Mark A. Rasmussen

Environmental and Food Safety Aspects of Escherichia coli O157:H7 Infections in Cattle

The presence of E. coli O157:H7 in cattle illustrates the complex, interrelated nature of the environment, livestock production practices, food safety, and the science of microbiology, particularly microbial ecology. Enterohemorrhagic E. coli, including E. coli O157:H7, can cause severe human diseases that can be debilitating and life threatening. Cattle are currently considered to be the definitive source for E. coli O157:H7 in the food supply, but this view may be simplistic and incomplete. E. coli O157:H7, appears widespread among U.S. cattle herds, while individual animal prevalence is low and transient. Most individual animals appear to be a transient reservoir for E. coli O157:H7 although the issue of carrier animals still remains unresolved. Epidemiological studies of the cattle production system have not clearly identified risk factors or management practices that affect E. coli O157:H7 prevalence in cattle feces. The problem of E. coli O157:H7 increases during the summer and fall months, but the environmental factors that contribute to this increase are poorly understood. Possible environmental factors that may influence E. coli O157:H7 shedding in cattle include livestock feed and waste handling practices as well as insects and microbial interactions in soil and water. Studies of E. coli O157:H7 ecology in cattle and the environment have been limited, but they suggest that a consideration of other independent, environmental sources of this microbe seems appropriate. The natural ecology of cholera may serve as a useful environmental model for pursuing additional environmental research on the occurrence and transmission of E. coli O157:H7 in nature.

Exposure to Rumen Protozoa Leads to Enhancement of Pathogenicity of and Invasion by Multiple-Antibiotic-Resistant Salmonella enterica Bearing SGI1

ABSTRACT Multiple-antibiotic-resistant Salmonella enterica serotype Typhimurium is a food-borne pathogen that has been purported to be more virulent than antibiotic-sensitive counterparts. The paradigm for this multiresistant/hyperpathogenic phenotype is Salmonella enterica serotype Typhimurium phage type DT104 (DT104). The basis for the multiresistance in DT104 is related to an integron structure designated SGI1, but factors underlying hyperpathogenicity have not been completely identified. Since protozoa have been implicated in the alteration of virulence in Legionella and Mycobacterium spp., we attempted to assess the possibility that protozoa may contribute to the putative hypervirulence of DT104. Our study reveals that DT104 can be more invasive, as determined by a tissue culture invasion assay, after surviving within protozoa originating from the bovine rumen. The enhancement of invasion was correlated with hypervirulence in a bovine infection model in which we observed a more rapid progression of disease and a greater recovery rate for the pathogen. Fewer DT104 cells were recovered from tissues of infected animals when protozoa were lysed by preinfection chemical defaunation of the bovine or ovine rumen. The protozoan-mediated hypervirulence phenotype was observed only in DT104 and other Salmonella strains, including serovars Agona and Infantis, possessing SGI1.

Identification of Salmonella enterica Serovar Typhimurium Genes Important for Survival in the Swine Gastric Environment

ABSTRACT Since the stomach is a first line of defense for the host against ingested microorganisms, an ex vivo swine stomach contents (SSC) assay was developed to search for genes important for Salmonella enterica serovar Typhimurium survival in the hostile gastric environment. Initial characterization of the SSC assay (pH 3.87) using previously identified, acid-sensitive serovar Typhimurium mutants revealed a 10-fold decrease in survival for a phoP mutant following 20 min of challenge and no survival for mutants of rpoS or fur. To identify additional genes, a signature-tagged mutagenesis bank was constructed and screened in the SSC assay. Nineteen mutants were identified and individually analyzed in the SSC and acid tolerance response assays; 13 mutants exhibited a 10-fold or greater sensitivity in the SSC assay compared to the wild-type strain, but only 3 mutants displayed a 10-fold or greater decrease in survival following pH 3.0 acidic challenge. Further examination determined that the lethal effects of the SSC are pH dependent but that low pH is not the sole killing mechanism(s). Gas chromatography analysis of the SSC revealed lactic acid levels of 126 mM. Upon investigating the effects of lactic acid on serovar Typhimurium survival in a synthetic gastric fluid, not only was a concentration- and time-dependent lethal effect observed, but the phoP, rpoS, fur, and pnp genes were identified as involved in protection against lactic acid exposure. These studies indicate a role in gastric survival for several serovar Typhimurium genes and imply that the stomach environment is defined by more than low pH.

Diverse Tetracycline Resistance Genotypes of Megasphaera elsdenii Strains Selectively Cultured from Swine Feces

ABSTRACT A total of 30 Megasphaera elsdenii strains, selectively isolated from the feces of organically raised swine by using Me109 M medium, and one bovine strain were analyzed for tetracycline resistance genotypic and phenotypic traits. Tetracycline-resistant strains carried tet(O), tet(W), or a tet gene mosaic of tet(O) and tet(W). M. elsdenii strains carrying tet(OWO) genes exhibited the highest tetracycline MICs (128 to >256 μg/ml), suggesting that tet(O)-tet(W) mosaic genes provide the selective advantage of greater tetracycline resistance for this species. Seven tet genotypes are now known for M. elsdenii, an archetype commensal anaerobe and model for tet gene evolution in the mammalian intestinal tract.

Involvement of a Salmonella genomic island 1 gene in the rumen protozoan-mediated enhancement of invasion for multiple-antibiotic-resistant Salmonella enterica serovar typhimurium

ABSTRACT Multiple-antibiotic-resistant Salmonella enterica serotype Typhimurium is a food-borne pathogen that may be more virulent than related strains lacking the multiresistance phenotype. Salmonella enterica serotype Typhimurium phage type DT104 is the most prevalent of these multiresistant/hypervirulent strains. Multiresistance in DT104 is conferred by an integron structure, designated Salmonella genomic island 1 (SGI1), while we recently demonstrated DT104 hyperinvasion mediated by rumen protozoa (RPz) that are normal flora of cattle. Hyperinvasion was also observed in other Salmonella strains, i.e., other S. enterica serovar Typhimurium phage types and other S. enterica serovars, like S. enterica serovar Infantis, possessing SGI1, while DT104 strains lacking SGI1 were not hyperinvasive. Herein we attempted to identify SGI1 genes involved in the RPz-mediated hyperinvasion of Salmonella strains bearing SGI1. Transposon mutagenesis, coupled with a novel reporter system, revealed the involvement of an SGI1 gene previously designated SO13. Disruption of SO13 expression led to an abrogation of hyperinvasion as assessed by tissue culture invasion assays and by bovine challenge experiments. However, hyperinvasion was not observed in non-SGI1-bearing strains of Salmonella engineered to express SO13. That is, SO13 and another SGI1 gene(s) may coordinately upregulate invasion in DT104 exposed to RPz.

Generation of Fluorescent Adducts of Malondialdehyde and Amino Acids: Toward an Understanding of Lipofuscin¶

Lipofuscin is a yellow‐brown, highly fluorescent pigment that undergoes an age‐related progressive accumulation in animal cells, mainly in postmitotic cells. It is a heterogeneous, high‐molecular weight material associated with proteins, lipids and nucleic acids. Lipofuscin is implicated in many aspects of human health, including aging, oxidative stress, macular degeneration, lipid peroxidation, atherosclerosis, dementia (Alzheimers Disease) and diseases associated with prions. Although the fluorescent properties of lipofuscin have long been recognized, neither histologists nor chemists have yet isolated the pigments themselves or characterized their optical properties. We have prepared lipofuscinlike species by reacting malondialdehyde (MDA) with cysteine (Cys). MDA: Cys adducts 3:2 and 2:2 are two of those that have been identified among the many that were present in the reaction. Whereas previous attempts to synthesize lipofuscinlike species resulted in compounds that were either nonfluorescent or emitted principally in the blue, the MDA: Cys adducts reported in this study are not only fluorescent but also emit over a broader range.

Fluorescence-Based Method, Exploiting Lipofuscin, for Real-Time Detection of Central Nervous System Tissues on Bovine Carcasses

The removal of central nervous system (CNS) tissues as part of bovine spongiform encephalopathy (BSE) risk material is one of the highest priority tasks to avoid contamination of the human food chain with BSE. No currently available method enables the real-time detection of possible CNS tissue contamination on carcasses during slaughter. The fluorescent pigment lipofuscin is a heterogeneous, high-molecular weight material that has been shown to be enriched in high concentrations in neuronal tissues. In this study, lipofuscin fluorescence was investigated as a marker for real-time detection of CNS contamination. Front-faced fluorescence spectra of brain and spinal cord samples from 11 cattle gave identical, reproducible fluorescence signal patterns with high intensities. The specificity of these spectra was assessed by investigating 13 different non-CNS tissues enabling the differentiation of brain and spinal cord by signal intensity and structure of the spectra, respectively. Small quantities of bovine spinal cord were reliably detected in the presence of raw bovine skeletal muscle, fat, and vertebrae. The presented data are a fundamental basis for the development of a prototype device allowing real-time monitoring of CNS tissue contamination on bovine carcasses and meat cuts.

Monitoring the Accumulation of Lipofuscin in Aging Murine Eyes by Fluorescence Spectroscopy

The integrated fluorescence of murine eyes is collected as a function of age. This fluorescence is attributed to pigments generally referred to as lipofuscin and is observed to increase with age. No difference in fluorescence intensity is observed between the eyes of males or females. This work provides a benchmark for further studies that are planned in order to use such signatures as markers of central nervous system (CNS) tissue or even of diseased CNS tissue and provides a basis for determining the age of a healthy animal.

Comparisons of Salmonella conjugation and virulence gene hyperexpression mediated by rumen protozoa from domestic and exotic ruminants

Recent studies have identified a phenomenon in which ciliated protozoa engulf Salmonella and the intra-protozoal environment hyperactivates virulence gene expression and provides a venue for conjugal transfer of antibiotic resistance plasmids. The former observation is relegated to Salmonella bearing the SGI1 multiresistance integron while the latter phenomenon appears to be a more generalized event for recipient Salmonella. Our previous studies have assessed virulence gene hyperexpression only with protozoa from the bovine rumen while conjugal transfer has been demonstrated in rumen protozoa from cattle and goats. The present study examined virulence gene hyperexpression for Salmonella exposed to rumen protozoa obtained from cattle, sheep, goats, or two African ruminants (giraffe and bongo). Conjugal transfer was also assessed in these protozoa using Salmonella as the recipient. Virulence gene hyperexpression was only observed following exposure to the rumen protozoa from cattle and sheep while elevated virulence was also observed in these animals. Conjugal transfer events were, however, observed in all protozoa evaluated. It therefore appears that the protozoa-based hypervirulence is not universal to all ruminants while conjugal transfer is more ubiquitous.

Fluorescence Spectroscopy of the Retina for Diagnosis of Transmissible Spongiform Encephalopathies

The feasibility of exploiting fluorescence spectra of the eye for diagnosis of transmissible spongiform encephalopathies (TSEs) was examined. Retinas from scrapie-positive sheep were compared with scrapie-negative sheep using fluorescence spectroscopy, and distinct differences in the fluorescence intensity and spectroscopic signatures were observed. The characteristic fluorescent signatures are thought to be the result of an accumulation of lipofuscin in the retina. It appears that the eye, in particular the retina, is a useful tissue for noninvasive examination of some neurological pathologies such as scrapie. The development of procedures based on examinations of the eye that permit the detection of neurological disorders in animals holds great promise.