Once the stress condition was removed, a fraction of the cells recovered the capacity to grow in laboratory media, thereby indicating a potential of the E. coli O1O4:H4 cells to cause human disease. The formation of VBNC bacterial cells on plants also was previously described for Listeria monocytogenes on parsley. The number of viable L. monocytogenes cells was 1 to 2 log higher than the culturable cells recovered from parsley grown in greenhouses at 20uC under low relative humidity ; growth of the VBNC cells was not restored on the plants when the RH was increased to 100%. Although we did not examine for E. coli O157:H7 recovery from the VBNC-like state, future efforts might investigate whether those cells can recover and resume growth either under growth-conducive conditions on lettuce or after removal of the cells from the plants and prior to or after plating for viable cell enumeration. Overall, the toxigenic strain EC4045 survived in similar quantities as ATCC 700728 on lettuce. A recent study showed that certain lineages of E. coli are more commonly associated with plants and presumably have evolved the capacity to tolerate plant associated environments better than E. coli isolated from other sites. Because we only compared two strains, subsequent investigations should examine multiple attenuated and virulent O157:H7 strains isolated from different sites for their capacity to colonize and persist on lettuce under field relevant conditions. Survival of E. coli O157:H7 on lettuce also was measured in two field studies. Culturable amounts of strain ATCC 700728 declined shortly after inoculation onto plants in the field, as we reported previously. Rates of cell decline were similar to E coli O157:H7 on lettuce in the growth chamber directly inoculated in drops with a pipette. Real-time PCR estimates of E. coli O157:H7 ATCC 700728 in lettuce leaf washes showed that this strain was present on the plants immediately after inoculation and 2 h later in quantities equivalent to the inoculum levels. Importantly,flower plastic pots the rapid decline in culturable E. coli during the first hours after application onto plants in the field was not due to an inability to remove the organism from the lettuce or from dispersal and lack of strain attachment.
Rather, it appears that the majority of the E. coli cells in the inoculum either died shortly after application or entered a VBNC state. In contrast to the growth chamber experiment results, the numbers of E. coli O157:H7 cells were below detection by real time PCR within 2 days after inoculation onto field lettuce. These findings suggest that the E. coli O157:H7 cells and genomic DNA were degraded rapidly. Environmental parameters such as solar radiation, heat, and water stress could be responsible for the differences in the stability of E. coli O157:H7 DNA in the field compared with the laboratory. Alternatively, cell maintenance might depend on other microorganisms on the leaves. Also, it is notable that different lettuce cultivars were used in the field and growth chamber studies, which may have impacted survival. The potential for cultivar-dependent effects was shown for E. coli O157:H7 on lettuce cultivars grown under axenic conditions in the laboratory. Because the E. coli O157:H7 ATCC 700728 DNA was degraded on field-grown plants within 2 days after inoculation, it is unlikely that the organism developed a VBNC state, particularly over longer time scales . However, this possibility could not be directly addressed using the PMA real time PCR assay in the field trials. Viable cell amounts measured by culturing and PMA real-time PCR were in agreement immediately after application of ATCC 700728 onto laboratory grown lettuce, but PMA-mediated detection was impaired on plants from the field. For those plants, the viable cell number estimates for strain ATCC 700278 were 10-fold lower as measured by PMA real-time PCR than by colony enumeration and total cell numbers estimated by real-time PCR. These differences might have been due to the higher turbidity or opacity of the lettuce plant washes from the field samples, thereby preventing light from penetrating the suspension during the PMA photo inactivation step. This interference would prevent the inactivation of free PMA, resulting in sufficient quantities of the compound to bind genomic DNA released from viable cells during the subsequent DNA extraction and amplification steps. In addition, the PMA real-time PCR assay was unable to detect low numbers of cells. Attempts to detect the ATCC 700278 strain after concentrating the leaf washes were unsuccessful . Similarly, PMA real-time PCR was found to be more reliable for viable cell detection in diluted wastewater than in pooled and concentrated wastewater samples. Such factors strongly limit the overall usefulness of this approach for field-grown plants. However, this method is informative for examination of E. coli O157:H7 on ‘‘cleaner’’ plants grown in the growth chamber and not exposed to the biotic conditions that are common outdoors.
In conclusion, this study illustrates the similarities and differences between controlled studies of human pathogens on plants in a growth chamber and experiments examining the population dynamics of pathogens on plants under production-like conditions in the field. By applying relevant environmental conditions and droplet inoculation in the growth chamber, we were able to more closely mimic the rapid decline in E. coli O157:H7 culturability that was observed after inoculation of this organism onto lettuce plants in the Salinas Valley. Culture-independent assessments con firmed that the pathogen remains on the plant long after application. However, field studies showed that at least for the majority of E. coli O157:H7 cell inoculants, the loss in culturability was most likely due to cell death rather than an inability to form colonies on standard laboratory media. Hence, this work confirmed our observations that low numbers of E. coli O157:H7 persist on lettuce grown in the Salinas Valley and variations in pathogen survival among individual plants are dependent on other unknown factors .Ethnically diverse populations are disproportionately exposed to hazardous environmental materials by virtue of living in close proximity to toxic waste materials. One-half of the uranium in the US is found on American Indian lands, where mining, milling, processing,grow table and waste storage has commonly occurred. From the 1940s to the 1980s, northwestern New Mexico alone contributed 40% of the U.S. U production. The study setting was a prime target of U mining for military purposes from the 1940s to the 1980s. Diné lands were one of the prime targets for mining, contributing thirteen million tons of U ore for military use from 1945 to 1988 and leaving more than 1100 abandoned and partially unreclaimed U mines, mills, and waste piles. The extent of the health threats to the Diné community exposed to these sites is anticipated to be high. Uranium enters the body primarily by inhalation or ingestion , and then it enters the bloodstream and is deposited in tissues, primarily the kidneys and bones. Human and animal studies of those exposed to U have shown kidney toxicity, as well as damage to the liver, muscles, cardiovascular, and nervous systems. Arsenic is a teratogen. Cadmium can accumulate in organs and impair renal function; Lead is associated with adverse effects on the nervous, developmental, renal and reproductive systems. Selenium toxicosis can cause neurological and gastrointestinal problems and endocrine function disruption and is a teratogen in several species of animals; Molybdenum has been shown to be a male reproductive toxicant in animals and humans.
The Dine Network for Environmental Health study worked closely with 20 Navajo chapters or communities to address the concerns of the community and leaders regarding the health effects of environmental exposures to unreclaimed U mines and mill sites. The DiNEH cohort found that 40% of participants lived within 3.2 km of an abandoned U mine, 16% lived near a U mill, and 12.6% of children played on tailing piles or waste dumps. In self-reported data of past exposure, 15.4% utilized materials from abandoned U mine sites to build homes or other structures, 1.8% sheltered livestock in abandoned mines, 12.7% herded their livestock near contaminated sites, and 12.8% said their livestock came into contact with contaminated water. In this population, surface and groundwater utilization is important for human and livestock consumption as well as agriculture. In affected areas, greater than half of the Diné people continue to drink from unregulated water sources. DiNEH data indicate that more than 80% of Diné people haul water for all uses, including irrigation and livestock watering, despite having regulated water in the home. Mutton or lamb meat and organs are primary food staples in this community, and all aspects of the animal are used; there are important cultural uses for the animal. The purpose of this study was to determine if sheep, a harvested primary dietary food staple on Diné lands in northwestern NM, were contaminated with U and other associated heavy metals. Past studies of these areas in NM demonstrated that the consumption of U contaminated food may occur through the ingestion of locally raised livestock and by way of their forage. Food chain contamination in locally harvested food in the Diné community in NM was reported as a plausible exposure pathway . The current study was undertaken to reexamine and contribute more recent data and introduce data not previously reported . This was a descriptive, comparative study examining contamination levels in locally harvested O. aries, their forage, and associated soil and water from reservation areas within a 3.2 km radius of previously mined areas. Data obtained from the DiNEH study cohort served as one of the sources for identifying subjects and samples of food, herbs, water, forage, and soil. Additional participants were recruited by word-of-mouth, home visits, and advertising at public tribal community events. Of the DiNEH cohort respondents, those individuals who reported harvesting sheep were recruited for potential participation in the present study. Sheep chosen represented a range of ages , their proximity to mining structures, and a variety of water sources. Three ewes were included in this study. The individual sheep data are compared and reported to reflect an accurate measure of heavy metal uptake in O. aries tissue with respect to the associated forage, water consumption, and their environment.The study area is a semi-arid to arid region of the American Southwest in northwestern NM on Diné reservation lands . The average precipitation was less than 25 cm per year according to meteorological data for NM for the study period. Despite several decades of longstanding drought in the area, community members still participated in subsistence activities. Two “Chapters” provided sheep and associated samples. The Mariano Lake Chapter is 272 km2 of land mass and the Churchrock Chapter is 233 km2 . Recruitment was initiated on May 2012 and enrollment began in July 2012. All samples were collected from 10 November to 13 December 2012. This study focused on sheep as a food staple and was part of a larger “parent” research project that examined subsistence farming on the reservation, including the metal contamination of herbs.Three sheep came from two different chapters. From 10 November to 13 December 2012, three ewes were contributed to the study. The O. aries tissue samples were collected in the field immediately after slaughter and included muscle, bone, intestine, lung, liver, kidney, and wool. Upon collection, all samples were placed on dry ice and shipped to the University of New Mexico Analytical Chemistry Laboratory Earth and Planetary Sciences Department for storage and analysis. The 13th cortical rib bone samples were sheared from the proximal, middle, and distal portions and composited together after the removal of excess tissue. The proximal, medial, and distal portions of the small intestine were collected and composited. For lung tissue, the samples were derived from each anatomical lobe and composited. Both kidneys were sampled, and the cortex and medulla were composited separately. Composited muscle samples were from the proximal, medial, and distal portions of the gastrocnemius. Of the wool fiber samples, the area over the neck, middle section, and posterior portions of the animal were sampled and composited. All tissues were representative of 1 g of dried tissue. For coupled organs, the tissues collected from the right side of the sheep were labeled as the sample, and one duplicate was obtained for each tissue type from the left side of the animal. A composited duplicate or replicate was obtained for non-dual type organs .