ABSTRACT: A total of 110 broilers from 11 flocks were tested for Helicobacter pullorum by polymerase chain reaction; positive samples were reexamined with a conventional isolation method. H. pullorum isolates were examined by amplified fragment length polymorphism (AFLP) fingerprinting for interstrain genetic diversity and relatedness. Sixteen isolates from cecal samples from 2 different flocks were obtained. AFLP analysis showed that these isolates and 4 additional isolates from a different flock clustered according to their origin, which indicates that H. pullorum colonization may occur with a single strain that disseminates throughout the flock. Strains isolated from different hosts or geographic sources displayed a distinctive pattern. H. pullorum is present in approximately one third of live chickens in Belgium and may represent a risk to human health.
Project description:Helicobacter pullorum represents a potential food-borne pathogen, and avian species appear to be a relevant reservoir of this organism. In this study, the prevalence of H. pullorum was investigated at 30 conventional farms where 169 ceca from 34 flocks were tested, at eight organic farms where 39 ceca from eight flocks were tested, and at seven free-range farms where 40 ceca from eight flocks were tested. All of the ceca were obtained from healthy broiler chickens. Moreover, amplified fragment length polymorphism, pulsed-field gel electrophoresis, and automated ribotyping were employed to estimate the levels of genetic variability of H. pullorum broiler isolates within and between flocks. Overall, Gram-negative, slender, curved rods, identified as H. pullorum by PCR, were isolated at 93.3% of the farms tested. The percentage of positive free-range farms (54.2%) was significantly lower than that of conventional (100%) or organic (100%) farms (P < 0.001). The level of within-flock genetic variability, calculated as the number of flocks colonized by isolates genetically different by all of the typing methods, was 34.9%. Isolates showing identical profiles by each typing method were observed in 11.6% of the flocks, but they were never detected between flocks. However, groups of isolates clustered together with an overall similarity level of ?85%. Our results suggest that even though a high level of genetic variability is attributable to H. pullorum broiler isolates, their hierarchical genotyping produces data useful for epidemiological investigations.
Project description:BACKGROUND:Mycoplasma synoviae (MS) is known to cause Eggshell Apex Abnormality (EAA) syndrome characterized by an altered shell surface with increased translucency on the apex. However, no large-scale studies have been conducted to obtain prevalence data of EAA and MS isolates associated to this syndrome. This manuscript reports the results of two field studies performed in the French poultry industry (2015-2017): focusing mainly on investigation of presence and prevalence of EAA in different types of laying hen flocks (phase 1), and isolation of MS strains from EAA-infected flocks (phase 2). RESULTS:The first survey included 77 farms of commercial layers in three French egg-production regions, hosting 40 flocks in alternative systems (ALT) and 56 in furnished cages (FC). Seven flocks (4 FC and 3 ALT) presented EAA clinical signs, giving a prevalence of 7.3% in this studied sample. A second independent field study was conducted to identify MS by in vitro cultivation and PCR in samples from 28 flocks with clinical signs of EAA. Different types of biological specimens were collected in EAA-affected flocks and submitted to the laboratory. M. synoviae was detected in 25/28 flocks, from both production systems (5/5 ALT and 20/23 FC). Detection of MS was significantly higher in tracheal swabs (59%) than in cloacal (10.5%), albumen (3.6%) and egg yolk (1.1%) swabs. It is worth to mention that attempts to clone MS from positive samples were often hampered by the presence of another Mycoplasma species, which showed fast growing behaviour in the selective media used in this study (Frey Medium 4 and Frey Medium 4 supplemented with erythromycin). The use of MALDI-TOF mass spectrometry in combination with next-generation sequencing (NGS) results allowed the identification of this fast growing mycoplasma as Mycoplasma pullorum, which was detected in 14 of the 25 (56%) MS-positive flocks. CONCLUSIONS:These results confirmed the presence of the EAA syndrome in MS-positive flocks of layers in France, reared in different regions and in different production systems (ALT and FC). Studies need to be conducted to test whether M. pullorum may influence the expression of clinical signs of EAA in MS-infected layer farms.
Project description:BACKGROUND:Pullorum disease, caused by Salmonella enterica serovar Pullorum (S. Pullorum), is one of the most important bacterial infections in the poultry industry in developing countries, including China. To examine the prevalence and characteristics of S. Pullorum, the Multilocus Sequence Typing (MLST) genotypes, fluoroquinolones resistance, and biofilm-forming abilities of S. Pullorum isolates were investigated, collected from 2011 to 2016 in China. RESULTS:Thirty S. Pullorum isolates collected from 2011 to 2016 were analyzed. Quinolones susceptibility testing showed that 90% of the isolates were resistant to the first generation of quinolines nalidixic acid, but the resistance rates to different fluoroquinolones agents were lower than 13.3%; for some there was even no resistance. Multilocus sequence typing (MLST) showed that ST-92 was the dominating genotype, accounting for 90.0% of all S. pullorum strains. The remaining three isolates were of the new reported sequence type ST-2151. Interestingly, the Asp87Gly substitution in quinolone resistance-determining regions (QRDR) of GyrA was only observed in the three strains of ST-2151, suggesting a potential correlation between Asp87Gly substitution and sequence type (p?<?0.05). However, Asp87Gly substitution could not confer the resistant to ofloxacin and ciprofloxacin of these isolates. The plasmid-mediated quinolone resistance (PMQR) gene was not found in any of the tested isolates. Furthermore, an assay measuring biofilm-forming abilities showed that 46.7% of the isolates were non-biofilm producers, while 53.3% could form very weak biofilms, which might explain the relatively lower resistance to fluoroquinolones. CONCLUSIONS:We reported a high resistance rate to the first generation of quinolines nalidixic acid and relatively low resistance rates to fluoroquinolones in S. Pullorum isolates. In addition, weak biofilm-forming abilities were found, which might be an important reason of the low fluoroquinolones resistance rates of S. Pullorum isolates. ST-92 was the dominating genotype demonstrated by MLST, and the new sequence type ST-2151 showed a potential correlation with Asp87Gly substitution in QRDR of GyrA. We believe the characterization of these S. Pullorum isolates will be helpful to develop prevention and control strategies.
Project description:BACKGROUND:Footrot and interdigital dermatitis are endemic infectious diseases in all sheep farming regions, impairing welfare and production. The development of efficacious vaccines against the primary causative pathogen has been hampered by the extensive antigenic diversity of Dichelobacter nodosus. Understanding the heterogeneity of the pathogen within and between flocks is essential if the feasibility of bespoke vaccine production is to be assessed for use in the U.K. RESULTS:In this study 56 ewe and lamb isolates from 9 flocks were compared by D. nodosus serogroup and Multi Locus Sequence Type which provides significantly enhanced discriminatory power for molecular epidemiology. Serogroup heterogeneity between flocks ranged from two to five unique serogroups per flock. Three flocks contained isolates of two serogroups, two flocks contained isolates of three serogroups and one flock included isolates of five serogroups. Analysis of 25 isolates from one flock with high prevalence of lameness, identified that serogroup and sequence type was significantly correlated with age. Significantly higher proportion of lambs were infected with serogroup B (principally ST85) as opposed to serogroup H (principally ST86), which predominated amongst adult sheep. CONCLUSIONS:Genomic heterogeneity of the pathogen was significantly lower within flock compared to heterogenicity observed between flocks. Furthermore, this study indicates that within a flock, the host-pathogen dynamics and susceptibility to particular D. nodosus strains may be age dependent.
Project description:The diseases caused by Salmonella Gallinarum and S. Pullorum in chickens known as fowl typhoid and pullorum disease, respectively, pose a great threat to the poultry industry mainly in developing countries, since they have already been controlled in the developed ones. These bacteria are very similar at the genomic level but develop distinct host-pathogen relationships with chickens. Therefore, a deep understanding of the molecular mechanisms whereby S. Gallinarum and S. Pullorum interact with the host could lead to the development of new approaches to control and, perhaps, eradicate both diseases from the chicken flocks worldwide. Based on our previous study, it was hypothesised that metabolism-related pseudogenes, fixed in S. Pullorum genomes, could play a role in the distinct host-pathogen interaction with susceptible chickens. To test this idea, three genes (idnT, idnO and ccmH) of S. Gallinarum str. 287/91, which are pseudogenes on the S. Pullorum chromosomes, were inactivated by mutations. These genetically engineered strains grew well on the solid media without any colony morphology difference. In addition, similar growth curves were obtained by cultivation in M9 minimal medium containing D-gluconate as the sole carbon source. Infection of chickens with idnTO mutants led to increased numbers of bacteria in the livers and spleens at 5 days post-infection, but with slightly decreased heterophil infiltration in the spleens when compared to the wild-type strain. On the other hand, no significant phenotypic change was caused by mutation to ccmH genes. Apart from the above-mentioned alterations, all S. Gallinarum strains provoked similar infections, since mortality, clinical signs, macroscopic alterations and immune response were similar to the infected chickens. Therefore, according to the model applied to this study, mutation to the idnTO and ccmH genes showed minor impact on the fowl typhoid pathogenesis and so they may be relics from the ancestor genome. Our data hints at a more complex mechanism driving the distinct host-pathogen interaction of S. Gallinarum/Pullorum with chickens than differential inactivation of a few genes.
Project description:Salmonella enterica subspecies enterica serovar Gallinarum biovar Pullorum (S Pullorum) is the etiological agent of pullorum disease, causing white diarrhea with high mortality in chickens. There are many unsolved issues surrounding the epidemiology of S Pullorum, including its origin and transmission history as well as the discordance between its phenotypic heterogeneity and genetic monomorphism. In this paper, we report the results of whole-genome sequencing of a panel of 97 S Pullorum strains isolated between 1962 and 2014 from four countries across three continents. We utilized 6,795 core genome single nucleotide polymorphisms (SNPs) to reconstruct a phylogenetic tree within a spatiotemporal Bayesian framework, estimating that the most recent common ancestor of S Pullorum emerged in ?914 CE (95% confidence interval [95%CI], 565 to 1273 CE). The extant S Pullorum strains can be divided into four distinct lineages, each of which is significantly associated with geographical distribution. The intercontinental transmissions of lineages III and IV can be traced to the mid-19th century and are probably related to the "Hen Fever" prevalent at that time. Further genomic analysis indicated that the loss or pseudogenization of functional genes involved in metabolism and virulence in S Pullorum has been ongoing since before and after divergence from the ancestor. In contrast, multiple prophages and plasmids have been acquired by S Pullorum, and these have endowed it with new characteristics, especially the multidrug resistance conferred by two large plasmids in lineage I. The results of this study provide insight into the evolution of S Pullorum and prove the efficiency of whole-genome sequencing in epidemiological surveillance of pullorum disease.IMPORTANCE Pullorum disease, an acute poultry septicemia caused by Salmonella Gallinarum biovar Pullorum, is fatal for young chickens and is a heavy burden on poultry industry. The pathogen is rare in most developed countries but still extremely difficult to eliminate in China. Efficient epidemiological surveillance necessitates clarifying the origin of the isolates from different regions and their phylogenic relationships. Genomic epidemiological analysis of 97 S Pullorum strains was carried out to reconstruct the phylogeny and transmission history of S Pullorum. Further analysis demonstrated that functional gene loss and acquisition occurred simultaneously throughout the evolution of S Pullorum, both of which reflected adaptation to the changing environment. The result of our study will be helpful in surveillance and prevention of pullorum disease.
Project description:Salmonella enterica serovar Pullorum/Gallinarum is an important infectious pathogen that has caused widespread problems for chicken industry. Traditional Salmonella serotyping is an expensive and time-consuming process. In this study, we developed a rapid one-step polymerase chain reaction (PCR) method to identify S. Pullorum/Gallinarum. The PCR-based assay focuses on flhB, which shows a deficient region only in S. Pullorum/Gallinarum, compared with that of other serovars. The specificity and sensitivity of the PCR system were evaluated. The developed PCR method could identify S. Pullorum/Gallinarum from 27 different Salmonella serovars and eight non-Salmonella pathogens. The minimum limit of DNA and the lowest number of cells of S. Pullorum for the PCR detection were no less than 5.85 pg/?L and 10 CFU, respectively. The method was applied to the analysis of Salmonella strains isolated from the chicken farm. The PCR-based testing results of the farm isolates were in concordance with those obtained using traditional serotyping method. This newly developed PCR-based system could be used to accurately screen for the presence of S. Pullorum/Gallinarum, and support traditional serotyping methods, especially in high-throughput screening situations.
Project description:Some life-threatening, foodborne, and zoonotic infections are transmitted through poultry birds. Inappropriate and indiscriminate use of antimicrobials in the livestock industry has led to an increased prevalence of multidrug-resistant bacteria with epidemic potential. Here, we present a functional molecular epidemiological analysis entailing the phenotypic and whole-genome sequence-based characterization of 11 H. pullorum isolates from broiler and free-range chickens sampled from retail wet markets in Hyderabad City, India. Antimicrobial susceptibility tests revealed all of the isolates to be resistant to multiple antibiotic classes such as fluoroquinolones, cephalosporins, sulfonamides, and macrolides. The isolates were also found to be extended-spectrum ?-lactamase producers and were even resistant to clavulanic acid. Whole-genome sequencing and comparative genomic analysis of these isolates revealed the presence of five or six well-characterized antimicrobial resistance genes, including those encoding a resistance-nodulation-division efflux pump(s). Phylogenetic analysis combined with pan-genome analysis revealed a remarkable degree of genetic diversity among the isolates from free-range chickens; in contrast, a high degree of genetic similarity was observed among broiler chicken isolates. Comparative genomic analysis of all publicly available H. pullorum genomes, including our isolates (n = 16), together with the genomes of 17 other Helicobacter species, revealed a high number (8,560) of H. pullorum-specific protein-encoding genes, with an average of 535 such genes per isolate. In silico virulence screening identified 182 important virulence genes and also revealed high strain-specific gene content in isolates from free-range chickens (average, 34) compared to broiler chicken isolates. A significant prevalence of prophages (ranging from 1 to 9) and a significant presence of genomic islands (0 to 4) were observed in free-range and broiler chicken isolates. Taken together, these observations provide significant baseline data for functional molecular infection epidemiology of nonpyloric Helicobacter species such as H. pullorum by unraveling their evolution in chickens and their possible zoonotic transmission to humans.Globally, the poultry industry is expanding with an ever-growing consumer base for chicken meat. Given this, food-associated transmission of multidrug-resistant bacteria represents an important health care issue. Our study involves a critical baseline approach directed at genome sequence-based epidemiology and transmission dynamics of H. pullorum, a poultry pathogen having established zoonotic potential. We believe our studies would facilitate the development of surveillance systems that ensure the safety of food for humans and guide public health policies related to the use of antibiotics in animal feed in countries such as India. We sequenced 11 new genomes of H. pullorum as a part of this study. These genomes would provide much value in addition to the ongoing comparative genomic studies of helicobacters.
Project description:Meat and meat products are important sources of human intestinal infections. We report the isolation of Helicobacter pullorum strains from chicken meat. Bacteria were isolated from 4 of the 17 analyzed fresh chicken meat samples, using a membrane filter method. MIC determination revealed that the four strains showed acquired resistance to ciprofloxacin; one was also resistant to erythromycin, and another one was resistant to tetracycline. Whole-genome sequencing of the four strains and comparative genomics revealed important genetic traits within the H. pullorum species, such as 18 highly polymorphic genes (including a putative new cytotoxin gene), plasmids, prophages, and a complete type VI secretion system (T6SS). The T6SS was found in three out of the four isolates, suggesting that it may play a role in H. pullorum pathogenicity and diversity. This study suggests that the emerging pathogen H. pullorum can be transmitted to humans by chicken meat consumption/contact and constitutes an important contribution toward a better knowledge of the genetic diversity within the H. pullorum species. In addition, some genetic traits found in the four strains provide relevant clues to how this species may promote adaptation and virulence.
Project description:We sequenced the 16S rRNA and groEL genes of Aegyptianella pullorum, a small bacterium that infects and replicates only in avian red blood cells. A specific PCR test was developed to analyze A. pullorum DNA. Phylogenic analysis revealed A. pullorum is most closely related to Anaplasma spp.