R. D. Ellender

Detection of the nifH gene of Methanobrevibacter smithii : a potential tool to identify sewage pollution in recreational waters

Aims:  The goal of this study was to develop and test the efficacy of a PCR assay for the environmental detection of the nifH gene of Methanobrevibacter smithii, a methanogen found in human faeces and sewage.

Development of a Swine-Specific Fecal Pollution Marker Based on Host Differences in Methanogen mcrA Genes

ABSTRACT The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10−6 g of wet pig feces in 500 ml of phosphate-buffered saline and 10−4 g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker.

Methods To Increase Fidelity of Repetitive Extragenic Palindromic PCR Fingerprint-Based Bacterial Source Tracking Efforts

ABSTRACT The goal of the study was to determine which similarity coefficient and statistical method to use to produce the highest rate of correct assignment (RCA) in repetitive extragenic palindromic PCR-based bacterial source tracking. In addition, the use of standards for deciding whether to accept or reject source assignments was investigated. The use of curve-based coefficients Cosine Coefficient and Pearsons Product Moment Correlation yielded higher RCAs than the use of band-based coefficients Jaccard, Dice, Jeffreys x, and Ochiai. When enterococcal and Escherichia coli isolates from known sources were used in a blind test, the use of maximum similarity produced consistently higher RCAs than the use of average similarity. We also found that the use of a similarity value threshold and/or a quality factor threshold (the ratio of the average fingerprint similarity within a source to the average similarity of this sources isolates to an unknown) to decide whether to accept source assignments of unknowns increases the reliability of source assignments. Applying a similarity value threshold improved the overall RCA (ORCA) by 15 to 27% when enterococcal fingerprints were used and 8 to 29% when E. coli fingerprints were used. Applying the quality factor threshold resulted in a 22 to 32% improvement in the ORCA, depending on the fingerprinting technique used. This increase in reliability was, however, achieved at the expense of decreased numbers of isolates that were assigned a source.

Natural Enterovirus and Fecal Coliform Contamination of Gulf Coast Oysters

The numbers of fecal coliforms and enteroviruses present in oysters and/or their growing waters of two Mississippi reefs were determined over a 12-month period. Bacterial and viral levels reflected the classification of the waters at each location as set by the Mississippi State Board of Health in compliance with the National Shellfish Sanitation Program, but statistically significant correlations between these levels were not observed. Twelve viral isolates were found at an approved oyster harvesting location, eight of which were identified as poliovirus type 1. At the prohibited site, 146 viruses were isolated including poliovirus types 1 and 2, echovirus type 24 and several isolates which remain to be identified. The number of virus isolates from samples from each location represented approximately 35% of the number of plaques observed; however, no consistent ratio of plaque to confirmed virus was demonstrated. The results suggest that the fecal coliform levels in oyster growing waters do not reflect the level of virus contaminaton in either approved or prohibited waters.

OBSERVATIONS ON THE PRIMARY CULTURE OF HEMOCYTES OF PENAEUS

ABSTRACT A study of the primary culture of hemocytes of Penaeus was conducted to examine growth and maintenance of these cellular populations. Hemocytes rapidly attached to flask surfaces and could be maintained for approximately one month. A medium consisting of 2 × L-15, 20% Cellect Gold fetal calf serum, 20% AKN salt solution, and other supplements was suitable for long term hemocyte maintenance. Initial cell counts were found to be more representative of monolayer confluency than volume of hemolymph collected. Bacterial contamination of cell cultures was common, but could be controlled by the addition of antibiotics to the growth medium. Contaminants were identified as members of the genera Vibrio, Pseudomonas, Flavobacterium, Aeromonas, or Proteus. Attempts to subculture hemocyte monolayers using enzymatic and mechanical methods were unsuccessful. Additional investigations concluded that hemocyte cryoprotection by DMSO was poor when cells were frozen at –20 or -70°C, that monolayer integrity was optimal at a temperature range of 25–32°C, that hemocyte populations appear to contain heterologous surface residues, and that only 1-2% of fresh hemocytes are in an active proliferation cycle at the time of collection.

Methanobrevibacter ruminantium as an Indicator of Domesticated-Ruminant Fecal Pollution in Surface Waters

ABSTRACT A PCR-based assay (Mrnif) targeting the nifH gene of Methanobrevibacter ruminantium was developed to detect fecal pollution from domesticated ruminants in environmental water samples. The assay produced the expected amplification product only when the reaction mixture contained DNA extracted from M. ruminantium culture, bovine (80%), sheep (100%), and goat (75%) feces, and water samples from a bovine waste lagoon (100%) and a creek contaminated with bovine lagoon waste (100%). The assay appears to be specific and sensitive and can distinguish between domesticated- and nondomesticated-ruminant fecal pollution in environmental samples.

Analysis of Shrimp Hemolymph and Ionic Modification of a Penaeus Cell Culture Formulation

Abstract The levels of 19 constituents of penaeid shrimp hemolymph were analytically determined. Ionic components of hemolymph were influenced by water salinity but not by water pH or temperature. Levels of hemolymph metabolites varied from sample to sample; the differences were possibly generated by variations in diet and stress. Enzymatic measurements were reported for possible comparison with future studies on stress, health, or immune status of cultured penaeid populations. Data were also used to determine an appropriate ionic mixture for shrimp cell culture.

Relaying to Decrease the Concentration of Oyster-Associated Pathogens

Oysters experimentally contaminated with indicator bacteria, Salmonella and poliovirus were used in relaying studies designed to measure microbial elimination under a variety of environmental conditions. Two factors, level of microorganism in the oyster and temperature of the water, were important in determining the length of time necessary to purge the contaminating organisms. Oysters under physiological stress cleansed at a slower rate than did healthy oysters. Based on the expected level of pathogen contamination in naturally polluted oysters, healthy relaid oysters were capable of cleansing in a 7-d period provided the temperature was above 10°C. These results were verified by following the elimination of indicator bacteria and poliovirus in commercially relaid oysters. Fecal indicator bacteria and enteric pathogenic bacteria were eliminated at similar rates but fecal coliform levels did not correlate with virus elimination. Relaying waters may contain some indicator bacteria and this study suggested that fecal coliforms may not be useful as end-point indicators for this method of oyster purification.

Fish cell culture: Characteristics of a cell line from the silver perch,Bairdiella chrysura

SummaryA cell line designated SP-1 was established from tissue of the silver perch,Bairdiella chrysura. Cells were fibroblast-like and grew best at 26°C in Leibovitz medium (L-15) containing 15% fetal bovine serum and 0.150m sodium chloride. Passage 1 to passage 9 SP-1 cells contained a chromosome number of 48; at passages 27 and 50 the modal numbers were 51 and 54, respectively. Confirmation of the origin of SP-1 cells was made by the cytotoxic antibody dye-exclusion test. This cell line supported the growth of lymphocystis virus from the silver perch but was not found to replicate various other fish and mammalian viruses.

Lack of Correlation Between Enterococcal Counts and the Presence of Human Specific Fecal Markers in Mississippi Creek and Coastal Waters

The objective of this study was to determine whether statistically valid correlations could be shown between enterococcal counts of samples from creek and coastal sites and the presence of two molecular, library-independent markers that specify human and/or sewage pollution. Four hundred ninety samples were collected between August 2007 and April 2009 to determine enterococcal counts and the presence of genetic markers for the sewage indicator organisms Methanobrevibacter smithii and Bacteroidales. The presence of human/sewage markers and enterococcal counts were higher in creek samples than coastal samples, but the higher creek levels did not statistically correlate with the either enterococcal count or the presence of the markers present in coastal samples. Furthermore, there was no correlation between enterococcal counts in coastal samples and either marker at any of the beach sites tested. The results of this investigation in Mississippi coastal waters suggest that human/sewage markers are unlikely to correlate with enterococci counts in the nearshore environment and that enterococcal counts may be indicative of other animal or environmental sources. Additionally, a study comparing conventional gel electrophoresis with capillary electrophoresis did not convincingly establish that one method was better than the other in regard to the results obtained. The capillary method does allow reproducibility of results and the ability to analyze multiple samples in a short period of time; however, the operational expenditures exceed the cost of traditional gel electrophoresis.