Karen B. Register

Role of the Dermonecrotic Toxin of Bordetella bronchiseptica in the Pathogenesis of Respiratory Disease in Swine

ABSTRACT Bordetella bronchiseptica is one of the etiologic agents causing atrophic rhinitis and pneumonia in swine. It produces several purported virulence factors, including the dermonecrotic toxin (DNT), which has been implicated in the turbinate atrophy seen in cases of atrophic rhinitis. The purpose of these experiments was to clarify the role of this toxin in respiratory disease by comparing the pathogenicity in swine of two isogenic dnt mutants to their virulent DNT+ parent strains. Two separate experiments were performed, one with each of the mutant-parent pairs. One-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally with the parent strain, the dnt mutant strain, or phosphate-buffered saline. Weekly nasal washes were performed to monitor colonization of the nasal cavity, and the pigs were euthanized 4 weeks after inoculation to determine colonization of tissues and to examine the respiratory tract for pathology. There was evidence that colonization of the upper respiratory tract, but not the lower respiratory tract, was slightly greater for the parent strains than for the dnt mutants. Moderate turbinate atrophy and bronchopneumonia were found in most pigs given the parent strains, while there was no turbinate atrophy or pneumonia in pigs challenged with the dnt mutant strains. Therefore, production of DNT by B. bronchiseptica is necessary to produce the lesions of turbinate atrophy and bronchopneumonia in pigs infected with this organism.

Prevalence and Sequence Variants of IS481 in Bordetella bronchiseptica: Implications for IS481-Based Detection of Bordetella pertussis

ABSTRACT We report the prevalence in Bordetella bronchiseptica of IS481, a frequent target for diagnosis of Bordetella pertussis, as approximately 5%. However, PCR amplicons of the predicted size were detectable in 78% of IS481-negative strains. Our results suggest that PCR targeting IS481 may not be sufficiently specific for reliable identification of B. pertussis.

Use of ribotyping to distinguish Bordetella bronchiseptica isolates

A total of 113 Bordetella bronchiseptica strains, isolated from 11 different host species worldwide, were characterized by ribotyping with restriction enzyme PvuII. Sixteen distinct ribotypes were identified, and each ribotype contained five to seven restriction fragments ranging in size from 1.8 to 5.6 kb. Approximately 88% of the swine isoaltes were identified as ribotype 3 strains. Isolates from dogs also displayed little variation; 74.1% were found to be ribotype 4 strains. Strains obtained from the remaining nine host species belonged to 15 different ribotypes. There was no association between geographic location and ribotype. The technique which we used may be useful for epidemiologic studies in which the transmission of B. bronchiseptica, both within and between species, is investigated.

Characterization and Comparative Analysis of the Genes Encoding Haemophilus parasuis Outer Membrane Proteins P2 and P5

Haemophilus parasuis is a swine pathogen of significant industry concern, but little is known about how the organism causes disease. A related human pathogen, Haemophilus influenzae, has been better studied, and many of its virulence factors have been identified. Two of these, outer membrane proteins P2 and P5, are known to have important virulence properties. The goals of this study were to identify, analyze, and compare the genetic relatedness of orthologous genes encoding P2 and P5 proteins in a diverse group of 35 H. parasuis strains. Genes encoding P2 and P5 proteins were detected in all H. parasuis strains evaluated. The predicted amino acid sequences for both P2 and P5 proteins exhibit considerable heterogeneity, particularly in regions corresponding to predicted extracellular loops. Twenty-five variants of P2 and 17 variants of P5 were identified. The P2 proteins of seven strains were predicted to contain a highly conserved additional extracellular loop compared to the remaining strains and to H. influenzae P2. Antigenic-site predictions coincided with predicted extracellular loop regions of both P2 and P5. Neighbor-joining trees constructed using P2 and P5 sequences predicted divergent evolutionary histories distinct from those predicted by a multilocus sequence typing phylogeny based on partial sequencing of seven housekeeping genes. Real-time reverse transcription-PCR indicated that both genes are expressed in all of the strains.

Bordetella bronchiseptica in a paediatric cystic fibrosis patient: possible transmission from a household cat.

Bordetella bronchiseptica is a zoonotic respiratory pathogen commonly found in domesticated farm and companion animals, including dogs and cats. Here, we report isolation of B. bronchiseptica from a sputum sample of a cystic fibrosis patient recently exposed to a kitten with an acute respiratory illness. Genetic characterization of the isolate and comparison with other isolates of human or feline origin strongly suggest that the kitten was the source of infection.

Reduced Virulence of a Bordetella bronchiseptica Siderophore Mutant in Neonatal Swine

ABSTRACT One means by which Bordetella bronchiseptica scavenges iron is through production of the siderophore alcaligin. A nonrevertible alcaligin mutant derived from the virulent strain 4609, designated DBB25, was constructed by insertion of a kanamycin resistance gene into alcA, one of the genes essential for alcaligin biosynthesis. The virulence of the alcA mutant in colostrum-deprived, caesarean-delivered piglets was compared with that of the parent strain in two experiments. At 1 week of age, piglets were inoculated with phosphate-buffered saline, 4609, or DBB25. Two piglets in each group were euthanatized on day 10 postinfection. The remainder were euthanatized at 21 days postinfection. Clinical signs, including fever, coughing, and sneezing, were present in both groups. Nasal washes performed 7, 14, and 21 days postinoculation demonstrated that strain DBB25 colonized the nasal cavity but did so at levels that were significantly less than those achieved by strain 4609. Analysis of colonization based on the number of CFU per gram of tissue recovered from the turbinate, trachea, and lung also demonstrated significant differences between DBB25 and 4609, at both day 10 and day 21 postinfection. Mild to moderate turbinate atrophy was apparent in pigs inoculated with strain 4609, while turbinates of those infected with strain DBB25 developed no or mild atrophy. We conclude from these results that siderophore production by B. bronchiseptica is not essential for colonization of swine but is required for maximal virulence. B. bronchiseptica mutants with nonrevertible defects in genes required for alcaligin synthesis may be candidates for evaluation as attenuated, live vaccine strains in conventionally reared pigs.

Analytical Verification of a PCR Assay for Identification of Bordetella avium

ABSTRACT Bordetella avium is the etiologic agent of turkey coryza or bordetellosis, a respiratory disease responsible for substantial economic losses to the turkey industry. At present, identification of this bacterium relies on isolation and biochemical testing. Although a PCR for the detection of B. avium was proposed a number of years ago (P. H. Savelkoul, L. E. de Groot, C. Boersma, I. Livey, C. J. Duggleby, B. A. van der Zeijst, and W. Gaastra, Microb. Pathog. 15:207-215, 1993), lack of analytical verification precludes its use as a diagnostic tool. Furthermore, a number of details pertaining to the reaction conditions used are missing or unclear. In the present study we have identified an optimal set of PCR conditions for use with the previously described primer pair and determined the limit of detection under these conditions to be approximately 20 pg. Assay sensitivity is 100%, based on an analysis of 72 B. avium isolates from diverse geographic locations and covering a time span of at least 25 years. Evaluation of a separate group of 87 bacterial isolates from poultry, comprising both gram-positive and gram-negative commensals and pathogens representing 11 genera, demonstrated an assay specificity of 98.8%. Reproducibility is 100% using either purified genomic DNA or boiled cell lysates less than 3 days old. Sequence analysis of the B. avium PCR amplicons identified only three occasional sequence polymorphisms. These data indicate the B. avium PCR assay can provide clinically significant results.

Virulence, Transmission, and Heterologous Protection of Four Isolates of Haemophilus parasuis

ABSTRACT Haemophilus parasuis causes Glässers disease, a syndrome of polyserositis, meningitis, and arthritis in swine. Previous studies with H. parasuis have revealed virulence disparity among isolates and inconsistent heterologous protection. In this study, virulence, direct transmission, and heterologous protection of 4 isolates of H. parasuis (SW114, 12939, MN-H, and 29755) were evaluated using a highly susceptible pig model. In an initial experiment, isolates 12939, MN-H, and 29755 caused Glässers disease, while strain SW114 failed to cause any clinical signs of disease. One pig from each group challenged with MN-H or 29755 failed to develop clinical disease but was able to transmit H. parasuis to noninfected pigs, which subsequently developed Glässers disease. Pigs colonized with SW114, 29755, or MN-H that were free of clinical disease were protected from a subsequent challenge with isolate 12939. In a following experiment, pigs vaccinated with strain SW114 given as either a bacterin intramuscularly or a live intranasal vaccine were protected from subsequent challenge with isolate 12939; however, some pigs given live SW114 developed arthritis. Overall these studies demonstrated that pigs infected with virulent isolates of H. parasuis can remain healthy and serve as reservoirs for transmission to naive pigs and that heterologous protection among H. parasuis isolates is possible. In addition, further attenuation of strain SW114 is necessary if it is to be used as a live vaccine.

Optimized ribotyping protocol applied to Hungarian Bordetella bronchiseptica isolates: identification of two novel ribotypes.

We reported previously that ribotype patterns generated with PvuII and a probe derived from the Escherichia coli rrnB gene could be used to differentiate isolates of Bordetella bronchiseptica. In the present study we report modifications made to the original ribotyping procedure that permit detection in the formerly characterized isolates of an additional 8 fragments with homology to rrnB. Ribotypes were redefined to include these fragments. Although this modification did not permit the detection of novel ribotypes from the previously characterized isolates, it did result in a more accurate reclassification of five of these isolates to other existing ribotypes. It was hypothesized that the additional fragments could form the basis for novel ribotypes in future analyses, and this was supported by the subsequent evaluation of 101 previously uncharacterized pig, rabbit, and dog B. bronchiseptica isolates from Hungary. A total of six different patterns were detected from this group, including two previously not identified that were designated ribotypes 17 and 18. The profile of ribotype 17 includes a novel fragment not associated with any other ribotype. A subset of the fragments constituting ribotype 18, essential for its differentiation from other ribotypes, is only detectable under the modified conditions reported here. Hungarian swine isolates are highly clonal, since 98.2% were identified as ribotype 3. Similarly, 83.7% of rabbit isolates from Hungary are also ribotype 3. Cluster analysis revealed that despite the existence of numerous ribotypes, B. bronchiseptica isolates display limited heterogeneity. The ability to detect additional ribotypes under the modified conditions described in this study strengthens the usefulness of ribotyping as an epidemiologic tool.

Phenotypic Modulation of the Virulent Bvg Phase Is Not Required for Pathogenesis and Transmission of Bordetella bronchiseptica in Swine

ABSTRACT The majority of virulence gene expression in Bordetella is regulated by a two-component sensory transduction system encoded by the bvg locus. In response to environmental cues, the BvgAS regulatory system controls expression of a spectrum of phenotypic phases, transitioning between a virulent (Bvg+) phase and a nonvirulent (Bvg−) phase, a process referred to as phenotypic modulation. We hypothesized that the ability of Bordetella bronchiseptica to undergo phenotypic modulation is required at one or more points during the infectious cycle in swine. To investigate the Bvg phase-dependent contribution to pathogenesis of B. bronchiseptica in swine, we constructed a series of isogenic mutants in a virulent B. bronchiseptica swine isolate and compared each mutant to the wild-type isolate for its ability to colonize and cause disease. We additionally tested whether a BvgAS system capable of modulation is required for direct or indirect transmission. The Bvg− phase-locked mutant was never recovered from any respiratory tract site at any time point examined. An intermediate phase-locked mutant (Bvgi) was found in numbers lower than the wild type at all respiratory tract sites and time points examined and caused limited to no disease. In contrast, colonization of the respiratory tract and disease caused by the Bvg+ phase-locked mutant and the wild-type strain were indistinguishable. The Bvg+ phase-locked mutant transmitted to naïve pigs by both direct and indirect contact with efficiency equal to that of the wild-type isolate. These results indicate that while full activation of the BvgAS regulatory system is required for colonization and severe disease, it is not deleterious to direct and indirect transmission. Overall, our results demonstrate that the Bvg+ phase is sufficient for respiratory infection and host-to-host transmission of B. bronchiseptica in swine.