Isolation of Campylobacter spp. from pigeon feces by a combined enrichment-filtration technique.

A technique combining enrichment in Preston enrichment broth and direct filtration onto chocolate agar was used to isolate Campylobacter species from pigeon feces. Campylobacter jejuni was isolated from 106 of 200 samples tested; 105 strains were isolated by enrichment-filtration, and 84 strains were isolated by direct plating. Most of the strains grew after 48 h at 37 degrees C.     

Survival of Escherichia coli, Enterococci, and Campylobacter spp. in sheep feces on pastures

The survival of enteric bacteria in 10 freshly collected sheep fecal samples on pastures was measured in each of four seasons. Ten freshly collected feces were placed on pasture, and concentrations of Escherichia coli, enterococci, and Campylobacter spp. were monitored until exhaustion of the fecal samples. In all four seasons, there was an increase in enterococcal concentrations by up to 3 orders of magnitude, with peak concentrations recorded between 11 and 28 days after deposition. E. coli concentrations increased in three out of four seasons by up to 1.5 orders of magnitude, with peak concentrations recorded between 8 and 14 days after deposition. The apparent growth of E. coli and enterococci was strongly influenced by the initial water content of the feces and the moisture gained during periods of rehydration following rainfalls. Conversely, the results suggested that dehydration promoted inactivation. Campylobacter spp. did not grow and were rapidly inactivated at a rate that tended to be faster at higher temperatures. Pulsed-field gel electrophoresis (PFGE) of a selection of Campylobacter spp. suggested that these survival data are applicable to a range of Campylobacter spp., including the most frequently isolated PFGE genotype from sheep in New Zealand, and to genotypes previously observed to cause disease in humans. The results of this study are currently being incorporated into a fecal microbe reservoir model that is designed to assist water managers' abilities to estimate microbial loads on pastures grazed by sheep, including the influence of factors such as rainfall and temperature.     

Isolation of Methanobrevibacter smithii from human feces.

Fecal specimens from nine adults were examined for the presence of methanogenic bacteria. Enrichment cultures of five specimens produced methane in 5 days. Of these five specimens, three were tested and produced methane during a short-term incubation. Four specimens did not produce methane in either short-term incubation or in enrichment culture. Each methanogenic culture contained methanogens similar in morphology to organisms of the genus Methanobrevibacter and showed factor-420 fluorescence by fluorescence microscopy. Pure cultures were obtained from four of the five methanogenic enrichment cultures. Each isolate grew and formed methane from either H2-CO2 or formate, but growth obtained with formate was poor. None of the isolates used acetate, methanol, or trimethylamine. All isolates grew in the presence of bile salts. In immunological studies, each isolate was closely related to the type strain of Methanobrevibacter smithii, a finding consistent with the physiological and morphological similarities between the isolates and the type strain.     

Colonization of chicks by non-culturable Campylobacter spp.

Six suspensions of non-culturable Campylobacter spp. were administered by gavage to day-of-hatch chicks. Four non-culturable isolates of Campylobacter spp. were found to colonize low numbers (5/79) of 1-week-old chicks, while two isolates did not (0/30). The original and recovered Campylobacter spp. isolates were serotyped and examined by restriction enzyme analysis. Evidence of clonality of two Camp, jejuni isolates was demonstrated.     

Quantification of Campylobacter spp. in pig feces by direct real-time PCR with an internal control of extraction and amplification

The rapid and direct quantification of Campylobacter spp. in complex substrates like feces or environmental samples is crucial to facilitate epidemiological studies on Campylobacter in pig production systems. We developed a real-time PCR assay for detecting and quantifying Campylobacter spp. directly in pig feces with the use of an internal control. Campylobacter spp. and Yersinia ruckeri primers-probes sets were designed and checked for specificity with diverse Campylobacter, related organisms, and other bacterial pathogens before being used in field samples. The quantification of Campylobacter spp. by the real-time PCR then was realized on 531 fecal samples obtained from experimentally and naturally infected pigs; the numeration of Campylobacter on Karmali plate was done in parallel. Yersinia ruckeri, used as bacterial internal control, was added to the samples before DNA extraction to control DNA-extraction and PCR-amplification. The sensitivity of the PCR assay was 10 genome copies. The established Campylobacter real-time PCR assay showed a 7-log-wide linear dynamic range of quantification (R² = 0.99) with a detection limit of 200 Colony Forming Units of Campylobacter per gram of feces. A high correlation was found between the results obtained by real-time PCR and those by culture at both qualitative and quantitative levels. Moreover, DNA extraction followed by real-time PCR reduced the time needed for analysis to a few hours (within a working day). In conclusion, the real-time PCR developed in this study provides new tools for further epidemiological surveys to investigate the carriage and excretion of Campylobacter by pigs.     

Isolation of Arcobacter spp. from a brackish environment

Arcobacter spp. were isolated from water and mussels of two brackish lakes near Messina (Italy). The isolates were phenotypically characterized on the basis of a large battery of cultural and biochemical tests. By comparison with the reference strains Arcobacter butzleri ATCC 49616 and A. cryaerophilus ATCC 43157 they may be considered Arcobacter butzleri-like bacteria. The current isolation suggests that the brackish environment may play an important role in the survival and transmission of Arcobacter spp. also by seafood cultured in the examined waters.     

Isolation of Campylobacter jejuni from retail mushrooms.

Campylobacter jejuni was isolated from 3 (1.5%) of 200 retail, polyvinyl chloride film-wrapped, fresh mushrooms. These results indicate that fresh mushrooms may indeed be a source of C. jejuni and support previously reported epidemiological data (Seattle-King County Department of Public Health, Surveillance of the Flow of Salmonella and Campylobacter in a Community, 1984) which revealed an an elevated relative risk of developing campylobacter enteritis in individuals who consume mushrooms.     

Isolation of Campylobacter jejuni from raw milk.

Campylobacter jejuni was isolated from raw milk by a method that can routinely detect less than or equal to 1 organism per ml. This procedure was used in a survey of 195 separate farms and showed a 1.5% incidence of C. jejuni in milk from bulk tanks.     

Isolation of NAD-kinase from pigeon heart

A procedure of isolation of pigeon breast muscle NAD-kinase resulting in a 100--130-fold purification of the enzyme with a total yield of 30--35% is described. The enzyme is electrophoretically homogenous; its molecular weight as determined by SDS-electrophoresis is 45 000. The partially purified preparation contains multiple enzymic forms with molecular weights varying from 45 000 to 270 000, which represent an equilibrious system of structurally different oligomers. At the last purification step, i. e. ion-exchange chromatography, the enzyme loses its ability for oligomerization. Possible causes of disappearance of the enzyme multiple forms during purification are discussed.     

Isolation and enumeration of Campylobacter jejuni from poultry products by a selective enrichment method.

A direct selective enrichment procedure was developed for the isolation of Campylobacter jejuni from poultry products. The selective enrichment medium (ATB) consisted of (per liter) tryptose (20 g), yeast extract (2.5 g), sodium chloride (5 g), FBP supplement (ferrous sulfate [0.25 g], sodium metabisulfite [0.25 g], sodium pyruvate [0.25 g]), bicine (10 g), and agar (1 g). Hematin solution (6.25 ml; prepared by dissolving 0.032 g of bovine hemin in 10 ml of 0.15 N sodium hydroxide solution and autoclaving at 0.35 kg/cm2 for 30 min), rifampin (25 mg), cefsulodin (6.25 mg), and polymyxin B sulfate (20,000 IU) were added after the medium was sterilized. The pH was adjusted to 8.0. Samples were enriched in the above medium at 42 degrees C for 48 h under an atmosphere of 5% O2, 10% CO2, and 85% N2. Enrichment cultures were streaked on a plating medium composed of Brucella agar, hematin solution, FBP supplement, and the above antibiotics. Plates were incubated under the same conditions as above. Suspect colonies from the plates were confirmed to be C. jejuni by morphological examination, growth characteristics, and biochemical tests. The above method yielded 25 isolates of C. jejuni from 50 samples of retail cut-up chicken and chicken parts, whereas a more complex method involving filtration, centrifugation, selective enrichment under a flowing atmosphere, and membrane filtration yielded only 6 positives from the same samples. The new isolation procedure was particularly effective in isolating C. jejuni in the presence of large numbers of Pseudomonas aeruginosa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of Aeromonas spp. from water by using anaerobic incubation.

A membrane filtration method incorporating a combination of anaerobic and aerobic incubation has been developed for the enumeration of Aeromonas spp. in drinking water. The use of anaerobic incubation improved the detection of Aeromonas spp. by reducing the growth of nonaeromonads. The confirmation rate of presumptive Aeromonas spp. identified on the initial isolation agar exceeded 92%.     

Isolation of an antigenically unique methanogen from human feces.

A methanogenic bacterium with the morphological and physiological properties of the genus Methanobrevibacter was isolated from the feces of a Japanese man who excreted methane in his breath. Indirect immunofluorescence staining revealed that the isolate had an antigenicity unrelated to that of any known members of the genus Methanobrevibacter.     

Genotyping of Campylobacter spp. from retail meats by pulsed-field gel electrophoresis and ribotyping

AIMS: To determine the genetic relatedness of Campylobacter spp. from retail meat products, and compare the discriminatory power of pulsed-field gel electrophoresis (PFGE) and automatic ribotyping. METHODS AND RESULTS: A total of 378 Campylobacter isolates recovered from 159 raw meats (130 chicken, 25 turkey, three pork and one beef) sampled from 50 retail grocery stores of four supermarket chains in the Maryland suburban area from August 1999 to July 2000 were analysed by PFGE with SmaI, 120 isolates of which were also characterized by ribotyping with PstI using RiboPrinter system. A total of 148 unique PFGE patterns were identified, 91 of which were present in multiple Campylobacter isolates and 24 in multiple meat samples. Nineteen Campylobacter clones with identical PFGE patterns recurred frequently (up to nine times) throughout the sampling period. Comparing ribotyping with PFGE, we identified 44 PFGE patterns and 22 RiboGroups among the 120 isolates tested. Multiple PFGE patterns within one RiboGroup were commonly observed, as well as multiple RiboGroups within one PFGE pattern. CONCLUSIONS: Although Campylobacter present in retail meats were genetically diverse, certain clones persisted in poultry meats. PFGE had a greater discriminatory power than ribotyping, and the two methods were complementary in genotyping Campylobacter. SIGNIFICANCE AND IMPACT OF THE STUDY: Genomic DNA fingerprinting of Campylobacter confirmed diverse and recurrent Campylobacter clones in the retail meats, which provides additional data for a better understanding of the epidemiological aspect of Campylobacter infection.     

Isolation of Aeromonas spp. from water by using anaerobic incubation

A membrane filtration method incorporating a combination of anaerobic and aerobic incubation has been developed for the enumeration of Aeromonas spp. in drinking water. The use of anaerobic incubation improved the detection of Aeromonas spp. by reducing the growth of nonaeromonads. The confirmation rate of presumptive Aeromonas spp. identified on the initial isolation agar exceeded 92%.     

Isolation ofErwinia spp. from human sources

FiveErwinia strains were isolated from human wounds; their characteristics are reported. Their pathogenicity for man appeared doubtful.     

Isolation and detection of Listeria spp, Salmonella spp and Yersinia spp using a simultaneous enrichment step followed by a surface adhesion immunofluorescent technique

There has been a proliferation of techniques and methods reported for analysis of water samples to determine the presence of the protozoan pathogens Cryptosporidium parvum and Giardia lamblia. Many of the proposed methods are presented as complete procedures, which include sampling, processing, staining, or detection steps while other methods are not complete. Some proposed methods have been extensively tested in multi-laboratory settings, however, others are still in the developmental stage. A set of evaluation criteria has been developed to evaluate the many proposed methods. These criteria have been applied as an example, to an existing method. These criteria should be useful to individuals attempting to evaluate methods developed for detecting protozoa in water, and conversely, they should serve as a guideline for individuals interested in developing methods, allowing them to gather data with and about their methods, and present this data in a manner that is both logical and easily evaluated.