lacZ Reporter System for Use in Borrelia burgdorferi

Regulation of gene expression is critical for the ability of Borrelia burgdorferi to adapt to different environments during its natural infectious cycle. Reporter genes have been used successfully to study gene regulation in multiple organisms. We have introduced a lacZ gene into B. burgdorferi, and we show that B. burgdorferi produces a protein with detectable β-galactosidase activity in both liquid and solid media when lacZ is expressed from a constitutive promoter. Furthermore, when lacZ is expressed from the ospC promoter, β-galactosidase activity is detected only in B. burgdorferi clones that express ospC, and it accurately monitors endogenous gene expression. The addition of lacZ to the repertoire of genetic tools available for use in B. burgdorferi should contribute to a better understanding of how B. burgdorferi gene expression is regulated during the infectious cycle.Borrelia burgdorferi sensu lato, the pathogen that causes Lyme disease (7), alternates between two distinct environments, an arthropod vector and a vertebrate host. As B. burgdorferi moves from one milieu to the other, its ability to adapt and survive requires dramatic changes in gene expression. Many studies have shown that different B. burgdorferi gene products are upregulated or downregulated at specific times during the infectious cycle (19, 31) and in response to host and environmental signals (6, 8a, 15, 24, 25). Although it is clear that B. burgdorferi alters gene expression to adapt to different environments, the genetic tools for studying gene regulation in B. burgdorferi are limited.Within the last 2 decades, the complete genomic sequence of B. burgdorferi strain B31 was published (10, 14) and techniques for basic genetic manipulation of B. burgdorferi became available (5, 11, 13, 27-29, 36). A chloramphenicol acetyltransferase (CAT) gene was the first reporter gene that was fused to B. burgdorferi promoters for analysis of promoter strength (33). The development of luciferase (4) and multiple fluorescent proteins (9, 11, 30) as reporter systems in B. burgdorferi followed. Although these systems have value, there are limitations with each. β-Galactosidase, encoded by lacZ, has been used extensively as a convenient reporter gene in Escherichia coli and is still applicable to a broad range of organisms, both prokaryotic and eukaryotic, but has not yet been used with B. burgdorferi. β-Galactosidase activity can be monitored easily and quickly by simple colorimetric assays in both liquid and solid media, neither of which require expensive or specialized equipment. Additionally, a wide variety of substrates for β-galactosidase allow for different levels of sensitivity in either in vitro or in vivo detection formats (17). Having lacZ available as a genetic tool for B. burgdorferi would enhance investigation of the complex regulatory events that are integral to the spirochete''s infectious cycle. To this end, we developed lacZ as a reporter gene in B. burgdorferi and demonstrated its utility.     

Factors facilitating research: a survey of zoo and aquarium professionals

The purpose of this survey was to assess the background, training, and perceptions of professionals conducting and coordinating research at North American zoos and aquariums accredited by the Association of Zoos and Aquariums (AZA) and to identify the factors they considered critical to successful scientific programs. We analyzed responses to a 57‐item questionnaire from 231 professionals at AZA zoos and aquariums. The majority of those surveyed conducted behavioral research, conducted research only in a captive setting, held curatorial positions, had their salaries supported by their institutions' operating budget, and considered themselves part of a successful scientific program. About 30% of those we surveyed possessed a doctoral research degree in comparison to 55% possessing lesser level degrees—19% with master's, 34% with bachelor's, and 2% with other degrees. Support from the chief executive officer and personnel dedicated to conducting scientific programs were judged as the two most important factors contributing to the successful scientific programs. The information provided in this report may be used to develop and improve both established and newly initiated scientific programs in zoos and aquariums. Zoo Biol 29:663–675, 2010. © 2010 Wiley‐Liss, Inc.     

Preparing chimpanzees for laboratory research

The chimpanzee is the only representative of the Great Apes that is extensively involved in biomedical research in primate laboratories. These apes are used as animal models in a variety of studies, including research on infectious disease, parasitic disease, pharmacokinetic studies, neuroscience, cognition, and behavior. Chimpanzees used in biomedical research in the United States reside largely in six specialized research and holding facilities, and most of the research with them is conducted at these sites. Given the relatively small population of chimpanzees and its importance to biomedical research, it is imperative that we carefully manage the care, production, and use of these animals in biomedical research studies. Selection criteria and preparation techniques are reviewed in this article in an effort to begin a discussion on best practices for choosing and handling chimpanzees participating in biomedical research. The use of routine health assessment information is described for subject selection, as are behavioral issues to be considered. Due to the relatively small number of chimpanzees available, issues related to experimental design and multiple uses of chimpanzees are discussed. Practices related to the transportation and acclimation of chimpanzees are described. Finally, behavioral conditioning procedures are discussed, including habituation, desensitization, and positive reinforcement training that have been applied to reduce animal distress and improve the quality of the science being conducted with chimpanzee subjects.     

The use of positive reinforcement training techniques to enhance the care, management, and welfare of primates in the laboratory

Handled frequently and subjected to a wide range of medical procedures that may be particularly invasive, nonhuman animals in a laboratory setting have unique needs. To produce the most reliable research results and to protect and enhance the well-being of the animals, it is desirable to perform these procedures with as little stress for the animals as possible. Positive reinforcement training can use targeted activities and procedures to achieve the voluntary cooperation of nonhuman primates. The benefits of such work include diminished stress on the animals, enhanced flexibility and reliability in data collection, and a reduction in the use of anesthesia. Training also provides the means to mitigate social problems, aid in introductions, reduce abnormal behavior, enhance enrichment programs, and increase the safety of attending personnel. This article describes the application of operant conditioning techniques to animal management.     

Multiple trophic pathways support fish on floodplains of California's Central Valley

We used compound-specific isotope analysis of carbon isotopes in amino acids (AAs) to determine the biosynthetic source of AAs in fish from major tributaries to California's Sacramento-San Joaquin river delta (i.e., the Sacramento, Cosumnes and Mokelumne rivers). Using samples collected in winter and spring between 2016 and 2019, we confirmed that algae are a critical component of floodplain food webs in California's Central Valley. Results from bulk stable isotope analysis of carbon and nitrogen in producers and consumers were adequate to characterize a general trophic structure and identify potential upstream and downstream migration into our study site by American shad Alosa sapidissima and rainbow trout Oncorhynchus mykiss, respectively. However, owing to overlap and variability in source isotope compositions, our bulk data were unsuitable for conventional bulk isotope mixing models. Our results from compound-specific carbon isotope analysis of AAs clearly indicate that algae are important sources of organic matter to fish of conservation concern, such as Chinook salmon Oncorhynchus tshawytscha in California's Central Valley. However, algae were not the exclusive source of energy to metazoan food webs. We also revealed that other sources of AAs, such as bacteria, fungi and higher plants, contributed to fish as well. While consistent with the well-supported notion that algae are critical to aquatic food webs, our results highlight the possibility that detrital subsidies might intermittently support metazoan food webs.     

The Environment and the Microbial Ecology of Human Skin

Microbial flora of the skin of three human population groups representing different natural environments was examined quantitatively and qualitatively to determine whether environmental differences in temperature and humidity can influence the microbial flora of normal skin. Five anatomical skin sites - hands, back, axillae, groin, and feet - were sampled from 10 subjects working in a high-humidity, high-temperature environment, 10 subjects from a low-temperature, high-humidity environment, and 10 subjects working in a moderate-temperature and low-humidity environment. Bacterial populations were significantly larger from the back, axillae, and feet in individuals from the high-temperature and high-humidity environment as compared to the moderate-temperature, low-humidity environment. High humidity and low temperature had no significant effect on total populations, but this group showed a higher frequency of isolation of fungi, and gram-negative bacteria from the back and feet. Although there was an indication that increase in the environmental humidity could result in an increased frequency of isolation of gram-negative bacteria, there was no evidence that an increase in either temperature or humidity altered the relative proportions of gram-negative bacteria in the predominantly gram-positive microbial flora found on normal skin. It was concluded that, although climatic changes may cause fluctation in microbial populations from certain sites, they are not a major influence on the ecology of the microbial flora of normal skin in the natural environment. The variables introduced by studying individuals in their natural environment and the influence of these on the results are discussed.     

Effects of human activity on chimpanzee wounding

Reducing the frequency and/or severity of aggression and wounding is a major concern of people managing socially housed chimpanzees. One factor that has not been investigated intensively for its effect on captive chimpanzee agonism is the presence of humans. Therefore, we examined an archival database of wounding incidents among 88 adult and adolescent members of 8 social groups of chimpanzees (42 females, 46 males) to determine whether variable levels of human activity (higher weekday versus lower weekend-day activity levels) in the colony was associated with changes in chimpanzee wounding patterns. Wounding was tabulated for each group for periods of 38 - 118 months. A series of Chi-square tests indicated that there were a greater than expected number of wounding episodes on weekdays but that day of the week did not affect the age or sex distribution of wounding. Together, these results suggest that the presence of personnel completing routine activities is associated with chimpanzee agonism. Zoo Biol 16:327–333, 1997. © 1997 Wiley-Liss, Inc.     

Nitric oxide effect on coloncyte metabolism: Co-action of sulfides and peroxide

Luminal levels of nitric oxide/nitrite are high in colitis. Whether nitric oxide is injurious or protective to human colonocytes is unknown and the role of nitric oxide in the genesis of colitis unclear. The aims were to establish whether nitric oxide was injurious to oxidation of substrates (n-butyrate and D-glucose) in isolated human and rat colonocytes both alone and in the presence of hydrogen sulfide and hydrogen peroxide, agents implicated in cell damage of colitis. Nitric oxide generation from S-nitrosoglutathione was measured by nitrite appearance. Colonocytes were isolated and incubated with [1-¹⁴C] butyrate or [6-¹⁴C] glucose and 2.6 M nitric oxide, 1.5 mM sodium hydrogen sulfide or 2.5 mM hydrogen peroxide. Acyl-CoA esters were measured by high performance liquid chromatography, ¹⁴CO₂ radiochemically and lactate/ketones by enzymic methods. Results indicate that nitric oxide very significantly (p < .001) reduced acyl-CoA formation but did not impair ¹⁴CO₂ generation. Peroxide and sulfide with nitric oxide resulted in significant reduction (p < 0.01) of substrate oxidation to CO₂. Sulfide significantly stimulated release of nitric oxide from S-nitrosoglutathione. The principal conclusion is that nitric oxide diminishes CoA metabolism in colonocytes. CoA depletion has been observed in chronic human colitis for which a biochemical explanation has been lacking. For acute injurious action in human colonocytes nitric oxide requires co-action of peroxide and sulfide to impair oxidation of substrates in cells. From current observations treatment of colitis should aim to reduce simultaneously nitric oxide, peroxide and sulfide generation in the colon.