No abstract

The purpose of this study was to develop a multiplex polymerase chain reaction (PCR) protocol useful in the virulence genotyping of Salmonella spp. with the idea that genotyping could augment current Salmonella characterization and typing methods. Seventeen genes associated with Salmonella invasion, fimbrial production, toxin production, iron transport, and intramacrophage survival were targeted by three PCR reactions. Most of these genes are required for full Salmonella virulence in a murine model, and many are also located onSalmonella pathogenicity islands (PAIs) and are associated with type III secretion systems (TTSSs). Once the success of procedures that used positive and negative control strains was verified, the genotypes of 78 Salmonella isolates incriminated in avian salmonellosis (primarily from sick, commercially reared chickens and turkeys) and 80 Salmonella isolates from apparently healthy chickens or turkeys were compared. Eleven of the 17 genes tested (invA, orgA, prgH, tolC, spaN [invJ], sipB, sitC, pagC, msgA, spiA, and iroN) were found in all of the isolates. Another (sopB) was present in all isolates from sick birds and all but one isolate from healthy birds. The remaining five genes (lpfC, cdtB, sifA, pefA, and spvB) were found in 10%-90% of the isolates from sick birds and 3.75%-90% of the healthy birds. No significant differences in the occurrence of these genes between the two groups of isolates were detected. These results suggest that these virulence genes, and presumably the PAIs and TTSSs with which they are associated, are widely distributed among Salmonella isolates of birds, regardless of whether their hosts of origin have been identified as having salmonellosis. SUMMARY. The purpose of this study was to develop a multiplex polymerase chain reaction (PCR) protocol useful in the virulence genotyping of Salmonella spp. with the idea that genotyping could augment current Salmonella characterization and typing methods. Seventeen genes associated with Salmonella invasion, fimbrial production, toxin production, iron transport, and intramacrophage survival were targeted by three PCR reactions. Most of these genes are required for full Salmonella virulence in a murine model, and many are also located on Salmonella pathogenicity islands (PAIs) and are associated with type III secretion systems (TTSSs). Once the success of procedures that used positive and negative control strains was verified, the genotypes of 78 Salmonella isolates incriminated in avian salmonellosis (primarily from sick, commercially reared chickens and turkeys) and 80 Salmonella isolates from apparently healthy chickens or turkeys were compared. Eleven of the 17 genes tested (invA, orgA, prgH, tolC, spaN [invJ], sipB, sitC, pagC, msgA, spiA, and iroN) were found in all of the isolates. Another (sopB) was present in all isolates from sick birds and all but one isolate from healthy birds. The remaining five genes (lpfC, cdtB, sifA, pefA, and spvB) were found in 10%-90% of the isolates from sick birds and 3.75%-90...

We have found an avian pathogenic Escherichia coli (APEC) plasmid, pAPEC-O2-ColV, which contains many of the genes associated with APEC virulence and also shows similarity in content to a plasmid and pathogenicity island of human uropathogenic E. coli (UPEC). To test the possible role of this plasmid in virulence, it was transferred by conjugation along with a large R plasmid, pAPEC-O2-R, into a commensal avian E. coli strain. The transconjugant was compared to recipient strain NC, UPEC strain HE300, and donor strain APEC O2 using various assays, including lethality for chicken embryos, growth in human urine, and ability to cause urinary tract infection in mice. The transconjugant killed significantly more chicken embryos than did the recipient. In human urine, APEC O2 grew at a rate equivalent to that of UPEC strain HE300, and the transconjugant showed significantly increased growth compared to the recipient. The transconjugant also significantly outcompeted the recipient in colonization of the murine kidney. These findings suggest that APEC plasmids, such as pAPEC-O2-ColV, contribute to the pathogenesis of avian colibacillosis. Moreover, since avian E. coli and their plasmids may be transmitted to humans, evaluation of APEC plasmids as possible reservoirs of urovirulence genes for human UPEC may be warranted.

Escherichia coli strains that cause disease outside the intestine are known as extraintestinal pathogenic E. coli (ExPEC) and include human uropathogenic E. coli (UPEC) and avian pathogenic E. coli (APEC). Regardless of host of origin, ExPEC strains share many traits. It has been suggested that these commonalities may enable APEC to cause disease in humans. Here, we begin to test the hypothesis that certain APEC strains possess potential to cause human urinary tract infection through virulence genotyping of 1,000 APEC and UPEC strains, generation of the first complete genomic sequence of an APEC (APEC O1:K1:H7) strain, and comparison of this genome to all available human ExPEC genomic sequences. The genomes of APEC O1 and three human UPEC strains were found to be remarkably similar, with only 4.5% of APEC O1's genome not found in other sequenced ExPEC genomes. Also, use of multilocus sequence typing showed that some of the sequenced human ExPEC strains were more like APEC O1 than other human ExPEC strains. This work provides evidence that at least some human and avian ExPEC strains are highly similar to one another, and it supports the possibility that a food-borne link between some APEC and UPEC strains exists. Future studies are necessary to assess the ability of APEC to overcome the hurdles necessary for such a food-borne transmission, and epidemiological studies are required to confirm that such a phenomenon actually occurs.Escherichia coli is among the world's most well-studied organisms and is often found at the forefront of advancing technology. Not surprisingly, E. coli is on the leading edge of an ongoing shift in the field of genomics (3,6,65). Now that at least one representative organism per species has been sequenced for most pathogens of interest, the focus in genomics has reoriented towards obtaining multiple sequences within a species. With more genomic sequences available for E. coli than for any other species, it leads this trend (3). Thus far, all of the pathogenic E. coli strains sequenced have originated from human hosts (6,8,10,24,47,68). This bias has left a gap in our knowledge, as various E. coli strains cause significant and widespread disease in animals, including in those raised for human consumption (2, 13, 41). Consequently, while the genomic analysis of E. coli strains from animals can be justified solely on the basis of E. coli's detrimental impact on animal agriculture, a broader justification would also include the potential link between animal-source E. coli and human disease.Links between human and animal disease caused by E. coli are well established in some instances but remain speculative in others. For instance, recent reports of outbreaks of human urinary tract infections (UTIs) have stimulated interest in the potential that E. coli from animals has to cause human UTIs via the food supply (28,41,49). Since UTIs are among the world's most common bacterial infections (20), cause significant morbidity, and cost the health care system of the United States over a billion dolla...

Zoonotic transmission of brucellosis often results from exposure to Brucella-infected livestock, feral animals, or wildlife or frequently via consumption of unpasteurized milk products or raw meat. Since natural infection of humans often occurs by the oral route, mucosal vaccination may offer a means to confer protection for both mucosal and systemic tissues. Significant efforts have focused on developing a live brucellosis vaccine, and deletion of the znuA gene involved in zinc transport has been found to attenuate Brucella abortus. A similar mutation has been adapted for Brucella melitensis and tested to determine whether oral administration of ⌬znuA B. melitensis can confer protection against nasal B. melitensis challenge. A single oral vaccination with ⌬znuA B. melitensis rapidly cleared from mice within 2 weeks and effectively protected mice upon nasal challenge with wild-type B. melitensis 16M. In 83% of the vaccinated mice, no detectable brucellae were found in their spleens, unlike with phosphate-buffered saline (PBS)-dosed mice, and vaccination also enhanced the clearance of brucellae from the lungs. Moreover, vaccinated gamma interferon-deficient (IFN-␥ ؊/؊ ) mice also showed protection in both spleens and lungs, albeit protection that was not as effective as in immunocompetent mice. Although IFN-␥, interleukin 17 (IL-17), and IL-22 were stimulated by these live vaccines, only RB51-mediated protection was codependent upon IL-17 in BALB/c mice. These data suggest that oral immunization with the live, attenuated ⌬znuA B. melitensis vaccine provides an attractive strategy to protect against inhalational infection with virulent B. melitensis.Brucellae are Gram-negative intracellular bacterial pathogens of both humans and animals. Brucellosis, caused primarily by Brucella melitensis, Brucella abortus, Brucella ovis, and Brucella suis (12,22

Brucella spp. are zoonotic, facultative intracellular pathogens, which cause animal and human disease. Animal disease results in abortion of fetuses; in humans, it manifests flu-like symptoms with an undulant fever, with osteoarthritis as a common complication of infection. Antibiotic regimens for human brucellosis patients may last several months and are not always completely effective. While there are no vaccines for humans, several licensed live Brucella vaccines are available for use in livestock. The performance of these animal vaccines is dependent upon the host species, dose, and route of immunization. Newly engineered live vaccines, lacking well-defined virulence factors, retain low residual virulence, are highly protective, and may someday replace currently used animal vaccines. These also have possible human applications. Moreover, due to their enhanced safety and efficacy in animal models, subunit vaccines for brucellosis show great promise for their application in livestock and humans. This review summarizes the progress of brucellosis vaccine development and presents an overview of candidate vaccines.

Brucellosis, caused by the intracellular bacterial pathogen is a zoonotic disease for which arthritis is the most common focal complication in humans. Here we investigated the role of inflammasomes and their effectors, including IL-1, IL-18 and pyroptosis, on inflammation and control of infection during-induced arthritis. Early in infection, both caspase-1 and caspase-11 were found to initiate joint inflammation and pro-inflammatory cytokine production. However, by one week post-infection caspase-1 and caspase-11 also contributed to control of joint infection. Inflammasome dependent restriction of joint burdens did not require AIM2 or NLRP3. IL-1R had a modest effect on -induced joint swelling, but mice lacking IL-1R were not impaired in their ability to control infection of the joint by In contrast, IL-18 contributed to the initiation of joint swelling and control of joint infection. Caspase1/11-dependent cell death was observed, and studies demonstrated caspase-1 and caspase-11 both induce pyroptosis which limited infection in macrophages. LPS alone was also able to induce caspase-11 dependent pyroptosis. Collectively these data demonstrate inflammasomes induce inflammation in an IL-18 dependent manner, and inflammasome-dependent IL-18 and pyroptosis restrict infection.

Human brucellosis exhibits diverse pathological manifestations that can affect almost any organ. In particular, osteoarticular complications are the most common focal manifestation of brucellosis and occur in 40-80% of patients. In immunocompetent mice, Brucella replication is generally restricted to the spleen, liver, and to a lesser extent, LNs, thereby limiting their use for study of focal inflammation often found in brucellosis. Here, we report that nasal, oral, or peritoneal infection of IFN-γ(-/-) mice with WT Brucella melitensis or Brucella abortus results in joint and periarticular tissue inflammation. Histological analysis of the affected joints revealed inflammatory infiltrates and debris within the joint space colocalizing with Brucella antigen. Osteoarthritis, necrosis, periarticular soft tissue inflammation, and substantial brucellae burdens were observed. Oral rifampicin was effective in clearing infection and halting further progression of focal inflammation from infected IFN-γ(-/-) mice, although some symptoms and swelling remained. Elevated IL-1 β, but not TNF-α, IL-6, or IL-17, was detected in joint homogenates from infected IFN-γ(-/-) mice. Whereas more susceptible to systemic infection, IL-1R(-/-) mice depleted of IFN-γ were more resistant to focal inflammation than WT mice similarly depleted of IFN-γ. Collectively, these results show IFN-γ(-/-) mice represent a potential model for study of focal inflammation attributed to Brucella infection and will allow evaluation of intervention strategies targeting IL-1, IL-1R, or other inflammatory mediators, with the potential to complement antibiotic-based therapies.