Nifuratel reduces Salmonella survival in macrophages by extracellular and intracellular antibacterial activity.

Salmonella are intracellular bacterial pathogens for which, as with many of the other Enterobacteriaceae, antibiotic resistance is becoming an increasing problem. New antibiotics are being sought as recommended by the World Health Organization and other international institutions. These must be able to penetrate macrophages, and infect the major host cells and the Salmonella-containing vacuole. This study reports screening a small library of Food and Drug Administration (FDA)-approved drugs for their antibacterial effect in macrophages infected with a rapid-multiplying mutant of Salmonella Enteritidis. The most effective drug that was least toxic for macrophages was Nifuratel, a nitrofuran antibiotic already in use for parasitic infections. In mice, it provided 60% protection after oral infection with a lethal S. Enteritidis dose with reduced bacterial numbers in the tissues. It was effective against different serovars, including multidrug-resistant strains of Salmonella Typhimurium, and in macrophages from different host species and against Listeria monocytogenes and Shigella flexneri. It reduced IL-10 and STAT3 production in infected macrophages which should increase the inflammatory response against Salmonella. IMPORTANCE Salmonella can keep long-term persistence in host's macrophages to evade cellular immune defense and antibiotic attack and exit in some condition and reinfect to cause salmonellosis again. In addition to multidrug resistance, this infection circle causes Salmonella clearance difficult in the host, and so there is a great need for new antibacterial agents that reduce intramacrophage Salmonella survival to block endogenous Salmonella reinfection.

Safety of the Salmonella enterica serotype Dublin strain Sdu189-derived live attenuated vaccine-A pilot study.

Salmonella enterica serovar Dublin (S. Dublin) is an important zoonotic pathogen with high invasiveness. In the prevention and control of the Salmonella epidemic, the live attenuated vaccine plays a very important role. To prevent and control the epidemic of S. Dublin in cattle farms, the development of more effective vaccines is necessary. In this study, we constructed two gene deletion mutants, Sdu189ΔspiC and Sdu189ΔspiCΔaroA, with the parental strain S. Dublin Sdu189. The immunogenicity and protective efficacy were evaluated in the mice model. First, both mutant strains were much less virulent than the parental strain, as determined by the 50% lethal dose (LD50) for specific pathogen-free (SPF) 6-week-old female BALB/c mice. Second, the specific IgG antibody level and the expression level of cytokine TNF-α, IFN-γ, IL-4, and IL-18 were increased significantly in the vaccinated mice compared to the control group. In addition, the deletion strains were cleared rapidly from organs of immunized mice within 14 d after immunization, while the parental strain could still be detected in the spleen and liver after 21 d of infection. Compared with the parental strain infected group, no obvious lesions were detected in the liver, spleen, and cecum of the deletion strain vaccinated groups of mice. Immunization with Sdu189ΔspiC and Sdu189ΔspiCΔaroA both provided 100% protection against subsequent challenges with the wild-type Sdu189 strain. These results demonstrated that these two deletion strains showed the potential as live attenuated vaccines against S. Dublin infection. The present study established a foundation for screening a suitable live attenuated Salmonella vaccine.

The Loss of focA Gene Increases the Ability of Salmonella Enteritidis to Exit from Macrophages and Boosts Early Extraintestinal Spread for Systemic Infection in a Mouse Model.

Salmonella Enteritidis (SE) can spread from the intestines to cause systemic infection, mainly involving macrophages. Intramacrophage Salmonella exits and reinfects neighboring cells, leading to severe disease. Salmonella genes involved in exiting from macrophages are not well understood or fully identified. A focA::Tn5 mutant was identified by an in vitro assay, with increased ability to exit from macrophages. A defined SEΔfocA mutant and its complemented derivative strain, SEΔfocA::focA, were constructed to confirm this phenotype. Although the lethal ability of focA mutants was similar to that of the parental SE in mice, it was isolated earlier from the liver and spleen than the parental SE. focA mutants induced higher levels of proinflammatory IL-12 and TNF-α compared with the parental SE and SEΔfocA::focA. focA mutants showed higher cytotoxicity and lower formate concentrations than SE and SEΔfocA::focA, whereas there was no change in pyroptosis, apoptosis and flagella formation ability. These current data suggest that the focA gene plays an important role in regulating intramacrophage Salmonella exiting and extraintestinal spread in mice, although the specific mechanism requires further in-depth studies.

A phage for the controlling of Salmonella in poultry and reducing biofilms.

As a natural alternative to traditional antimicrobials, phages are being recognised as highly effective control agents for Salmonella and other foodborne bacteria. Due to the high diversity of Salmonella serotypes and the emergence of phage-resistant strains, attempting to isolate more widespread, strictly lytic Salmonella phages is highly warranted. In this study, a lytic phage, LP31, was isolated from poultry faecal samples. Transmission electron microscopy revealed that the phage had a polyhedral head and a retraction-free tail, indicative of the Siphoviridae family. Adsorption rate experiments showed that LP31 required the participation of lipopolysaccharides, but not flagella, during phage adsorption. Host profile identification showed that LP31 could lyse most Salmonella Enteritidis (S. Enteritidis) (96.15%, N = 104) and Salmonella Pullorum (S. Pullorum) (96.67%, N = 60). Initial applications found that LP31 reduced the concentration of static S. Enteritidis on metal surfaces (0.951 log10 cfu/ml) and in the faeces of chicks (2.14 log10 cfu/g). Notably, LP31 could almost completely remove biofilms formed by S. Enteritidis and S. Pullorum in 1 h. These findings suggest that LP31 has a good prevention and control effect against biofilms and planktonic antibiotic-resistant Salmonella, and is therefore a potentially promising biocontrol agent for controlling the spread of Salmonella in the poultry and food processing industries.

Safety and protective efficacy of Salmonella Pullorum spiC and rfaH deletion rough mutant as a live attenuated DIVA vaccine candidate.

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.

Revisiting Persistent Salmonella Infection and the Carrier State: What Do We Know?

One characteristic of the few Salmonella enterica serovars that produce typhoid-like infections is that disease-free persistent infection can occur for months or years in a small number of individuals post-convalescence. The bacteria continue to be shed intermittently which is a key component of the epidemiology of these infections. Persistent chronic infection occurs despite high levels of circulating specific IgG. We have reviewed the information on the basis for persistence in S. Typhi, S. Dublin, S. Gallinarum, S. Pullorum, S. Abortusovis and also S. Typhimurium in mice as a model of persistence. Persistence appears to occur in macrophages in the spleen and liver with shedding either from the gall bladder and gut or the reproductive tract. The involvement of host genetic background in defining persistence is clear from studies with the mouse but less so with human and poultry infections. There is increasing evidence that the organisms (i) modulate the host response away from the typical Th1-type response normally associated with immune clearance of an acute infection to Th2-type or an anti-inflammatory response, and that (ii) the bacteria modulate transformation of macrophage from M1 to M2 type. The bacterial factors involved in this are not yet fully understood. There are early indications that it might be possible to remodulate the response back towards a Th1 response by using cytokine therapy.

Salmonella Pullorum spiC mutant is a desirable LASV candidate with proper virulence, high immune protection and easy-to-use oral administration.

Live attenuated Salmonellavaccine (LASV) is considered to be an effective contributory measure during the control of Salmonella infection. A Salmonella Pullorum spiC mutant was evaluated comprehensively as a LASV candidate (LASV-p) for broilers in terms of safety and immunogenicity. LASV-p was adminstered to 3-day broilers by intramuscular injection. The LD50 increased 126 fold, and no tissue lesions were observed in the liver, spleen and cecum, in comparison with the control group inoculated with PBS and a passive group by wild-type Salmonella. Growth rates of all broilers were normal and not affected. LASV-p persisted in vivo until 21 days in liver, 28 days in spleen and 35 days in feces, and induced high levels of humoral IgG and mucosal IgA. Cellular immunity was also stimulated in the form of antigen-specific lymphocyte proliferation and higher counts of CD3+CD8+ T cells and increased expression of mRNA of Th1 cytokines, IFN-γ and IL-2, in the early stage, and Th2 cytokines, IL-4 and IL-10, in the later stages. LASV-p provided at least 90% immuneprotection against a wild-type Salmonella Pullorum and cross-protection in different degree against other Salmonella searovars. Oral vaccine could also offer high immune protection of 87.5%. These results indicated that LASV-p vaccine candidate had a high level of safety and immune protection and it might be developed as a novel easy-to-use oral vaccine to improve poultry health in the future.

High genetic similarity of Salmonella Enteritidis as a predominant serovar by an independent survey in 3 large-scale chicken farms in China.

Salmonella Enteritidis (SE) are important zoonotic pathogens, and can be easily transferred to humans by contaminated animal products. Epidemic surveys of SE are necessary in current modern large-scale chicken farms. In this study, Salmonella strains were isolated from possibly infected samples collected at 3 independent farms, and their serotype, drug resistances, virulence genes, and genetic similarity were analyzed by molecular genetic analysis technologies including multilocus sequence typing (MLST), clustered regularly interspaced short palindromic repeats (CRISPR), pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). A total of 346 Salmonella strains were isolated from 3,598 samples (9.61%); 329 isolates were identified as SE (95.09%) and 308 isolates were multidrug resistant (93.62%). Virulotyping based on 6 virulence genes showed high similarity in SE isolates of each farm, with the exception of 2 isolates. All SE isolates were found to be the same ST11 type by MLST, and 22 strains of 150 SE isolates selected at random were found to belong to 1 cluster by PFGE and the same SET1 type by CRISPR. WGS results further revealed that these isolates belonged to the same clonal cluster, with high genetic similarity of 99.80 to 100.00%. All these results indicated that these SE isolates were overwhelmingly dominant and demonstrated high genetic similarity, which revealed that the same SE clone might be transmitted in these farms.

WbaP is required for swarm motility and intramacrophage multiplication of Salmonella Enteritidis spiC mutant by glucose use ability.

Salmonella spp. can survive and replicate in macrophage cells to cause persistent infection, SpiC is a necessary T3SS effector, but its pathogenic mechanism is still not known completely. In our study, Salmonella Enteritidis spiC mutant (SEΔspiC) was found to have stronger swarming motility and intramacrophage hyperproliferation which was closely related to glucose metabolism. SEΔspiC wbaP::Tn5 mutant was screened out by transposon mutagenesis, which had weaker swarming motility and intramacrophage replication ability than SEΔspiC in the presence of glucose. Bioinformatics displayed that undecaprenyl-phosphate galactose phosphotransferase (Wbap), encoded by wbaP gene, was a key enzyme for glucose metabolism and Lipopolysaccharide(LPS) synthesis, which confirmed our outcome that Wbap was involved in intramacrophage replication ability by glucose use in addition to swarming motility based on SEΔspiC. This discovery will further promote the understanding of the interaction between wbaP gene and spiC gene and the intracellular Salmonella replication mechanism.

Multiple PCR assay based on the cigR gene for detection of Salmonella spp. and Salmonella Pullorum/Gallinarum identification.

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.

A McAb-Based Direct Competitive ELISA to Detect O:9 Salmonella Infection in Chicken.

Salmonella enteritidis and Salmonella pullorum belonging to Group O9 Salmonella are major causative agents of infectious diseases in chicken. O9 antigen as a part of lipopolysaccharide (LPS) is a predominant detected target for Salmonella infection. To identify the infection, an anti-O9 monoclonal antibody (McAb)-based direct competitive enzyme-linked assay (O9 Dc-ELISA) was developed after constraints were optimized; the establishment and application of O9 Dc-ELISA, compared to two commercial kits and plate agglutination test (PAT), showed that O9 Dc-ELISA could screen out more positive samples than the PAT method could and produce the same agreement rates with commercial kits in terms of sensitivity in addition to strong specificity to clinical serum samples.

Evaluation of the Safety and Protection Efficacy of spiC and nmpC or rfaL Deletion Mutants of Salmonella Enteritidis as Live Vaccine Candidates for Poultry Non-Typhoidal Salmonellosis.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a host-ranged pathogen that can infect both animals and humans. Poultry and poultry products are the main carriers of S. Enteritidis, which can be transmitted to humans through the food chain. To eradicate the prevalence of S. Enteritidis in poultry farms, it is necessary to develop novel vaccines against the pathogen. In this study, we constructed two vaccine candidates, CZ14-1∆spiC∆nmpC and CZ14-1∆spiC∆rfaL, and evaluated their protective efficacy. Both mutant strains were much less virulent than the parental strain, as determined by the 50% lethal dose (LD50) for three-day-old specific-pathogen free (SPF) White Leghorns and Hyline White chickens. Immunization with the mutant candidates induced highly specific humoral immune responses and expression of cytokines IFN-γ, IL-1ß, and IL-6. In addition, the mutant strains were found to be persistent for almost three weeks post-infection. The survival percentages of chickens immunized with CZ14-1∆spiC∆nmpC and CZ14-1∆spiC∆rfaL reached 80% and 75%, respectively, after challenge with the parental strain. Overall, these results demonstrate that the two mutant strains can be developed as live attenuated vaccines.

Immunogenic potential and protective efficacy of a sptP deletion mutant of Salmonella Enteritidis as a live vaccine for chickens against a lethal challenge.

Salmonella Enteritidis (SE) is a highly adapted pathogen causing severe economic losses in the poultry industry worldwide. Chickens infected by SE are a major source of human food poisoning. Vaccination is an effective approach to control SE infections. This study evaluated the immunogenicity and protective efficacy of a SE sptP deletion mutant (C50336ΔsptP) as a live attenuated vaccine (LAV) candidate in chickens. 14 day-old specific pathogen-free (SPF) chickens were intramuscularly immunized with various doses of C50336ΔsptP. Several groups of chickens were challenged with the virulent wild-type SE strain Z-11 via the same route at 14 days post vaccination. Compared to the control group, the groups vaccinated with 1 × 106, 1 × 107 and 1 × 108 colony-forming units (CFU) of C50336ΔsptP exhibited no clinical symptoms after immunization. Only slight pathological changes occurred in the organs of the 1 × 109 CFU vaccinated group. C50336ΔsptP bacteria were cleared from the organs of immunized chickens within 14 days after vaccination. Lymphocyte proliferation and serum cytokine analyses indicated that significant cellular immune responses were induced after the vaccination of C50336ΔsptP. Compared to the control group, specific IgG antibody levels increased significantly in vaccinated chickens, and the levels increased markedly after the challenge. The 1 × 107, 1 × 108, and 1 × 109 CFU vaccinated chickens groups showed no clinical symptoms or pathological changes, and no death after the lethal challenge. Whereas severe clinical signs of disease and pathological changes were observed in the control group chickens after the challenge. These results suggest that a single dose of C50336ΔsptP could be an effective LAV candidate to against SE infection in chickens.

Controversy Surrounding the Function of SpiC Protein in Salmonella: An Overview.

Salmonella is an important pathogenic microorganism that can infect humans and animals and has been studied globally as a model microorganism for its pathogenesis. The SpiC protein of T3SS2 is a significant factor that has been studied for almost 20 years, but to date, the function/effect of SpiC in the pathogenesis of Salmonella has not been completely understood. There is controversy over the functions of SpiC protein in the literature. Thus, an overview of the literature on SpiC protein is provided here which highlights expression features of SpiC protein and its various functions and effect.

Salmonella Coiled-Coil- and TIR-Containing TcpS Evades the Innate Immune System and Subdues Inflammation.

Toll-like receptors (TLRs) activate innate immunity via interactions between their Toll/interleukin-1 (IL-1) receptor (TIR) domain and downstream adaptor proteins. Here we report that Salmonella Enteritidis produces a secreted protein (TcpS) that contains both a TIR domain and a coiled-coil domain. TcpS blocks MyD88- and TRIF-mediated TLR signaling, inhibits inflammatory responses, and promotes bacterial survival. Early-stage immune evasion by TcpS results in severe tissue damage in the late stage of infection and contributes to Salmonella virulence. TcpS-derived peptides inhibit nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation and reduce lipopolysaccharide (LPS)-elicited systemic inflammation. Therapeutic peptide administration alleviates weight loss of mice infected with H1N1 influenza. Importantly, maximal TcpS-mediated TLR inhibition requires the critical TIR-TcpS residues Y191 and I284, as well as TcpS homodimerization via its N-terminal coiled-coil domain. Our study unveils a mechanism in which TcpS suppresses innate immunity via both its homodimerization and interaction with MyD88. TcpS is also a potential therapeutic agent for inflammation-associated diseases.

Molecular cloning and functional analysis of TRAF6 from Yangzhou great white goose Anser anser.

TNF receptor-associated factor 6 (TRAF6) is an adaptor protein and an E3 ubiquitin ligase mediating multiple cell signaling pathway activation in a context-dependent manner. TRAF6 plays critical roles in innate immune response and regulates function of antigen-presenting cells. Here, we cloned the goose TRAF6 (goTRAF6) gene from a healthy Yangzhou great white goose (Anser anser), which had a typical TRAF structure and shared a high-sequence identity with TRAF6 of other birds. Quantitative real-time PCR revealed that goTRAF6 mRNA was broadly expressed in all the studied tissues, with highest expression in the heart and pectoral muscle. Overexpression of goTRAF6 caused NF-κB activation in a dose-dependent manner and substantially upregulated IFN-ß expression in HEK293T cells. Following Toll-like receptor (TLR) ligand stimulation of goose peripheral blood mononuclear cells, goTRAF6 and downstream inflammatory cytokine mRNA levels considerably up-regulated, especially at early stages. Salmonella Enteritidis challenge caused overexpression of goTRAF6 and cytokine mRNA in all the examined organs. These findings demonstrated that goTRAF6 played a substantial role in TLR-TRAF6 signaling cascade, and further contributed to the antibacterial-responses in host.

The SseL protein inhibits the intracellular NF-κB pathway to enhance the virulence of Salmonella Pullorum in a chicken model.

To persist in the host, Salmonella is known to facultatively parasitize cells to escape the immune response. Intracellular Salmonella enterica can replicate using effector proteins translocated across the Salmonella-containing vacuolar membrane via a type III secretion system (T3SS) encoded by Salmonella pathogenicity island-2 (SPI-2). One of these factors, Salmonella secreted factor L (SseL), is a deubiquitinase that contributes to the virulence of Salmonella Typhimurium in mice by inhibiting the cellular NF-κB inflammatory pathway. However, the nature of its effect on the NF-κB pathway is controversial, and little research has been performed in other animal models. In this study, the SseL of Salmonella Pullorum was studied, and chickens were used as an infection model. An sseL gene deletion strain, a complementation strain and a eukaryotic expression plasmid were used to clarify the means by which SseL regulates Salmonella virulence and the cellular inflammatory response. SseL significantly enhanced the virulence of Salmonella Pullorum in chickens and suppressed activation of the cellular NF-κB pathway, thus inhibiting cellular inflammatory cytokine expression.

Induction of arthritis in chickens by infection with novel virulent Salmonella Pullorum strains.

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.

A new PCR assay based on the new gene-SPUL_2693 for rapid detection of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum.

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.

Analyses of prevalence and molecular typing reveal the spread of antimicrobial-resistant Salmonella infection across two breeder chicken farms.

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.