Evaluation of different live Salmonella enteritidis vaccine schedules administered during layer hen rearing to reduce excretion, organ colonization, and egg contamination.

Salmonellosis caused by Salmonella Enteritidis is a widespread zoonosis and poultry products are an important source of infection. This study was carried out to evaluate the protection of different vaccination schedules in layers using a live commercial attenuated Salmonella Enteritidis vaccine based on strain Sm24/Rif12/Ssq (AviPro® Salmonella Vac E, ELANCO) during rearing and egg production. Three hundred and fifty Salmonella-free chickens were distributed into 7 vaccinated groups and 1 unvaccinated group. Different vaccination schedules were performed combining either 1, 2, or 3 oral gavage doses. Chickens from Group A, B, and C were vaccinated once, either at the first day, at 7 or 16 wk old, respectively. Chickens from Group D were vaccinated twice-at the first day and 7 wk old. Chickens from Group E were vaccinated twice-at the first day and 16 wk old. Chickens from Group F were vaccinated twice-at 7 and 16 wk old. Chickens from Group G were vaccinated 3 times, following the manufacturer's recommendation: at the first day, 7 and 16 wk old. Chickens from Group H remained unvaccinated. Five challenge trials numbered 1 to 5 were carried out at 8, 12, 16, 29, and 55 wk old, respectively. After challenge, chickens were sampled by cloacal swabbing and, after euthanasia, livers, ovaries, spleens, and cecal contents were cultured to isolate S. Enteritidis. Additionally, eggs were collected after challenge and cultured to isolate S. Enteritidis on egg shells (Trials 4 and 5). Protection against experimental infection with a virulent nalidixic acid resistant S. Enteritidis strain K285/94, was evaluated by measuring reduction of excretion, colonization, invasion into organs, eggshell contamination, and egg production. The live S. Enteritidis vaccine protected the hens by reducing S. Enteritidis excretion, isolation from organs, and egg contamination. Higher protection throughout laying period was afforded after administration of three vaccine doses during rearing period.

Assessing the intra-species genetic variability in the clonal pathogen Campylobacter fetus: CRISPRs are highly polymorphic DNA markers.

Campylobacter fetus is a Gram-negative, microaerophilic bacterium that infects animals and humans. The subspecies Campylobacter fetus subsp. fetus (Cff) affects a broad range of vertebrate hosts and induces abortion in cows and sheep. Campylobacter fetus subsp. venerealis (Cfv) is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus subsp. testudinum (Cft) has been isolated mostly from apparently healthy reptiles belonging to different species but also from ill snakes and humans. Genotypic differentiation of Cff and Cfv is difficult, and epidemiological information is scarce because there are few methods to study the genetic diversity of the strains. We analyze the efficacy of MLST, ribosomal sequences (23S gene and internal spacer region), and CRISPRs to assess the genetic variability of C. fetus in bovine and human isolates. Sequences retrieved from complete genomes were included in the analysis for comparative purposes. MLST and ribosomal sequences had scarce or null variability, while the CRISPR-cas system structure and the sequence of CRISPR1 locus showed remarkable diversity. None of the sequences here analyzed provided evidence of a genetic differentiation of Cff and Cfv in bovine isolates. Comparison of bovine and human isolates with Cft strains showed a striking divergence. Inter-host differences raise the possibility of determining the original host of human infections using CRISPR sequences. CRISPRs are the most variable sequences analyzed in C. fetus so far, and constitute excellent representatives of a dynamic fraction of the genome. CRISPR typing is a promising tool to characterize isolates and to track the source and transmission route of C. fetus infections.

A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences.

BACKGROUND: Campylobacter fetus is a pathogen of major concern for animal and human health. The species shows a great intraspecific variation, with three subspecies: C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. testudinum. Campylobacter fetus fetus affects a broad range of hosts and induces abortion in sheep and cows. Campylobacter fetus venerealis is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus testudinum has been proposed recently based on genetically divergent strains isolated from reptiles and humans. Both C. fetus fetus and C. fetus testudinum are opportunistic pathogens for immune-compromised humans. Biochemical tests remain as the gold standard for identifying C. fetus but the fastidious growing requirements and the lack of reliability and reproducibility of some biochemical tests motivated the development of molecular diagnostic tools. These methods have been successfully tested on bovine isolates but fail to detect some genetically divergent strains isolated from other hosts. The aim of the present study was to develop a highly specific molecular assay to identify and quantify C. fetus strains. RESULTS: We developed a highly sensitive real-time PCR assay that targets a unique region of the 16S rRNA gene. This assay successfully detected all C. fetus strains, including those that were negative for the cstA gene-based assay used as a standard for molecular C. fetus identification. The assay showed high specificity and absence of cross-reactivity with other bacterial species. The analytical testing of the assay was determined using a standard curve. The assay demonstrated a wide dynamic range between 102 and 107 genome copies per reaction, and a good reproducibility with small intra- and inter-assay variability. CONCLUSIONS: The possibility to characterize samples in a rapid, sensitive and reproducible way makes this assay a good option to establish a new standard in molecular identification and quantification of C. fetus species.

Genomic evidence for the emergence and evolution of pathogenicity and niche preferences in the genus Campylobacter.

The genus Campylobacter includes some of the most relevant pathogens for human and animal health; the continuous effort in their characterization has also revealed new species putatively involved in different kind of infections. Nowadays, the available genomic data for the genus comprise a wide variety of species with different pathogenic potential and niche preferences. In this work, we contribute to enlarge this available information presenting the first genome for the species Campylobacter sputorum bv. sputorum and use this and the already sequenced organisms to analyze the emergence and evolution of pathogenicity and niche preferences among Campylobacter species. We found that campylobacters can be unequivocally distinguished in established and putative pathogens depending on their repertory of virulence genes, which have been horizontally acquired from other bacteria because the nonpathogenic Campylobacter ancestor emerged, and posteriorly interchanged between some members of the genus. Additionally, we demonstrated the role of both horizontal gene transfers and diversifying evolution in niche preferences, being able to distinguish genetic features associated to the tropism for oral, genital, and gastrointestinal tissues. In particular, we highlight the role of nonsynonymous evolution of disulphide bond proteins, the invasion antigen B (CiaB), and other secreted proteins in the determination of niche preferences. Our results arise from assessing the previously unmet goal of considering the whole available Campylobacter diversity for genome comparisons, unveiling notorious genetic features that could explain particular phenotypes and set the basis for future research in Campylobacter biology.

Complete Genome Sequence of Campylobacter fetus subsp. venerealis Biovar Intermedius, Isolated from the Prepuce of a Bull.

Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, a sexually transmitted disease distributed worldwide. Campylobacter fetus subsp. venerealis biovar Intermedius strains differ in their biochemical behavior and are prevalent in some countries. We report the first genome sequence for this biovar, isolated from bull prepuce.

Application of a multiplex PCR assay for Campylobacter fetus detection and subspecies differentiation in uncultured samples of aborted bovine fetuses.

Campylobacter (C.) fetus (epsilonproteobacteria) is an important veterinary pathogen. This species is currently divided into C. fetus subspecies (subsp.) fetus (Cff) and C. fetus subsp. venerealis (Cfv). Cfv is the causative agent of bovine genital Campylobacteriosis, an infectious disease that leads to severe reproductive problems in cattle worldwide. Cff is a more general pathogen that causes reproductive problems mainly in sheep although cattle can also be affected. Here we describe a multiplex PCR method to detect C. fetus and differentiate between subspecies in a single step. The assay was standardized using cultured strains and successfully used to analyze the abomasal liquid of aborted bovine fetuses without any pre-enrichment step. Results of our assay were completely consistent with those of traditional bacteriological diagnostic methods. Furthermore, the multiplex PCR technique we developed may be easily adopted by any molecular diagnostic laboratory as a complementary tool for detecting C. fetus subspecies and obtaining epidemiological information about abortion events in cattle.