Human skin in organ culture and human skin cells (keratinocytes and fibroblasts) in monolayer culture for assessment of chemically induced skin damage.

Human skin cells (epidermal keratinocytes and dermal fibroblasts) in monolayer culture and human skin in organ culture were exposed to agents that are known to produce irritation (redness, dryness, edema and scaly crusts) when applied topically to skin. Among the agents used were three well accepted contact irritants (i.e., all-trans retinoic acid [RA], sodium lauryl sulfate [SLS] and benzalkonium chloride) as well as the corrosive organic mercury compound, aminophenyl mercuric acetate (APMA), and 5 contact sensitizers (oxazolone, nickel sulfate, eugenol, isoeugenol and ethylene glycol dimethacrylate [EGDM]). As a group, the contact irritants (including the corrosive mercuric compound) were cytotoxic for keratinocytes and fibroblasts and suppressed growth at lower concentrations than the contact sensitizers. The contact irritants also produced histological changes (hyperplasia, incomplete keratinization, loss of the granular layer, acantholysis and necrosis) in organ-cultured skin at dose levels at which the contact sensitizers appeared to be inert. Finally, the profile of secreted molecules from organ-cultured skin was different in the presence of contact irritants versus contact sensitizers. Taken together, these data suggest that the use of organ-cultured skin in conjunction with cells derived from the skin in monolayer culture may provide an initial approach to screening agents for deleterious changes in skin.

Patterns of antimicrobial susceptibility in Michigan wildlife and bovine isolates of Mycobacterium bovis.

The state of Michigan has recognized the presence of Mycobacterium bovis in its free-ranging white-tailed deer population since 1994. This endemic infection is primarily located in a 12-county area in the northeastern lower peninsula of Michigan. A statewide surveillance and eradication program of the disease has been in effect since 1994. Worldwide, Mycobacterium tuberculosis complex organisms have a known predilection toward development of antimicrobial resistance. The objective of this study was to investigate the antimicrobial susceptibility of M. bovis isolates from white-tailed deer in Michigan and detect any changes in susceptibility over time. M. bovis isolates from 2 fall hunting seasons (1999 and 2004) were used in this study. The fall season of 2004 marked the first documented case of direct transmission of M. bovis from a wild deer to a human in Michigan. Since M. bovis is a zoonotic disease, knowledge of susceptibility can expedite treatment options in humans. M. bovis isolates were obtained from 58 deer, 4 coyotes, 3 cattle, 2 raccoons, and 1 human case from the 2 years combined. Methods of susceptibility testing included 1% proportion agar plates and Bactec radiometric broth testing. M. bovis was found to be uniformly resistant to the antibiotic pyrazinamide; this resistance is common to all M. bovis isolates. No other antimicrobial resistance was found in any of the tested M. bovis isolates, which may be, in part, attributed to the lack of any significant treatment pressure in wildlife.

Experimental aerosol inoculation of Mycobacterium bovis in North American opossums (Didelphis virginiana).

The goal of this study was to evaluate the susceptibility of North American opossums (Didelphis virginiana) to aerosol inoculation of Mycobacterium bovis at two dose levels in order to gain information on disease pathogenesis, fecal shedding of the organism, and the potential role that opossums play in the spread of this disease in nature. Six opossums received high dose (1 x 10(7) colony forming units (cfu) by aerosol inoculation, six opossums received low dose (1 x 10(3) cfu inoculation, and six opossums were sham-inoculated with sterile water and served as controls. Lungs were the most frequently infected tissues, with nine of 12 inoculated opossums positive for M. bovis on culture. Gross lesions consisted of multifocal pneumonia and enlarged lymph nodes. Microscopically, granulomatous pneumonia and granulomatous lymphadenitis associated with acid-fast bacilli were present in eight of 12 inoculated opossums. Fecal shedding of M. bovis was uncommon at both inoculation doses. While opossums were highly susceptible to aerosol inoculation of M. bovis, they did not become emaciated or develop widely disseminated lesions. From this study, opossums may transmit tuberculosis by aerosol infection to other opossums in close contact and serve as a source of infection to carnivores that feed upon them, however, transmission of the disease to large herbivores by fecal shedding or direct contact may be less likely.

Experimental inoculation of North American opossums (Didelphis virginiana) with Mycobacterium bovis.

Eight North American opossums (Didelphis virginiana) were inoculated with 1 x 10(5) colony forming units of Mycobacterium bovis to investigate their potential as reservoir hosts for bovine tuberculosis in Michigan. Four animals received this dose orally and four were inoculated intramuscularly (i.m.). In each group, two animals were euthanized 1 mo postinoculation (PI) and two at 2 mo PI. Four control animals were housed separately and sacrificed in the same manner as those inoculated. One of four orally inoculated opossums and three of four i.m.-inoculated opossums were positive for M. bovis by culture of tissues obtained at necropsy. The oral recipient had positive cultures from intestine and pooled lymphoid samples. Pooled lymphoid samples were positive in three i.m.-inoculated animals and two of these also had positive liver and lung cultures. One animal with gross and histologic lesions compatible with tuberculosis had negative tissue cultures. The findings suggest that opossums are susceptible to M. bovis infection by multiple routes, although their relative susceptibility compared to true reservoir hosts appears to be low.

Epidemiology of Mycobacterium bovis in free-ranging white-tailed deer, Michigan, USA, 1995-2000.

An endemic area of bovine tuberculosis (TB) (Mycobacterium bovis) currently affecting wild white-tailed deer (Odocoileus virginianus) in northern lower Michigan, USA, constitutes the first self-sustaining outbreak of the infection in free-ranging North American cervids. Given this precedent, epidemiologic insights gained from the outbreak afford the opportunity to guide not only current surveillance and intervention but also control efforts for future outbreaks involving wildlife reservoirs. Our specific objectives were to evaluate retrospective data from field surveillance conducted from 1995 to 2000 to determine apparent prevalence, trends in apparent prevalence, and the effects of various factors on the odds of being M. bovis positive. Data were gathered from post-mortem examinations of 62,560 wild deer collected from all 83 Michigan counties. Records of survey method, sex, age, geographic area and infection status as determined by mycobacterial culture were subjected to trend analysis and multivariable logistic regression. Apparent prevalence for the period was 0.54% (336/62,560) statewide. Prevalence varied widely with geographic area, but significantly decreased since 1995 in the core area of the outbreak-which coincided with implementation of control strategies. Significant risk factors were geographic area, sex, age, and the sex-by-age interaction. The survey method by which deer were obtained for testing was not a predictor of infection. Our results to date suggest an outbreak characterized by broad areas of very low prevalence surrounding focal areas where prevalence is sometimes orders-of-magnitude higher (e.g., deer originating from the core area were up to 147 times more likely to be TB positive than deer from other areas). Our results also identify older male deer as most likely to be M. bovis positive (OR=11.3, 95% CI 3.2, 40.3 for bucks > or =5 years vs. does < or =1.5 years)-an observation consistent with the biology and behavior of the species. Synthesizing these results with those of other ongoing investigations, we hypothesize a two-stage model of disease transmission where TB is maintained at very low prevalence in matriarchal groups, with primary dissemination of the disease attributable to the dispersal and movements of bucks (as well as to the large aggregations of animals created by human activities).