David Ammons

Comparison of polymerase chain reaction and bacterial culture for Salmonella detection in the Muscovy duck in Trinidad and Tobago

OBJECTIVES The purpose of this study was to investigate the presence and serovar identity of Salmonella, at the national level, in farmed Muscovy ducks (Cairina moschata) in Trinidad and Tobago, and to compare the relative benefits of bacterial culture to those of polymerase chain reaction (PCR) for use in the routine detection and surveillance of Salmonella in these ducks. METHODS From March-September 2003, 110 fecal samples were collected from 82 farms across the islands of Trinidad and Tobago. Salmonella was isolated from fresh and frozen samples and the serotype of each was determined through bacterial culture. An in-house, nested PCR that detects all pathogenic Salmonella species was utilized in analyzing the samples. RESULTS Five samples were positive for Salmonella by bacterial culture, whereas 44 were positive by the nested PCR. Serovars isolated were Kiambu, Orion, Uganda, and two isolates from Group E1 whose H antigens could not be fully characterized. Of the samples, 87 (79%) gave equivalent PCR results for both enrichment broths-28 were positive for both and 59 were negative for both). However, 16 samples were positive for one broth, but not for the other, with the majority (14 of the 16) resulting positive for Selenite broth. PCR results for seven samples were inconclusive due to ambiguous band size or multiple bands near the expected band size. CONCLUSIONS In Trinidad and Tobago, the Muscovy duck does not appear to be a significant source of S. typhimurium or S. enteritidis, but it does harbor other Salmonella species. In-house, nested PCR represents a simple, relatively inexpensive and potentially more sensitive method than bacterial culture for the routine surveillance of pathogenic Salmonella in the Muscovy duck.

VERSATILE BLOOD BAGS FOR LABORATORY FEEDING OF MOSQUITOES

ABSTRACT Research programs in mosquito-borne diseases have necessitated the bloodfeeding of mosquitoes in captivity. Parafilm has been shown to be an effective membrane through which mosquitoes can access blood, and numerous feeding devices using parafilm membranes have been reported. However, these devices can be incompatible with experimental conditions, which may require small blood volumes, exact and constant blood temperatures, and inexpensive or disposable materials. We report herein on 2 methodologies that can be used to make parafilm blood bags with a support side and a feeding side made up of a thinner sheet of parafilm. Blood bags were easily and cheaply made to handle volumes as little as 150 µl or as large as 5 ml, to be disposable, and to be easily fastened to a heat source to provide constant and reproducible blood temperatures. Blood bags represent perhaps the most versatile and inexpensive approach to parafilm membrane feeding yet reported.

An Investigation of Bacillus thuringiensis in Rectal-Collected Fecal Samples of Cows

In order to better understand the range and role of Bacillus thuringiensis (Bt) and its toxins in nature, we have undertaken a study of Bt taken directly from the rectum of 117 cows from 37 farms on the Caribbean island of Trinidad. Thirty-seven fecal samples (32%) were found to contain at least one Bt. Generally only one or two isolates with a particular crystal morphology were isolated from any one sample, however, a few samples contained more, up to 11 isolates, suggesting post-ingestion amplification. Bioassays using larvae of Musca domestica, Caenorhabditis elegans and Tetrahymena pyriformis showed no observable toxicity in gross bioassays. Very small dot-like parasporal bodies, not generally characteristic of Bt, were isolated from 44% of the samples, which in many instances appeared unstable and whose relation to Bt Cry protein-containing parasporal bodies is unknown. In conclusion, we find little evidence for a host adapted strain of Bt in the cows examined, nor toxicity to organisms that might logically be associated with either the cow or its feces. The presence of a large number of isolates containing small dot-like parasporal bodies, possibly either poly-β-hydroxybutyrate storage bodies or Cry proteins, was unexpected and merits further investigation.

Effects of gamete concentration on the in vitro fertilization of manually extracted gametes of the oyster (Crassostrea rhizophorae)

A number of experiments were undertaken to better understand and characterize the effects of different gamete concentrations on the efficiency of in vitro fertilization in Crassostrea rhizophorae. Maximum fertilization efficiency was achieved with a broad range of sperm concentrations, thus minimizing the importance of sperm concentration in successful in vitro fertilizations. However, a linear and inverse relationship was found between egg concentration and fertilization efficiency. This suggests that egg concentration is an important parameter for successful in vitro fertilizations. No evidence was found to support oxygen depletion or the physical crowding of eggs as the cause for the deleterious effect of high egg concentrations. The deleterious effect was counteracted by continually washing the fertilized eggs, suggesting the presence of a water-soluble substance, originating from the zygote, as the cause of the egg effect. In addition, maximizing the total number of viable larvae/ml was found not to be strictly correlated with fertilization efficiency. Lastly, support is presented for the use of concentrations, instead of ratios, in determining optimal gamete numbers.

A novel Bacillus thuringiensis Cry-like protein from a rare filamentous strain promotes crystal localization within the exosporium

ABSTRACT Mutation of a novel cry-like gene (cry256) from Bacillus thuringiensis resulted in a protein crystal, normally located within the spores exosporium, being found predominately outside the exosporium. The cry256 gene codes for a 3-domain Cry-like protein that does not correspond to any of the known Cry protein holotypes.

An apparatus to control and automate the formation of continuous density gradients

A common problem associated with making a continuous density gradient is the unwanted mixing of liquids as the gradient solution is being delivered from the gradient mixer to the centrifuge tube. We show that by using a Styrofoam float on a thin drawn-out glass pipette, liquids from the gradient mixer will flow down the outside surface of the glass fiber and contact the float, after which the liquid spreads across the gradient surface without unwanted mixing. As the liquid in the tube rises, the float rises up the glass fiber, thereby allowing the creation of gradients without the need to monitor the process.

Rapid in vivo exploration of a 5S rRNA neutral network

A partial knockout compensation method to screen 5S ribosomal RNA sequence variants in vivo is described. The system utilizes an Escherichia coli strain in which five of eight genomic 5S rRNA genes were deleted in conjunction with a plasmid which is compensatory when carrying a functionally active 5S rRNA. The partial knockout strain is transformed with a population of potentially compensatory plasmids each carrying a randomly generated 5S rRNA gene variant. a The ability to compensate the slow growth rate of the knockout strain is used in conjunction with sequencing to rapidly identify variant 5S rRNAs that are functional as well as those that likely are not. The assay is validated by showing that the growth rate of 15 variants separately expressed in the partial knockout strain can be accurately correlated with in vivo assessments of the potential validity of the same variants. A region of 5S rRNA was mutagenized with this approach and nine novel variants were recovered and characterized. Unlike a complete knockout system, the method allows recovery of both deleterious and functional variants.. The method can be used to study variants of any 5S rRNA in the E. coli context including those of E. coli.

Cry-like genes, in an uncommon gene configuration, produce a crystal that localizes within the exosporium when expressed in an acrystalliferous strain of Bacillus thuringiensis

Cry proteins are pesticidal toxins produced by the bacterium Bacillus thuringiensis (Bt), which aggregate in sporulating cells to form a crystal. Except in a relatively few cases, these crystals are located outside the exosporium that surrounds the spore. Bt2-56 is a strain of Bt that has the relatively uncommon characteristic of locating its Cry protein-containing crystal within the exosporium, and in association with a long, multifiber filament. With the ultimate goal of both understanding and manipulating the localization of Cry proteins within the exosporium, we sought to identify the genes coding for the exosporium-localized Cry proteins in Bt2-56. Herein we show (i) that five cry-like genes are present in the genome of Bt2-56, (ii) that two pairs of these genes show organizational similarity to a relatively uncommon gene configuration that coexpress a cry gene along with a gene whose product aids crystal formation and (iii) that when one of these two gene pairs (cry21A-cdA) is expressed in an acrystalliferous strain of Bt, crystals are formed that localize within the exosporium. In Bt ssp. finitimus, the only other strain in which crystal localization has been studied, a Cry protein needed expression of two non-cry ORFs in order to localize within the exosporium, indicating that there are some mechanistic differences for crystal localization between Bt ssp. finitimus and Bt2-56.

The Use of Polystyrene Beads to Prepare Arrayed Samples of Bacillus thuringiensis for Microscopic Examination.

A common activity in the global search for useful Cry toxins is the microscopic screening of bacterial colonies for the presence of Bacillusthuringiensis. High-throughput screens require that aliquots from large numbers of colonies be arrayed on a microscopic slide. However, precisely placing a small amount of bacteria on a slide, and at a density that is useful for microscopic examination, is both difficult to achieve and time consuming. Herein we share a simple technique that utilizes a hooked wand and small polystyrene beads to quickly collect, and uniformly apply, aliquots of bacterial colonies onto gridded microscope slides in a manner optimal for viewing. If desired, libraries of examined bacteria can simultaneously be generated by discharging the beads into indexed multiwell plates. This simple and inexpensive method is robust, suitable for both light and phase contrast microscopy, and has been also used successfully to screen randomly mutated bacteria for phenotypic changes.

MicroTracker: a Data Management Tool for Facilitating the Education of Undergraduate Students in Laboratory Research Environments.

Many undergraduate laboratories are, too often, little more than an exercise in “cooking” where students are instructed step-by-step what to add, mix, and, most unfortunately, expect as an outcome. Although the shortcomings of “cookbook” laboratories are well known, they are considerably easier to manage than the more desirable inquiry-based laboratories. Thus the ability to quickly access, share, sort, and analyze research data would make a significant contribution towards the feasibility of teaching/mentoring large numbers of inexperienced students in an inquiry-based research environment, as well as facilitating research collaborations among students. Herein we report on a software tool (MicroTracker) designed to address the educational problems that we experienced with inquiry-based research education due to constraints on data management and accessibility.