Stephen W. Davis

Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing.

ABSTRACT A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.

Biodiversity of Clostridium botulinum Type E Associated with a Large Outbreak of Botulism in Wildlife from Lake Erie and Lake Ontario

ABSTRACT The genetic relatedness of Clostridium botulinum type E isolates associated with an outbreak of wildlife botulism was studied using random amplification of polymorphic DNA (RAPD). Specimens were collected from November 2000 to December 2008 during a large outbreak of botulism affecting birds and fish living in and around Lake Erie and Lake Ontario. In our present study, a total of 355 wildlife samples were tested for the presence of botulinum toxin and/or organisms. Type E botulinum toxin was detected in 110 samples from birds, 12 samples from fish, and 2 samples from mammals. Sediment samples from Lake Erie were also examined for the presence of C. botulinum. Fifteen of 17 sediment samples were positive for the presence of C. botulinum type E. Eighty-one C. botulinum isolates were obtained from plants, animals, and sediments; of these isolates, 44 C. botulinum isolates produced type E toxin, as determined by mouse bioassay, while the remaining 37 isolates were not toxic for mice. All toxin-producing isolates were typed by RAPD; that analysis showed 12 different RAPD types and multiple subtypes. Our study thus demonstrates that multiple genetically distinct strains of C. botulinum were involved in the present outbreak of wildlife botulism. We found that C. botulinum type E is present in the sediments of Lake Erie and that a large range of bird and fish species is affected.

Isolation of Avian Paramyxovirus 1 from a Patient with a Lethal Case of Pneumonia

ABSTRACT An unknown virus was isolated from a lung biopsy sample and multiple other samples from a patient who developed a lethal case of pneumonia following a peripheral blood stem cell transplant. A random PCR-based molecular screening method was used to identify the infectious agent as avian paramyxovirus 1 (APMV-1; a group encompassing Newcastle disease virus), which is a highly contagious poultry pathogen that has only rarely been found in human infections. Immunohistochemical analysis confirmed the presence of APMV-1 antigen in sloughed alveolar cells in lung tissue from autopsy. Sequence from the human isolate showed that it was most closely related to virulent pigeon strains of APMV-1. This is the most completely documented case of a systemic human infection caused by APMV-1 and is the first report of an association between this virus and a fatal disease in a human.

Laboratory-Confirmed Transmission of Vaccinia Virus Infection through Sexual Contact with a Military Vaccinee

ABSTRACT A laboratory-confirmed, inadvertent transmission of vaccinia virus from an unusual source highlights the importance of epidemiologic tracing, proper biosafety practices in the clinical diagnostic laboratories, and educating clinicians and laboratorians to potential bioterrorism-initiated outbreaks as well as look-alike disease discrimination.

Laboratory Confirmation of Generalized Vaccinia following Smallpox Vaccination

ABSTRACT The reinitiation of smallpox vaccination has renewed interest in implementing modern diagnostic methods to assess orthopoxvirus infection and adverse events following vaccination. We report here the laboratory confirmation of vaccinia virus in pustular lesions of a healthy adult vaccinee by use of a two-tier algorithm incorporating TaqMan PCR and electron microscopy.

Two cases of adult botulism caused by botulinum neurotoxin producing Clostridium baratii.

Type F botulism occurs rarely in clinical cases. Two cases of type F botulism in elderly patients that were clustered in time and space are described. Clostridium baratii producing type F botulinum neurotoxin was isolated from both patients; molecular typing of these isolates revealed that they were unrelated strains.

Genetically engineered poxviruses: a novel approach to the construction of live vaccines.

Gene splicing techniques have been used to modify the smallpox vaccine virus thus providing a generic approach for the construction of live vaccines directed against a variety of heterologous infectious disease agents. The technique involves translocating a particular gene from an infectious agent into the genetic material of the smallpox vaccine virus. This unique foreign gene, selected because it contains the information essential for the synthesis of an antigen important in immunity to that particular infectious disease agent, is now expressed under the regulation of the engineered smallpox vaccine virus. On immunization with this live recombinant vaccine, the body is fooled into thinking that it was infected by the foreign infectious disease agent and mounts a defensive attack resulting in immunity to that particular infectious agent. Three examples of this approach are provided. Thus, smallpox vaccine viruses were engineered to express genes encoding the hepatitis B virus surface antigen (HBsAg), the herpes simplex virus glycoprotein D (HSV-gD) and the haemagglutinin (HA) from influenza virus. These foreign gene products when synthesized in vitro under vaccinia virus regulation were shown to be antigenic by a variety of serological tests. When these recombinant vaccinia viruses were inoculated into laboratory animals, the heterologous gene products elicited the production of specific antibodies thus demonstrating that they were immunogenic. Serum neutralizing antibodies were demonstrated to be present for both influenza and herpes simplex viruses. Additional studies in mice showed that a recombinant smallpox vaccine virus expressing a gene from herpes simplex virus effectively protected the mice when subsequently challenged with what would normally be lethal doses of infectious herpes simplex virus.

Implementing the Bruker MALDI Biotyper in the Public Health Laboratory for C. botulinum Neurotoxin Detection

Currently, the gold standard method for active botulinum neurotoxin (BoNT) detection is the mouse bioassay (MBA). A Centers for Disease Control and Prevention-developed mass spectrometry (MS)-based assay that detects active BoNT was successfully validated and implemented in a public health laboratory in clinical matrices using the Bruker MALDI-TOF MS (Matrix-assisted laser desorption ionization–time of flight mass spectrometry) Biotyper. For the first time, a direct comparison with the MBA was performed to determine MS-based assay sensitivity using the Bruker MALDI Biotyper. Mice were injected with BoNT/A, /B, /E, and /F at concentrations surrounding the established MS assay limit of detection (LOD) and analyzed simultaneously. For BoNT/B, /E, and /F, MS assay sensitivity was equivalent or better than the MBA at 25, 0.3, and 8.8 mLD50, respectively. BoNT/A was detected by the MBA between 1.8 and 18 mLD50, somewhat more sensitive than the MS method of 18 mLD50. Studies were performed to compare assay performance in clinical specimens. For all tested specimens, the MS method rapidly detected BoNT activity and serotype in agreement with, or in the absence of, results from the MBA. We demonstrate that the MS assay can generate reliable, rapid results while eliminating the need for animal testing.