William J. Todd

Effects of Specimen Preparation on the Apparent Ultrastructure of Microorganisms

As microbiologists, we have available to us a variety of both established and newly developed electron microscopy (EM) techniques useful for detailed ultra-structural analysis of microorganisms. Although applications of the new methods will undoubtedly lead to a better understanding of microbial ultrastructure and function, the traditional approach of investigations using fixed, embedded, and sectioned specimens will remain as a central EM method for the foreseeable future. Among the many useful protocols for traditional processing of specimens, the procedures recommended by Ito and Rikihisa (1981) are consistently effective for preserving the ultrastructure of both microorganisms and host cells. These procedures have evolved through the input of many investigators and are summarized here.

Detection of stable diagnostic antigen from bile and feces of Fasciola hepatica infected cattle

Diagnostic antigens in bile and feces from Fasciola hepatica infected cattle were detected and characterized by enzyme-linked immunotransfer blot (EITB) techniques. As sources of antigen, samples of bile, intestinal contents and feces were collected from five uninfected calves and from 10 calves with known Fasciola hepatica burdens. A band detected by EITB using a densitometer in the area corresponding to 26 kDa reacted with rabbit anti-fresh fluke antigen and infected cattle sera but not with fluke-negative rabbit sera, rabbit anti-Fasciola hepatica egg sera, Fascioloides magna positive or negative cattle sera. This band was not detected by Coomassie blue in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels or by Ponceau-S stained nitrocellulose strips. Band groups located at 104-66, 66-42, 42-26 and 25-16 kDa reacted inconsistently with the above sera. Sera from mice hyperimmunized with Fasciola hepatica excretory-secretory (ES) products detected only the 26 kDa band by EITB, without cross-reactivity with bands in the other molecular weight (MW) ranges. The results suggest that the 26 kDa antigen may consist of a stable component of ES products and/or tegument-related worm antigen. Diagnosis of Fasciola hepatica through detection of specific, stable antigens in feces of infected animals offers potential advantages over serum-based tests of better sample accessibility, discrimination between previous and current infections, and possible semi-quantitation of fluke burdens.

Colloidal Gold Labels for Immunocytochemical Analysis of Microbes

In considering the spectrum of research involving biochemical and genetic analysis of microorganisms, it is apparent that understanding the location and spatial orientation of the components studied often accelerates problem solving. Research on membrane structure and function, cell growth and development processes, and mechanisms of microbial pathogenesis is particularly likely to benefit from a visual determination of the position and movements of cell constituents. The application of molecular genetics and monoclonal antibody methods to vaccine development requires a clear understanding of what components are exposed on the surface of microbial pathogens and how they must be presented to the host immune system to be exploited as vaccines. Easy and rapid methods to determine such positional or structural information are needed.

Immune response to lytic peptides conjugated to a βCG fragment in treated BALB/C mice

Hecate-betaCG and Phor14-betaCG(ala) are relatively short, amphipathic alpha-helical cationic peptides with the ability to destroy selectively breast, prostate and ovarian cancer cells. Treatment with proteins and peptides frequently initiated antibody formation. Short peptides may minimize the risk of the immune system mobilization after treatment but it is necessary to investigate whether Hecate-betaCG and Phor14-betaCG(ala) induce the immune system to produce antibody and whether they affect the reproductive organs in normal wild-type mice. The results of our experiments showed that specific antibodies, tested by the enzyme-immunoassay, were not detected in the group treated with Hecate-betaCG and Phor14-betaCG(ala). The blood concentrations of both peptides begun to decrease from 60 minutes after injection and after 240 minutes its levels were undetectable. Histopatho-logical examination exhibited degenerative changes in the prostate glands and testes in males and in the ovaries and uteri of females treated with both peptides. In conclusion, our results indicate that both relatively small and rapidly metabolized peptides are not immunogenic and can be used for further investigation as a potential cancer treatment.

Data Analyses and new Findings Indicate a Primordial Neurotropic Pathogen Evolved into Infectious Causes of Several CNS Neurodegenerative Diseases

The extraordinary genetic flexibility of microorganisms enables their evolution into diverse forms expressing unanticipated structures and functions. Typically, they evolve in response to selective pressures of challenging niches, enabling their evolution and survival in extreme environments wherein life forms were not thought to exist. Approaching the problem of persistent neurodegenerative CNS infections as a challenging niche for pathogen evolution led to uncovering microorganisms which expand concepts of microbial diversity. These organisms are proposed as hybrid pathogens. They express two separate sets of structures and functions: viruslike properties when intracellular, and yet also reproduce as unique prokaryotes when outside the host. Their recovery opens new opportunities to comprehend the remarkable diversity of pathogens and elucidate etiologies of unresolved CNS neurodegenerative infections. Cells infected with these agents produce virus-like particles, inclusions and cytopathic effects consistent with biopsy studies of multiple sclerosis (MS), the α-synucleinopathies, and the transmissible spongiform encephalopathies (TSE) or prion diseases. The principle agents described were recovered from sheep with scrapie and are available via the Biodefense and Emerging Infections Research Resources Repository. Comparative studies with SMCA, a tick isolate inducing neurodegeneration in lab animal models, are included as supportive evidence.