B. Helen Jost

Prevalence of cpb2, encoding beta2 toxin, in Clostridium perfringens field isolates: correlation of genotype with phenotype

Beta2 toxin, encoded by the cpb2 gene, has been implicated in the pathogenesis of porcine, equine and bovine enteritis by type A Clostridium perfringens. By incorporating primers to cpb2 into a multiplex genotyping PCR, we screened 3270 field isolates of C. perfringens. Of these, 37.2% were PCR positive for the cpb2 gene. The majority of isolates from cases of porcine enteritis were positive for cpb2 (>85%), and this was even more true for C. perfringens isolated from cases of porcine neonatal enteritis (91.8%). In contrast, isolates from normal pigs only contained cpb2 in 11.1% of cases. The correlation between enteritis in other animal species and the presence of cpb2 was not so strong. cpb2 was found in 21.4% of C. perfringens isolates from cattle with enteritis, and in 47.3% of isolates from calves with enteritis or abomastitis. The prevalence of cpb2 varied with genotype, with type A isolates being positive for this gene in 35.1% of cases. Furthermore, enterotoxigenic type D or type E strains almost always carried cpb2. We cloned a 6xHIS-tagged beta2 (HIS-beta2) and used this protein to raise antiserum against beta2. Culture supernatants from 68 cpb2-positive and 13 cpb2-negative strains were tested for the presence of beta2 by Western blotting. In cpb2-positive isolates of porcine origin, beta2 was almost always detected (96.9%). However, in cpb2-positive isolates from other animal species, only 50.0% expressed beta2 protein. The high rate of cpb2-positivity among strains from neonatal pigs with enteritis and the high correlation of genotype with phenotype, supports the contention that beta2 toxin plays a role in the pathogenesis of these infections. However, it may be important to consider the use of an additional method for the detection of beta2 toxin in non-porcine cpb2-positive isolates when making claims about the role of beta2 in enteritis in non-porcine species.

Characterization of Campylobacter jejuni Biofilms under Defined Growth Conditions

ABSTRACT Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries and is a source of public health and economic burden. C. jejuni, present as normal flora in the intestinal tract of commercial broiler chickens and other livestock, is probably the main source of human infections. The presence of C. jejuni in biofilms found in animal production watering systems may play a role in the colonization of these animals. We have determined that C. jejuni can form biofilms on a variety of abiotic surfaces commonly used in watering systems, such as acrylonitrile butadiene styrene and polyvinyl chloride plastics. Furthermore, C. jejuni biofilm formation was inhibited by growth in nutrient-rich media or high osmolarity, and thermophilic and microaerophilic conditions enhanced biofilm formation. Thus, nutritional and environmental conditions affect the formation of C. jejuni biofilms. Both flagella and quorum sensing appear to be required for maximal biofilm formation, as C. jejuni flaAB and luxS mutants were significantly reduced in their ability to form biofilms compared to the wild-type strain.

Atypical cpb2 Genes, Encoding Beta2-Toxin in Clostridium perfringens Isolates of Nonporcine Origin

ABSTRACT Beta2-toxin, encoded by cpb2, is implicated in the pathogenesis of Clostridium perfringens enteritis. However, cpb2 genes from nonporcine C. perfringens isolates were not always expressed, at least in vitro. Nucleotide sequencing identified atypical cpb2 genes with 70.2 to 70.7% DNA identity to previously identified (consensus) cpb2. Atypical beta2-toxin displayed 62.3% identity and 80.4% similarity to consensus beta2-toxin. No porcine type C isolates (n = 16) and only 3.3% of porcine type A isolates (n = 60) carried atypical cpb2 genes. However, 88.5% of nonporcine isolates carried atypical cpb2 (n = 78), but beta2-toxin was not expressed. Almost half of the nonporcine consensus cpb2 genes (44.4%) carried a frameshift mutation (n = 9), resulting in an absence of beta2-toxin expression. These findings strengthen the role of beta2-toxin in the pathogenesis of enteritis in neonatal pigs. However, the identification of apparently nonexpressed, atypical cpb2 genes raises the question of whether this protein plays the same role in enteritis in other animal species.

Species-Specific, Nested PCR-Restriction Fragment Length Polymorphism Detection of Single Cryptosporidium parvum Oocysts

ABSTRACT Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum,Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguishedC. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolatedC. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.

Clostridium perfringens Beta-Toxin Forms Potential-Dependent, Cation-Selective Channels in Lipid Bilayers

ABSTRACT Recombinant beta-toxin from Clostridium perfringenstype C was found to increase the conductance of bilayer lipid membranes (BLMs) by inducing channel activity. The channels exhibited a distribution of conductances within the range of 10 to 380 pS, with the majority of the channels falling into two categories of conductance at 110 and 60 pS. The radii of beta-toxin pores found for the conductance states of 110 and 60 pS were 12.7 and 11.1 Å, respectively. The single channels and the steady-state currents induced by beta-toxin across the BLMs exhibited ideal monovalent cation selectivity. Addition of divalent cations (Zn2+, Cd2+, or Mg2+) at a concentration of 2 mM increased the rate of beta-toxin insertion into BLMs and the single-channel conductance, while application of 5 mM Zn2+ to a beta-toxin-induced steady-state current decreased the inward current by approximately 45%. The mutation of arginine 212 of beta-toxin to aspartate, previously shown to increase the 50% lethal dose of beta-toxin for mice nearly 13-fold, significantly reduced the ability of beta-toxin to form channels. These data support the hypothesis that the lethal action of beta-toxin is based on the formation of cation-selective pores in susceptible cells.

Widespread Distribution of a Tet W Determinant among Tetracycline-Resistant Isolates of the Animal Pathogen Arcanobacterium pyogenes

ABSTRACT Tetracycline resistance is common among isolates of the animal commensal and opportunistic pathogen Arcanobacterium pyogenes. The tetracycline resistance determinant cloned from two bovine isolates of A. pyogenes was highly similar at the DNA level (92% identity) to the tet(W) gene, encoding a ribosomal protection tetracycline resistance protein, from the rumen bacterium Butyrivibrio fibrisolvens. The tet(W) gene was found in all 20 tetracycline-resistant isolates tested, indicating that it is a widely distributed determinant of tetracycline resistance in this organism. In 25% of tetracycline-resistant isolates, the tet(W) gene was associated with a mob gene, encoding a functional mobilization protein, and an origin of transfer, suggesting that the determinant may be transferable to other bacteria. In fact, low-frequency transfer of tet(W) was detected from mob+ A. pyogenes isolates to a tetracycline-sensitive A. pyogenes recipient. The mobile nature of this determinant and the presence of A. pyogenes in the gastrointestinal tract of cattle and pigs suggest that A. pyogenes may have inherited this determinant within the gastrointestinal tracts of these animals.

The Arcanobacterium pyogenes Collagen-Binding Protein, CbpA, Promotes Adhesion to Host Cells

ABSTRACT Arcanobacterium pyogenes is an opportunistic pathogen associated with suppurative diseases in economically important food animals such as cattle, pigs, and turkeys. A. pyogenes adheres to host epithelial cells, and adhesion is promoted by the action of neuraminidase, which is expressed by this organism. However, a neuraminidase-deficient mutant of A. pyogenes only had a reduced ability to adhere to host epithelial cells, indicating that other factors are involved in adhesion. Far Western blotting revealed the presence of an approximately 120-kDa A. pyogenes cell wall protein that binds collagen type I. The 3.5-kb gene that encodes the 124.7-kDa CbpA protein was cloned, and sequence analysis indicated that CbpA contains a typical MSCRAMM protein domain structure. Recombinant, six-His-tagged CbpA (HIS-CbpA) was capable of binding collagen types I, II, and IV but not fibronectin. In addition, CbpA was involved in the ability of A. pyogenes to adhere to HeLa and 3T6 cells, as a cbpA knockout strain had 38.2 and 57.0% of wild-type adhesion, respectively. This defect could be complemented by providing cbpA on a multicopy plasmid. Furthermore, HIS-CbpA blocked A. pyogenes adhesion to HeLa or 3T6 cells in a dose-dependent manner. cbpA was only present in 48% of the A. pyogenes strains tested (n = 75), and introduction of plasmid-encoded cbpA into a naturally cbpA-deficient strain increased the ability of this strain to bind to HeLa and 3T6 cells 2.9- and 5.7-fold, respectively. These data indicate that CbpA, a collagen-binding protein of A. pyogenes, plays a role in the adhesion of this organism to host cells.

Identification of a second Arcanobacterium pyogenes neuraminidase and involvement of neuraminidase activity in host cell adhesion.

ABSTRACT Arcanobacterium pyogenes, a common inhabitant of the upper respiratory and urogenital tracts of economically important animals, such as cattle and swine, is also an opportunistic pathogen associated with suppurative infections in these animals. A. pyogenes expresses neuraminidase activity encoded by the nanH gene, and previously, construction of a nanH mutant of A. pyogenes BBR1 indicated that a second neuraminidase is present in this strain. A 5,112-bp gene, nanP, was cloned and sequenced, and this gene conferred neuraminidase activity on an Escherichia coli host strain. The predicted 186.8-kDa NanP protein exhibited similarity to a number of bacterial neuraminidases and contained the RIP/RLP motif and five copies of the Asp box motif found in all bacterial neuraminidases. As expected, insertional inactivation of the nanP gene in A. pyogenes BBR1 resulted in a mutant with reduced neuraminidase activity. However, insertional inactivation of the nanP gene in the nanH mutant strain resulted in a complete lack of neuraminidase activity. Like NanH, NanP was localized to the A. pyogenes cell wall. However, unlike the nanH gene, which was present in 100% of the strains examined, nanP was present in only 64.2% of the isolates (n = 53). A. pyogenes adheres to HeLa cells, and a nanP mutant displayed a wild-type adhesion phenotype with these cells. In contrast, the ability of a nanHnanP double mutant to bind to HeLa cells was reduced by 53%. The wild-type adhesion phenotype was restored by providing nanP in trans. These data indicate that the neuraminidases of A. pyogenes play a role in adhesion of this organism to host epithelial cells.

Evaluation of a Test for Clostridium Difficile Toxins A and B for the Diagnosis of Neonatal Swine Enteritis

A commercially available 1-hour enzyme immunoassay (EIA) for detecting the presence of Clostridium difficile toxins A and B was evaluated for use in diagnosis of C. difficile infections in neonatal swine. This test was compared with a tissue culture cytotoxicity assay, which is considered to be the reference standard for the detection of C. difficile toxins. Twenty-seven samples of colonic contents and 23 fecal samples were collected from freshly euthanized neonatal swine with a history of scours. Of the 50 specimens tested, 20 were positive by the EIA test and tissue culture and 24 were negative by both tests, for an overall correlation of 88%. The sensitivity and specificity of the EIA were 91% and 86%, respectively, and the positive and negative predictive values were 84% and 86%, respectively. The EIA test is considered suitable as an aid for the diagnosis of C. difficile enteritis because of the high correlation between EIA results and those of the tissue culture cytotoxicity assay.

Tylosin Resistance in Arcanobacterium pyogenes Is Encoded by an Erm X Determinant

ABSTRACT Arcanobacterium pyogenes, a commensal on the mucous membranes of many economically important animal species, is also a pathogen, causing abscesses of the skin, joints, and visceral organs as well as mastitis and abortion. In food animals, A. pyogenes is exposed to antimicrobial agents used for growth promotion, prophylaxis, and therapy, notably tylosin, a macrolide antibiotic used extensively for the prevention of liver abscessation in feedlot cattle in the United States. Of 48 A. pyogenes isolates, 11 (22.9%) exhibited inducible or constitutive resistance to tylosin (MIC of ≥128 μg/ml). These isolates also exhibited resistance to other macrolide and lincosamide antibiotics, suggesting a macrolide-lincosamide resistance phenotype. Of the 11 resistant isolates, genomic DNA from nine hybridized to an erm(X)-specific probe. Cloning and nucleotide sequencing of the A. pyogenes erm(X) gene indicated that it was >95% similar to erm(X) genes from Corynebacterium and Propionibacterium spp. Eight of the erm(X)-containing A. pyogenes isolates exhibited inducible tylosin resistance, which was consistent with the presence of a putative leader peptide upstream of the erm(X) open reading frame. For at least one A. pyogenes isolate, 98-4277-2, erm(X) was present on a plasmid, pAP2, and was associated with the insertion sequence IS6100. pAP2 also carried genes encoding the repressor-regulated tetracycline efflux system determinant Tet 33. The repA gene from pAP2 was nonfunctional in Escherichia coli and at least one A. pyogenes isolate, suggesting that there may be host-encoded factors required for replication of this plasmid.