Biomedical subjects

W Burkhardt

Publications and source records attributed to W Burkhardt.

11 recordsLinked to original sources

PubMed2002

Applied technique for increasing calicivirus detection in shellfish extracts.

AIMS: Optimal detection of enteric RNA viruses in clinical, environmental, and food products using reverse transcription-PCR (RT-PCR) when inhibitory substances in extracted sample materials are present. METHODS AND RESULTS: We adapted a device for detection of RNA viruses in plant tissues and insects to detect a calicivirus strain (San Miguel sea lion virus, serotype 17) in water and oyster tissue extracts. This single, compartmentalized tube-within-a-tube (TWT) device for RT-PCR-nested PCR was compared to a conventional protocol of RT-PCR-nested PCR. In the presence of 100 mg of shellfish tissue extract equivalent, this TWT device decreases the calicivirus assay detection limit 10-fold over that of conventional RT-PCR-nested PCR while maintaining an identical detection limit of viral nucleic acid suspended in water. Both the conventional and TWT methods estimated the total particle-to-infectious particle ratio for this strain of calicivirus at approximately 40 : 1. CONCLUSIONS: We believe that the TWT device with appropriate RT-PCR primers will decrease the detection limit for other calicivirus strains and RNA viruses in shellfish tissue extracts. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe that the TWT approach is applicable to other situations where RT and/or PCR inhibitory materials are present or nucleic acid targets of bacteria or viruses are at low levels in extracts of food products or clinical specimens.

Animals↗

PubMed2000

Detection of norwalk-like virus in shellfish implicated in illness.

In the 1990s, Norwalk-like viruses (NLVs) were identified in patient specimens as the primary pathogen associated with shellfish-borne gastroenteritis in the United States. Identification of these viruses from implicated shellfish has been difficult due to inefficient recovery of viruses, natural polymerase chain reaction (PCR) inhibitors in shellfish, and low virus contamination. Recent improvements to the method of detecting NLVs in shellfish include enhanced processing of virus and shellfish samples, application of nested PCR and nucleotide sequencing, and increased knowledge of NLV genetic diversity. Using a newly developed and sensitive method, an NLV G2 strain was identified in 2 oyster samples implicated in a 1998 California outbreak involving 171 cases. NLV capsid primers demonstrated a greater specificity of PCR detection than did polymerase primers. The 175-base viral capsid nucleotide sequences derived from oysters were 100% identical to those derived from a patient stool sample. This finding supports the epidemiologic associations indicating that contaminated shellfish serve as the vehicle for NLV transmission.

Animals↗

PubMed2000

Selective accumulation may account for shellfish-associated viral illness.

From 1991 through 1998, 1,266 cases of shellfish-related illnesses were attributed to Norwalk-like viruses. Seventy-eight percent of these illnesses occurred following consumption of oysters harvested from the Gulf Coast during the months of November through January. This study investigated the ability of eastern oysters (Crassostrea virginica) to accumulate indicator microorganisms (i.e., fecal coliforms, Escherichia coli, Clostridium perfringens, and F(+) coliphage) from estuarine water. One-week trials over a 1-year period were used to determine if these indicator organisms could provide insight into the seasonal occurrence of these gastrointestinal illnesses. The results demonstrate that oysters preferentially accumulated F(+) coliphage, an enteric viral surrogate, to their greatest levels from late November through January, with a concentration factor of up to 99-fold. However, similar increases in accumulation of the other indicator microorganisms were not observed. These findings suggest that the seasonal occurrence of shellfish-related illnesses by enteric viruses is, in part, the result of seasonal physiological changes undergone by the oysters that affect their ability to accumulate viral particles from estuarine waters.

Animals↗

PubMed1998

Application and evaluation of male-specific bacteriophage as a process integrity or faecal contamination indicator in a pork slaughterhouse environment.

A male-specific bacteriophage plaque assay was evaluated as a faecal contamination or process integrity indicator for aspects of the pork slaughter process. Over 400 samples were tested including: sponge swabs from animal hauling trailer floors and dressed carcass surfaces; faecal material; water from slaughter sites; and water from each stage of wastewater treatment. Bacteriophage were observed in wastewater, trailers, slaughter process water and swine faeces. No bacteriophage were observed on dressed carcasses. Numbers of phage plaque-forming units per gram or millilitre showed greater variation and were usually lower than standard indicators, including total coliform or Escherichia coli counts. Among the applications studied, male-specific bacteriophage appear to be best suited for process control verification for wastewater treatment.

Abattoirs↗

PubMed1998

Occurrence of male-specific bacteriophage in feral and domestic animal wastes, human feces, and human-associated wastewaters.

Male-specific bacteriophage (MSB) densities were determined in animal and human fecal wastes to assess their potential impact on aquatic environments. Fecal samples (1,031) from cattle, chickens, dairy cows, dogs, ducks, geese, goats, hogs, horses, seagulls, sheep, and humans as well as 64 sewerage samples were examined for MSB. All animal species were found to harbor MSB, although the great majority excreted these viruses at very low levels. The results from this study demonstrate that in areas affected by both human and animal wastes, wastewater treatment plants are the principal contributors of MSB to fresh, estuarine, and marine waters.

Animals↗

PubMed1997

Identification of genetic variants of hepatitis A virus.

Although detection of hepatitis A virus (HAV) has been greatly aided by the development of polymerase chain reaction (PCR) technology, identification of genetic variants requires sequencing PCR products, which necessarily limits the length of the HAV genome (typically 2%) that can be analyzed. From a regulatory standpoint, identification of the specific strain detected by PCR is a prerequisite not only to overrule contamination of test samples in the diagnostic laboratory, but also to possibly locate the origin of the virus detected by PCR. We explored alternatives to sequencing PCR products to achieve these goals. The findings indicate that restriction fragment length polymorphism (RFLP) analysis of PCR products from two noncontiguous regions of the HAV genome encompassing 765 nucleotides (approximately 10% of the genome) by the restriction endonucleases HinfI and AluI, which cut frequently within the HAV genome, can distinguish the common tissue culture adapted strains of HAV from stool isolates. The resolution can be greatly enhanced by combining single strand conformation polymorphism (SSCP) analysis with restriction enzyme digestion, when most of the seventeen strains analyzed could be identified.

DNA, Viral↗

PubMed1996

Collaborative evaluation of a method for the detection of Norwalk virus in shellfish tissues by PCR.

A multicenter, collaborative trial was performed to evaluate the reliability and reproducibility of a previously described method for the detection of Norwalk virus in shellfish tissues with the PCR (R.L. Atmar, F. H. Neill, J. L. Romalde, F. Le Guyader, C. M. Woodley, T. G. Metcalf, and M. K. Estes, Appl. Environ. Microbiol. 61:3014-3018, 1995). Virus was added to the stomachs and hepatopancreatic tissues of oysters or hard-shell clams in the control laboratory, the samples were shipped to the participating laboratories, and viral nucleic acids were extracted and then detected by reverse transcription-PCR. The sensitivity and specificity of the assay were 85 and 91%, respectively, when results were determined by visual inspection of ethidium bromide-stained agarose gels; the test sensitivity and specificity improved to 87 and 100%, respectively, after confirmation by hybridization with a digoxigenin-labeled, virus-specific probe. We have demonstrated that this method can be implemented successfully by several laboratories to detect Norwalk virus in shellfish tissues.

Animals↗

PubMed1992

Seasonal effects on accumulation of microbial indicator organisms by Mercenaria mercenaria.

The ability of hard-shelled clams (Mercenaria mercenaria) to accumulate fecal coliforms and other microorganisms (Escherichia coli, Clostridium perfringens, and male-specific bacteriophages) was determined over a 1-year period. Twenty separate trails were conducted during different seasons to encompass a wide range of water temperatures. The greatest accumulation of microorganisms in hard-shelled clams occurred during certain periods in the spring, at temperatures ranging from 11.5 to 21.5 degrees C. These periods of hyperaccumulation did not always coincide for all organisms; the accumulation of bacteriophages was not predicted by the accumulation of either fecal coliforms or C. perfringens. Bacteriophages and C. perfringens showed significantly higher rates of accumulation than either the fecal coliform group or E. coli, especially during the spring. The higher incidence of human viral gastroenteritis associated with the consumption of shellfish during this period may be a result of the extraordinary concentration of certain microorganisms, including enteric viral pathogens.

Animals↗

PubMed1992

Survival and replication of male-specific bacteriophages in molluscan shellfish.

The survival and replication of male-specific bacteriophages in hard-shelled clams (Mercenaria mercenaria) and their homogenates were examined to further assess their potential utility as indicator organisms. Trials were conducted in the presence and absence of a suitable bacterial host, Escherichia coli HS[pFamp]R. Results of this study demonstrated that male-specific bacteriophages were unable to replicate in hard-shelled clams, with or without added host cells. In addition, the densities of these bacteriophages were stable for up to 7 days in shellfish held at ambient seawater temperatures (less than 25 degrees C). Evidence of replication, although not observed in live shellfish, was found to occur in temperature-abused shellfish homogenates and supernatants, but only when a suitable bacterial host was present.

Animals↗

PubMed1988

Novel compound for identifying Escherichia coli.

A new chromogenic compound, 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronide, was found to be useful for the rapid, specific, differential identification of Escherichia coli in the sanitary analysis of shellfish and wastewater. Of 1,025 presumptively positive colonies (blue) and 583 presumptively negative colonies (nonblue), only 1% false-negative and 5% false-positive results were found.

Animals↗

PubMed1976

Some peculiar synaptic complexes in the first visual ganglion of the fly, Musca domestica.

In the lamina ganglionaris, the first optic ganglion of the fly, the inventory of cell types as well as the patterns of their connections are well known from light microscopic investigations. Even the synaptic contacts are known with relative completeness. However, the structural details visible on electron micrographs are very difficult to interpret in functional terms. This paper concentrates on two aspects: 1) the synaptic complex between a retinula cell axon and four postsynaptic elements, arranged in a constant elongated array (it is suggested that all synapses in which the retinula cell is presynaptic are of this kind), and 2) the "gnarl" complex in which a presynaptic specialization in one neuron is separated from another neuron by a complicated glial invagination. The participation of glia at postsynaptic sites seems to be quite common in this ganglion. Occasionally it seems that a glia cell is the only postsynaptic partner facing a presynaptic specialization within a neuron.

Animals↗