Environmentally responsive synthesis of surface proteins represents a hallmark of the infectious cycle of the Lyme disease agent, Borrelia burgdorferi. Here we created and analyzed a B. burgdorferi mutant lacking outer-surface protein C (OspC), an abundant Osp that spirochetes normally synthesize in the tick vector during the blood meal and down-regulate after transmission to the mammal. We demonstrate that B. burgdorferi strictly requires OspC to infect mice but not to localize or migrate appropriately in the tick. The induction of a spirochetal virulence factor preceding the time and host in which it is required demonstrates a developmental sequence for transmission of this arthropod-borne pathogen.

This study demonstrates a strict temporal requirement for a virulence determinant of the Lyme disease spirochete Borrelia burgdorferi during a unique point in its natural infection cycle, which alternates between ticks and small mammals. OspC is a major surface protein produced by B. burgdorferi when infected ticks feed but whose synthesis decreases after transmission to a mammalian host. We have previously shown that spirochetes lacking OspC are competent to replicate in and migrate to the salivary glands of the tick vector but do not infect mice. Here we assessed the timing of the requirement for OspC by using an ospC mutant complemented with an unstable copy of the ospC gene and show that B. burgdorferi's requirement for OspC is specific to the mammal and limited to a critical early stage of mammalian infection. By using this unique system, we found that most bacterial reisolates from mice persistently infected with the initially complemented ospC mutant strain no longer carried the wild-type copy of ospC. Such spirochetes were acquired by feeding ticks and migrated to the tick salivary glands during subsequent feeding. Despite normal behavior in ticks, these ospC mutant spirochetes did not infect naive mice. ospC mutant spirochetes from persistently infected mice also failed to infect naive mice by tissue transplantation. We conclude that OspC is indispensable for establishing infection by B. burgdorferi in mammals but is not required at any other point of the mouse-tick infection cycle.

Clostridium perfringens isolates are currently classified into one of five biotypes on the basis of the differential production of alpha-, beta-, epsilon- and iota-toxins. Different biotypes are associated with different diseases of man and animals. In this study a multiple PCR assay was developed to detect the genes encoding these toxins. In addition, detection of the genes encoding the C. perfringens enterotoxin and beta2-toxin allowed subtyping of the bacteria. C. perfringens isolates taken from a variety of animals, including foals, piglets or lambs, were genotyped using this assay. Most of the isolates were found to be genotype A and the gene encoding beta2-toxin [corrected] was present in 50% of the isolates genotyped. A significant association between C. perfringens possessing the beta2-toxin gene and diarrhoea in piglets was identified, suggesting that beta2-toxin may play a key role in the pathogenesis of the disease.

No abstract

We previously demonstrated that outer surface protein C (OspC) of Borrelia burgdorferi is essential for establishing mammalian infection. However, the role of OspC in mammalian infection is unknown. Here, we report experiments designed to distinguish between two models of OspC function in the mammalian host: (i) OspC fulfills an essential physiological role for growth and host adaptation or (ii) OspC provides a protective role for evasion of components of the innate immune response. We found that a B. burgdorferi ospC mutant, previously demonstrated to be noninfectious in both immunocompetent and SCID mice, could survive in the relatively immune-privileged environment of dialysis membrane chambers implanted within the peritoneum of a rat. The ospC mutant also adapts to the mammalian environment, as determined by the protein profiles of the chamber-cultivated spirochetes. Therefore, OspC does not appear to provide a physiological function for the survival of B. burgdorferi within the mammalian host. The second model, evasion of the innate immune system, was tested by assessing the infectivity of the ospC mutant in mice deficient for myeloid differentiation protein 88 (MyD88). Recent studies have shown that B. burgdorferi is prevented from reaching high cell numbers in the mammalian host by MyD88-dependent signaling pathways. The ospC mutant was incapable of infecting MyD88-deficient mice, suggesting that the role of OspC cannot be related solely to evasion of MyD88-mediated innate immunity. These results reiterate the importance of OspC in mammalian infection and eliminate simple models of function for this enigmatic protein.

Although Clostridium perfringens is recognized as an important cause of clostridial enteric diseases, only limited knowledge exists concerning the association of particular C. perfringens toxinotypes (type A to E) with gastrointestinal (GI) diseases in domestic animals. Some C. perfringens isolates also produce the newly discovered beta2-toxin (CPB2). Recent epidemiological studies suggested that C. perfringens isolates carrying the gene encoding CPB2 (cpb2) are strongly associated with clostridial GI diseases in domestic animals, including necrotic enteritis in piglets and typhlocolitis in horses. These putative relationships, obtained by PCR genotyping, were tested in the present study by further genotyping and phenotyping of 29 cpb2-positive C. perfringens isolates from pigs with GI disease (pig GI disease isolates). PCR and restriction fragment length polymorphism analysis reconfirmed the presence of cpb2 gene sequences in all the disease isolates included in the study. Furthermore, genotyping by pulsed-field gel electrophoresis analyses showed that the pig GI disease isolates included in this study all carry a plasmid cpb2 gene, yet no clonal relationships were detected between the cpb2-positive pig GI disease isolates surveyed. Finally, CPB2-specific Western blotting demonstrated CPB2 expression by all of the cpb2-positive isolates surveyed. The CPB2 proteins made by five of these pig GI disease isolates were shown to have the same deduced amino acid sequences as the biologically active CPB2 protein made by the original type C isolate, CWC245. Collectively, our present results support a significant association between CPB2-positive C. perfringens isolates and diarrhea in piglets.

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.

Clostridium difficile was investigated as a possible cause of enteritis in calves. The organism and its toxins (TcdA and TcdB), respectively, were found in 25.3% and 22.9% of stool samples from diarrheic calves. Culture positive samples were more likely than culture negative samples to be toxin positive. However, toxin positive stools were more common among nondiarrheic calves, but diarrheic calves were nearly twice as likely to be culture positive. Ribotype 078 was dominant among isolates. Salmonella sp. was isolated from both diarrheic and nondiarrheic calves, but large numbers of E. coli were found more commonly in diarrheic calves than in nondiarrheic animals. Prevalence rates for coronavirus and Cryptosporidium sp. were substantially higher in nondiarrheic calves than in diarrheic, but rates of detection of rotavirus and Giardia sp. were more nearly equal between groups. Lesions in naturally infected calves included superficial mucosal erosion with associated fibrinous exudates. Neutrophils and eosinophils infiltrated lamina propria. Large Gram-positive rods morphologically compatible with C. difficile were abundant in the colonic lumen and the organism was isolated by bacteriologic culture. Toxins were found throughout the colon. Purified toxins A and B (individually and conjointly) caused comparable lesions, as well as fluid accumulation, in ligated intestinal loops. Our findings are in substantial agreement with those of others [Rodriguez-Palacios, A., Stampfli, H.R., Duffield, T., Peregrine, A.S., Trotz-Williams, L.A., Arroyo, L.G., Brazier, J.S., Weese, J.S., 2006. Clostridium difficile PCR ribotypes in calves, Canada. Emerg. Infect. Dis. 12, 1730-1736; Porter, M.C., Reggiardo, C., Bueschel, D.M., Keel, M.K., Songer, J.G., 2002. Association of Clostridium difficile with bovine neonatal diarrhea. Proc. 45th Ann. Mtg. Amer. Assoc. Vet. Lab. Diagn., St. Louis, MO, U.S.A.] and add strength to a working hypothesis that C. difficile infection and the accompanying intoxication can manifest as diarrhea in calves. It seems clear that calves serve as multiplying hosts for this organism.