Biovar Gallinarum Research Articles

Identification of Salmonella enterica biovars Gallinarum and Pullorum and their antibiotic resistance pattern in integrated crop-livestock farms and poultry meats

Due to consumer demand, many conventional poultry farms are now growing poultry without antibiotics or synthetic chemicals. In addition to this, pasture/organic poultry farms have increased significantly in the USA, and they are also antibiotic- and chemical-free. According to recent reports, both antibiotic-free conventional and pasture poultry farmers are facing the re-emergence of bacterial diseases. Bacterial diseases cause higher mortality rates in birds and lead to non-profitable poultry farming. This study investigated the prevalence of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum (S. Gallinarum), the causative agent of fowl typhoid, and Salmonella enterica subsp. enterica serovar Gallinarum biovars Pullorum (S. Pullorum), the causative agent of pullorum disease, within integrated crop-livestock/pasture farm environments and their processed products. Specifically, the study focused on both the pre-harvest period, which includes the conditions and practices on the farm before the crops and livestock are harvested, and the post-harvest period, which encompasses the handling, processing, and storage of the products after harvest. A total of 1286 samples were collected from six farms and adjacent 13 markets to determine the prevalence of S. Gallinarum and S. Pullorum by using both microbiological culture and molecular techniques, specifically PCR. Antimicrobial susceptibility testing was performed using the agar dilution method for the recommended antibiotics as described in the Clinical Laboratory Standards Institute (CLSI). S. Pullorum was detected in 11 samples (2.7%), while S. Gallinarum was found in six samples (1.5%) out of a total of 403 samples at the pre-harvest level. At the post-harvest level, only S. Gallinarum was identified in 14 meat samples out of 883(1.6%) recovered from samples collected from retail markets. Antibiogram showed S. Gallinarum and S. Pullorum to be highly resistant to cephradine, trimethoprim-sulfamethoxazole, amoxicillin, streptomycin, and ampicillin. This data demonstrates that both S. Pullorum and S. Gallinarum are commonly present in farm poultry environments as well as the products sold in the markets, which warrants implementation of regular surveillance and monitoring programmes, as well as potentially requiring future control strategies to reduce S. Pullorum and S. Gallinarum transmission.

Critical Involvement of the Thioredoxin Reductase Gene (trxB) in Salmonella Gallinarum-Induced Systemic Infection in Chickens.

Salmonella enterica serovar Gallinarum biovar Gallinarum (SG) causes fowl typhoid, a notifiable infectious disease in poultry. However, the pathogenic mechanism of SG-induced systemic infection in chickens remains unclear. Thioredoxin reductase (TrxB) is a redox protein crucial for regulating various enzyme activities in Salmonella serovar, but the role in SG-induced chicken systemic infection has yet to be determined. Here, we constructed a mutant SG strain lacking the trxB gene (trxB::Cm) and used chicken embryo inoculation and chicken oral infection to investigate the role of trxB gene in the pathogenicity of SG. Our results showed that trxB::Cm exhibited no apparent differences in colony morphology and growth conditions but exhibited reduced tolerance to H2O2 and increased resistance to bile acids. In the chicken embryo inoculation model, there was no significant difference in the pathogenicity of trxB::Cm and wild-type (WT) strains. In the chicken oral infection, the WT-infected group exhibited typical clinical symptoms of fowl typhoid, with complete mortality between days 6 and 9 post infection. In contrast, the trxB::Cm group showed a 100% survival rate, with no apparent clinical symptoms or pathological changes observed. The viable bacterial counts in the liver and spleen of the trxB::Cm-infected group were significantly reduced, accompanied by decreased expression of cytokines and chemokines (IL-1β, IL-6, IL-12, CXCLi1, TNF-α, and IFN-γ), which were significantly lower than those in the WT group. These results show that the pathogenicity of the trxB-deficient strain was significantly attenuated, indicating that the trxB gene is a crucial virulence factor in SG-induced systemic infection in chickens, suggesting that trxB may become a potentially effective target for controlling and preventing SG infection in chickens.

Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum in Poultry: Review of Pathogenesis, Antibiotic Resistance, Diagnosis and Control in the Genomic Era.

Salmonella enterica subsp. enterica serovar Gallinarum (SG) has two distinct biovars, Pullorum and Gallinarum. They are bacterial pathogens that exhibit host specificity for poultry and aquatic birds, causing severe systemic diseases known as fowl typhoid (FT) and Pullorum disease (PD), respectively. The virulence mechanisms of biovars Gallinarum and Pullorum are multifactorial, involving a variety of genes and pathways that contribute to their pathogenicity. In addition, these serovars have developed resistance to various antimicrobial agents, leading to the emergence of multidrug-resistant strains. Due to their economic and public health significance, rapid and accurate diagnosis is crucial for effective control and prevention of these diseases. Conventional methods, such as bacterial culture and serological tests, have been used for screening and diagnosis. However, molecular-based methods are becoming increasingly important due to their rapidity, high sensitivity, and specificity, opening new horizons for the development of innovative approaches to control FT and PD. The aim of this review is to highlight the current state of knowledge on biovars Gallinarum and Pullorum, emphasizing the importance of continued research into their pathogenesis, drug resistance and diagnosis to better understand and control these pathogens in poultry farms.

High prevalence of antibiotic resistance and biofilm formation in Salmonella Gallinarum.

Antibiotic resistance is an indicator of the passively acquired and circulating resistance genes. Salmonella Gallinarum significantly affects the poultry food industry. The present study is the first study of the S. Gallinarum biofilm in Iran, which is focused on the characterization of the S. Gallinarum serovars and their acquired antibiotic resistance genes circulating in poultry fields in central and northwestern Iran. Sixty isolates of S. Gallinarum serovar were collected from feces of live poultry. The bacteria were isolated using biochemical tests and confirmed by Multiplex PCR. Biofilm formation ability and the antibacterial resistance were evaluated using both phenotypic and genotypic methods. The data were analyzed using SPSS software. According to Multiplex PCR for ratA, SteB, and rhs genes, all 60 S. Gallinarum serovars were Gallinarum biovars. In our study, the antibiotic resistance rate among isolated strains was as follows: Penicillin (100%), nitrofurantoin (80%), nalidixic acid (45%), cefoxitin (35%), neomycin sulfate (30%), chloramphenicol (20%), and ciprofloxacin (5%). All isolates were susceptible to imipenem, ertapenem, ceftriaxone, ceftazidime, and ceftazidime+clavulanic acid. All sixty isolates did not express the resistance genes IMP, VIM, NDM, DHA, blaOXA48, and qnrA. On the other hand, they expressed GES (85%), qnrB (75%), Fox M (70%), SHV (60%), CITM (20%), KPC (15%), FOX (10%), MOXM (5%), and qnrS (5%). All S. Gallinarum isolates formed biofilm and expressed sdiA gene. Considering that the presence of this bacteria is equal to the death penalty to the herd, the distribution of resistance genes could be a critical alarm for pathogen monitoring programs in the region. This study showed a positive correlation between biofilm formation and 50% of tested resistance genes. Also, it was found that the most common circulating S. gallinarum biovars are multidrug-resistant.

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 gallinarum strains from outbreaks of fowl typhoid fever in Southern Africa closely related to SG9R vaccines.

Salmonella enterica subspecies enterica 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. 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. 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 (aceE) and/or lipopolysaccharide 1,2-glucosyltransferase (rfaJ) genes, which are known markers of attenuation, were found in four of the field isolates along with intact spv, 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 yihX gene, insertions in the ybjX and hydH genes, and deletions in the ftsK and sadA 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. 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 aceE and rfaJ genes. A possible new marker of attenuation was identified as a point mutation in the yihX 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.

Characterization of two-component system CitB family in Salmonella enterica serovar Gallinarum biovar Gallinarum

Salmonella enterica serovar Gallinarum biovar Gallinarum is an avian-adapted pathogen causing fowl typhoid and leading to enormous economic loss in the global poultry industry. Two-component systems (TCSs) are crucial for bacteria survival, virulence, sensing and responding to the environment. 23 pairs of TCSs classified into five families were found in S. Gallinarum strain 287/91, of which the CitB family contains three pairs of TCSs, namely CitA/CitB, DcuS/DcuR and DpiB/DpiA, whose functions remained unaddressed. Thus, four mutants of S. Gallinarum strain U20, ΔcitAB (Δcit), ΔdcuSR (Δdcu), ΔdpiBA (Δdpi) and ΔcitABΔdcuSRΔdpiBA (Δ3), were constructed. The results suggested that the CitB family did not affect the growth or the metabolic capacities tested, while different TCSs exerted various effects on biofilm formation and antimicrobial resistance against multiple drug classes. Furthermore, the CitB family negatively impacted the tolerance of environmental stress, contributing to compromised virulence in chicken embryos and in vivo survival of S. Gallinarum. Collectively, this research provided new knowledge of how the CitB family is involved in the pathogenicity of S. Gallinarum.

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.

A new multiplex PCR for the accurate identification and differentiation of Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum.

Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum cause severe chicken salmonellosis, a disease associated with high mortality and morbidity among chickens worldwide. The conventional serotyping and biochemical reactions have been used to identify Salmonella serovars. However, the conventional methods are complicated, time-consuming, laborious, and expensive. Furthermore, it is challenging to distinguish S. Gallinarum and S. Pullorum via biochemical assays and serotyping because of their antigenic similarity. Although various PCR methods were established, a PCR protocol to detect and discriminate S. Gallinarum and S. Pullorum simultaneously is lacking. Herein, a one-step multiplex PCR method was established for the accurate identification and discrimination of S. Pullorum and S. Gallinarum. Three specific genes were used for the multiplex PCR method, with the I137_14445 and ybgL genes being the key targets to identify and differentiate S. Gallinarum and S. Pullorum, and stn being included as a reference gene for the Salmonella genus. In silico analysis showed that the I137_14445 gene is present in all Salmonella serovars, except for S. Gallinarum, and could therefore be used for the identification of S. Gallinarum. A 68-bp sequence deficiency in ybgL was found only in S. Pullorum compared to other Salmonella serovars, and this could therefore be used for the specific identification of S. Pullorum. The developed PCR assay was able to distinguish S. Gallinarum and S. Pullorum among 75 various Salmonella strains and 43 various non-Salmonella pathogens with excellent specificity. The detection limit for the genomic DNA of S. Gallinarum and S. Pullorum was 21.4 pg./μL, and the detectable limit for bacterial cells was 100 CFU. The developed PCR method was used for the analysis of Salmonella isolates in a chicken farm. This PCR system successfully discriminated S. Gallinarum and S. Pullorum from other different Salmonella serovars. The PCR results were confirmed by the conventional serotyping method. The newly established multiplex PCR is a simple, accurate, and cost-effective method for the timely identification and differentiation of S. Pullorum and S. Gallinarum.

Characteristics of systemic infection and host responses in chickens experimentally infected with Salmonella enterica serovar Gallinarum biovar Gallinarum.

Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) is a host-specific pathogen causing systemic infection in poultry, which leads to significant economic losses due to high mortality. However, little is known about the dynamic process of systemic infection and pathogenic characteristics of S. Gallinarum in chickens. In the present study, we developed an oral infection model that reproduces the pathology of S. Gallinarum and clarified the host immune response of the infected chickens. Chickens at 20 days of age orally inoculated at a dose of 108 colony forming unit (CFU) showed typical clinical signs of fowl typhoid and died between 6 and 10 days post infection. The inoculated S. Gallinarum rapidly disseminated to multple organs and the bacterial counts increased in the liver and spleen at 3 days post infection. Pathological changes associated wirh inflammation in the liver and spleen became apparent at 4 days post infection, and increased expression of interferon (IFN)-γ and interleuikin (IL)-12 in the liver and spleen did not observed until 3 days post infection. These results indicate that S. Gallinarum rapidly spread to entire body through intestine, and the low-level of inflammatory responses in the liver during the early stage of infection may contribute to rapid, systemic dissemination of the bacteria. Our infection model and findings will contribute to the better understanding of the pathogenic mechanism of S. Gallinarum, and provide new insights into the prevention and control of fowl typhoid.

Development of an effective vaccination protocol to produce Salmonella-free layer fock

Salmonella infections are of prime importance in the layer chicken industry and pose a significant public health threat. Production of export quality poultry products requires Salmonella-free certification. We developed and applied a vaccination schedule based on a commercial live attenuated Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) vaccine in a layer flock. A flock of 1000 ISA Brown chickens has vaccinated with a lyophilized fowl typhoid live vaccine at definite time intervals. Isolation of Salmonella spp. in cloacal swabs and detection of serum antibody responses were performed using serum plate agglutination (SPA) test and ELISA. At the time of vaccination (16 weeks (w) of age), 50% of the tested birds carried Salmonella spp. in feces. Following booster immunization at 18 w, 21 w, and then every 12 weeks interval, the shedding of Salmonella decreased significantly over time with 40% at 21 w, 10% at 30 w, and 5% each at 42 and 54 w, and no shedding was detected at 66 and 78 w. Biochemical analysis of 32 Salmonella isolates revealed 15.6% (n=5) Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum), 37.5% (n=12) Salmonella Gallinarum, and 46.9% (n=15) paratyphoid causing Salmonella. Of note, the Salmonella spp. detected after 21 w of age belonged to the paratyphoid group. The decreased shedding of bacteria paralleled with increased antibody responses. The antibody level at vaccination (20% positive rate at 16 w) increased over time with a 50% positive rate at 18 w, 80% at 42 w, 90% at 54 w, and 100% at 66 w and 78 w by serum plate agglutination (SPA) test. Similarly, the serum antibody levels of chickens were also measured using ELISA and were similar to the SPA test. In conclusion, the vaccine schedule developed in this study confirmed a high seroconversion and prevented Salmonella shedding in feces. Therefore, a three-month interval vaccination protocol from the pre-laying stage to the last stage of laying is recommended to prevent Salmonella infections in laying flocks.

A one-step closed-tube enzyme-activated blocked probe assay based on SNP for rapid detection of Salmonella Pullorum

Salmonella enterica serovar Gallinarum biovars Pullorum (S. Pullorum) is an infectious bacterial pathogen in the poultry industry that causes systemic pullorum disease. This disease causes great losses in terms of the clinical production and quality of chicken products in breeding farms. However, an acknowledged usable rapid detection method for its specific identification has not been reported, and it is generally difficult to distinguish from fowl typhoid caused by Salmonella enterica serovar Gallinarum biovars Gallinarum. The development of a specific and rapid detection method for this pathogen is therefore needed. In the present study, we targeted the single-nucleotide mutation position 237 of the S. Pullorum rfbS gene to develop an enzyme-activated blocked probe for its clinical rapid detection. The method displayed robust specificity and reproducibility, and it achieved minimal detection limits of 21 copies/μL of copy number and 4.53 pg/μL of genomic DNA. Compared with traditional identification and PCR methods, this method performed better for the detection of 100 clinical actual samples and without false negative results. The entire process can be accomplished in a 1-step closed-tube operation, overcomes the difficulties currently associated with S. Pullorum detection, and provides a specific and rapid method with broad application potential for SNP detection.

Comparison of Humoral Immune Responses to Different Forms of Salmonella enterica Serovar Gallinarum Biovar Gallinarum.

Fowl typhoid is caused by Salmonella enterica serovar Gallinarum biovar Gallinarum (SG), and live attenuated, rough vaccine strains have been used. Both humoral and cellular immune responses are involved in protection, but the humoral responses to different forms of SG antigens are unclear. In this study, we compared humoral responses to a killed oil-emulsion (OE) smooth vaccine (SG002) and its rough mutant vaccine (SR2-N6) strains using proteomics techniques. We identified two immunogenic outer membrane proteins (OmpA and OmpX), and the selected linear epitopes were successfully applied in peptide-ELISA. Our peptide- and total OMP-ELISAs were used to compare the temporal humoral responses to various SG antigens: OE SG002 and SR2-N6; live, killed [PBS-suspension (PS) and OE)] and mixed (live and PS) formulations of another rough vaccine strain (SG 9R); and orally challenge with a field strain. Serum antibodies to the linear epitopes of OmpA and OmpX lasted only for the first 2 weeks, but serum antibodies against OMPs increased over time. The rough strain (SR2-N6) and mixed SG 9R induced higher serum antibody titers than the smooth strain (SG002) and single SG 9R (OE, live and PS SG 9R), respectively. Infection with the field strain delayed the serum antibody response by ~2 weeks. Mucosal immunity was not induced by any formulation, except for infection with the field strain after SG 9R vaccination. Thus, our results may be useful to understand humoral immunity against various SG antigens and to improve vaccine programs and serological diagnosis in the field.

Evidence of vector borne transmission of Salmonella enterica enterica serovar Gallinarum and fowl typhoid disease mediated by the poultry red mite, Dermanyssus gallinae (De Geer, 1778)

BackgroundThe poultry red mite Dermanyssus gallinae (De Geer, 1778) is a major ectoparasite of poultry. Infestations are found in most laying hen farms in Europe, and breeder flocks have also been reported to be affected. Mite infestation has detrimental effects on animal welfare, it causes significant economic losses, and, additionally, D. gallinae is often considered as a vector for pathogens. Despite suspicion of a close relationship between the poultry red mite and Salmonella enterica enterica serovar Gallinarum biovar Gallinarum (serovar Gallinarum), the causative agent of fowl typhoid disease (FT), there has been no definitive proof of mite-mediated transmission. Therefore, an investigation was conducted to determine if D. gallinae-mediated transmission of serovar Gallinarum could be demonstrated among four different hen groups.MethodsTwo groups of 8 hens (A and B) were experimentally infected with serovar Gallinarum in two isolators. After 7 days, when birds showed signs of FT, about 25,000 mites were introduced. After 3 days, mites were harvested and used to infest two other hen groups of 8 (C and D), in two separate isolators. The health status of hens was constantly monitored; detection and quantification of serovar Gallinarum were performed by PCR and qPCR from mites and organs of dead hens. The maximum likelihood estimation of the infection rate and mite vectorial capacity were calculated.ResultsClinical disease was observed in groups infected with serovar Gallinarum (A and B) and in hens of groups C and D infested with mites harvested from the isolators containing groups A and B. In all four groups, serovar Gallinarum was detected from liver, spleen, ovary, and cecum of hens, thus confirming the diagnosis of FT. Mite analysis demonstrated the presence of the pathogen, with an estimated infection rate ranging between 13.72 and 55.21 infected per thousand mites. Vectorial capacity was estimated to be 73.79.ConclusionsMites harvested from birds infected with serovar Gallinarum were shown to carry the mite, and then to transfer serovar Gallinarum to isolated groups of pathogen-free birds that subsequently showed signs of FT. Mite vectorial capacity was high, demonstrating that D. gallinae should be considered an effective vector of FT.

A Sensitive, Highly Specific Novel Isothermal Amplification Method Based on Single-Nucleotide Polymorphism for the Rapid Detection of Salmonella Pullorum.

S. Pullorum (Salmonella enterica serovar Gallinarum biovars Pullorum) is an infectious pathogen that causes the acute systemic disease called Pullorum disease in poultry. This disease causes huge losses to the poultry industry and seriously affects the yield and quality of the chicken product. It is not easily distinguishable with fowl typhoid caused by S. Gallinarum (Salmonella enterica serovar Gallinarum biovars Gallinarum), hence the development of a specific and rapid detection method for this pathogen is highly desired. In this study, we propose a novel single-nucleotide polymorphism (SNP) detection strategy termed loop primer probe-introduced loop-mediated isothermal amplification (LP-LAMP) for S. Pullorum detection. Based on the original primer sets, we targeted the nucleotide position 237 of the rfbS gene sequence to design a new modified loop-primer probe with a ribonucleotide insertion, where activity of the enzyme ribonuclease H2 (RNase H2) is only activated when the probe is perfectly complementary, leading to the hydrolytic release of a quencher moiety and thus an amplified signal. The method exhibits robust specificity and a low detection limit as the copy number and genomic DNA is 21 copies/μL and 4.92 pg/μL, respectively. This method showed great performance in real sample testing of 130 samples of embryos, livers, and anal swabs from chickens in poultry farms. The experimental results are mainly consistent with traditional identification methods and a PCR method reported in the past. However, the other two methods still contain some false negative results, while our method is without miss detection. The entire closed-tube reaction process can be accomplished within 40 min at a constant temperature (61°C) without the need for expensive instruments or a complicated operation. The LP-LAMP strategy established in this study not only overcomes the existing difficulties of S. Pullorum rapid detection, it also provides a novel, sensitive, and highly specific detection platform for SNPs that is suitable for clinical use.

Characterization of Salmonella spp. isolated from chickens in Central China

BackgroundSalmonella is an important zoonotic pathogen, and chickens are one of its main hosts. Every year, Salmonella infections pose a serious threat to the poultry industry in developing countries, especially China. In this study, a total of 84 Salmonella isolates recovered from sick and healthy-looking chickens in central China were characterized by serotyping, MLST-based strain typing, presence of potential virulence factors, and antimicrobial resistance profiles.ResultData showed that the main serotypes of Salmonella isolates in central China were Salmonella enterica serovar Gallinarum biovar Pullorum, Salmonella enterica serovar Gallinarum biovar Gallinarum, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium, and among them, S. Pullorum was the dominant type in both sick and healthy-looking chickens, accounting for 43.9 and 46.5%, respectively, while S. Enteritidis was only found in healthy-looking chickens. All isolates exhibited higher resistance rates to ampicillin (97.6%), tetracycline (58.3%) and colistin (51.2%), and among these isolates, 49.5% were resistant to more than three drugs in different combinations. S. Enteritidis was the most severe multidrug-resistant serotype, which showed higher resistance rates to colistin, meropenem and ciprofloxacin. Multilocus sequence typing (MLST) revealed that S. Gallinarum and S. Enteritidis isolates were clustered in clade 1, which belonged to two and one STs, respectively. All S. Typhimurium isolates were clustered in clade 3, and belonged to three STs. However, S. Pullorum were distributed in three clades, which belonged to 7 STs. Twenty-seven virulence-associated genes were detected, and expected cdtB, which was absent in all the isolates, the other 26 genes were conserved in the closely related Salmonella serogroup D (S. Enteritidis, S. Pullorum, and S. Gallinarum).ConclusionSalmonella serogroup D was the major subgroup, and S. Pullorum was the most common type in sick and healthy-looking chickens in central China. Drug resistance assays showed serious multiple antimicrobial resistances, and S. Enteritidis was the most severe drug-resistant serotype. MLST showed that there was correlation between serotypes and genotypes in most Salmonella isolates, except S. Pullorum, which showed complicated genetic diversity firstly. These results provide important epidemiological information for us to control Salmonella in chickens.

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.

Molecular Detection and Characterization of Salmonella Gallinarum From Poultry Farms in Brazil

SUMMARY Salmonella is a bacterium usually associated with mild gastroenteritis in chickens, but strains of the serotype Gallinarum can result in 2 severe systemic chicken diseases: fowl typhoid (by biovar Gallinarum) and pullorum disease (by biovar Pullorum). These diseases can be controlled by immunization with a specific vaccine (SG 9R). The aim of this study was to characterize recent Salmonella Gallinarum isolates by molecular analyses, as well as detect field and vaccine strains in clinical and environmental samples from poultry flocks. Field isolates of S. Gallinarum (n = 18) and other serotypes (n = 117) were obtained from Brazilian poultry farms between 2012 and 2015. In addition, clinical and environmental samples of poultry flocks (vaccinated or not) with fowl typhoid were obtained in a 3-year period (2013 to 2015). The results demonstrated that specific PCR targeted on speC and glgC genes and PCR-restriction fragment length polymorphisms based on fliC and sefA genes detected all the 18 isolates, 10 identified as field isolates of biovar Gallinarum and 8 as biovar Pullorum. All isolates of other serotypes presented negative results. In addition, PCR tests were performed directly from the pre-enrichment broth of the bacteriological procedures. Vaccine SG 9R and field S. Gallinarum strains were detected in samples with (n = 19) and without (n = 59) vaccination. These results demonstrate the use of PCR tests for detection of S. Gallinarum in clinical and environmental Brazilian poultry samples.

Molecular evolution of Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum in the field

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (SG) causes fowl typhoid (FT) and substantial economic loss in Korea due to egg drop syndrome and mortality. Despite the extensive use of vaccines, FT still occurs in the field. Therefore, the emergence of more pathogenic SG or the recovered pathogenicity of a vaccine strain has been suspected. SpvB, an ADP-ribosyl transferase, is a major pathogenesis determinant, and the length of the polyproline linker (PPL) of SpvB affects pathogenic potency. SG strains accumulate pseudogenes in their genomes during host adaptation, and pseudogene profiling may provide evolutionary information. In this study, we found that the PPL length of Korean SG isolates varied from 11 to 21 prolines and was longer than that of a live vaccine strain, SG 9R (9 prolines). According to growth competition in chickens, the growth of an SG isolate with a PPL length of 17 prolines exceeded that of an SG isolate with a PPL length of 15 prolines. We investigated the pseudogenes of the field isolates, SG 9R and reference strains in GenBank by resequencing and comparative genomics. The pseudogene profiles of the field isolates were notably different from those of the foreign SG strains, and they were subdivided into 7 pseudogene subgroups. Collectively, the field isolates had gradually evolved by changing PPL length and acquiring additional pseudogenes. Thus, the characterization of PPL length and pseudogene profiling may be useful to understand the molecular evolution of SG and the epidemiology of FT.

English

Salmonella enterica subsp. enterica ser. Gallinarum biovar. gallinarum (S. gallinarum) is the causative agent of fowl Typhoid, a bacterial disease that affects domestic and wild birds. For many years, S. gallinarum was defined as an aflagellated bacterium, that is, non-motile. However, the closely related strain Salmonella pullorum is capable of expressing a flagellum-like filament. Since these bacteria are practically identical, it was determined if S. gallinarum expresses flagellum-like filament under different culture conditions in order to increase the basic knowledge of this bacterium. The motility of S. gallinarum was evaluated in different culture media (GI, S and nutrient media), and visualized the bacterial filaments using electron microscopy. fliC was identified in some of the studied strains; subsequently, sequencing analysis of fliC gene shown 98% of identity with that in S. Typhimurium. In the present study, the presence of a flagellum-like structure was demonstrate in different strains of S. gallinarum using electron microscopy. In addition, the fliC gene was amplified, which allowed us to suggest that this bacterium is capable to shows a flagellum under certain culture conditions, similar to that reported for S. pullorum. Key words: fliC, flagella, S. gallinarum, motility, medium-dextrose.