytotoxicity of S. Enteritidis devoid of MST1Fs may be associated with crosstalk between T1Fs and other virulence factors.
Topics: Animals; Salmonella enteritidis; Mannose; Chickens; Glycoproteins; Salmonella Infections, Animal
PubMed: 37356296
DOI: 10.1016/j.psj.2023.102833 -
Polish Journal of Microbiology Jun 2023genus harbors five Type VI Secretion System (T6SS) gene clusters. The T6SS encoded in SPI-6 (T6SS) contributes to Typhimurium colonization of chickens and mice, while...
genus harbors five Type VI Secretion System (T6SS) gene clusters. The T6SS encoded in SPI-6 (T6SS) contributes to Typhimurium colonization of chickens and mice, while the T6SS encoded in SPI-19 (T6SS) of Gallinarum contributes to chicken colonization. Interestingly, the T6SS of Gallinarum complemented the defect in chicken colonization of a Typhimurium strain that lacks the T6SS, suggesting that both T6SSs are interchangeable. Here we show that the transfer of Gallinarum T6SS complemented the defect in mice colonization of a Typhimurium ΔT6SS strain, indicating that both T6SSs are functionally redundant during host colonization.
Topics: Animals; Mice; Salmonella typhimurium; Chickens; Multigene Family
PubMed: 37314360
DOI: 10.33073/pjm-2023-017 -
Veterinaria Italiana Dec 2022Non-typhoidal Salmonellae are important foodborne bacterial pathogens that can cause bacteremia, gastroenteritis, and subsequent infection. The aim of the study was to...
Prevalence and molecular characterization of typhoidal and non-typhoidal Salmonella isolated from meat and environmental samples of retail shops of Lahore Punjab, Pakistan.
Non-typhoidal Salmonellae are important foodborne bacterial pathogens that can cause bacteremia, gastroenteritis, and subsequent infection. The aim of the study was to determine the prevalence of Salmonella in the live bird market and retail shops of Lahore (Pakistan). A total of 720 samples of chicken meat, chopping board, cages, hands, and transportation vans were collected. Salmonella was recovered from 103 (14.36%) samples. The highest prevalence (33.33%) was found in transportation van samples followed by chicken meat samples (17.26%). In the towns of Lahore, the highest prevalence was found in Samanabad Town (19%) followed by Data Ganj Bakhsh Town (17%) with the lowest in Gulberg Town (6.9%). Salmonella Typhimurium was most common (35.92%) followed by S. Enteritidis (25.24%), S. Dublin (14.56%), S. Gallinarum biovar Gallinarum (8.74%), and untyped Salmonella species (15.53%). This was the first baseline study of the prevalence of non-typhoidal Salmonella at the live bird market and retail shops of Lahore. Implementation of appropriate control measures is required at both the human side and poultry food production chain to reduce the burden and transmission of the zoonotic Salmonellae.
Topics: Animals; Humans; Pakistan; Prevalence; Meat; Salmonella typhimurium; Transportation
PubMed: 37303147
DOI: 10.12834/VetIt.2392.14037.1 -
Colloids and Surfaces. B, Biointerfaces Jun 2023Salmonella strain is a prevalent pathogen, affecting poultry industry and hence human population around the world. Host-specific pathogen infections including fowl...
Salmonella strain is a prevalent pathogen, affecting poultry industry and hence human population around the world. Host-specific pathogen infections including fowl typhoid, pullorum disease and typhoid fever affects poultry birds, causing huge economic loss worldwide. This study explored the fabrication of immunochromatographic (ICG) strip by colorimetric method integrated with smartphone ColorGrab application for the detection of Salmonella using in-house generated antibodies (Abs) conjugated with gold nanoparticles. The developed point-of-care diagnostic platform was fabricated in-house and tested to detect the presence of Salmonella in a linear range of 10-10 CFU/mL with the limit of detection (LOD) of 10, 10 and 10 CFU/mL respectively, for Salmonella gallinarum (S.gal), Salmonella pullorum (S.pul) and Salmonella enteritidis (S.ent), which was further confirmed by smartphone-based ColorGrab application. The fabricated ICG strips were further validated using spiked fecal, meat, and milk samples which provided results in 10 mins with stability at 4 °C and 37 °C up to 28 days. Hence, the fabricated in-house ICG strip can be used as a portable, cost-effective diagnostic device for rapid detection of Salmonella strains in food samples.
Topics: Animals; Humans; Gold; Point-of-Care Systems; Smartphone; Salmonella Infections, Animal; Poultry Diseases; Metal Nanoparticles; Salmonella; Immunoassay; Chickens
PubMed: 37120932
DOI: 10.1016/j.colsurfb.2023.113319