Differentiation of Cryptosporidium parvum Isolates by a Simplified Randomly Amplified Polymorphic DNA Technique

Genomic DNA was isolated from Cryptosporidium parvum oocysts by a specific immunomagnetic separation-in vitro excystation procedure and subjected to randomly amplified polymorphic DNA analysis using sequence-independent primers. An estuary C. parvum isolate was easily differentiated from several bovine isolates, while five bovine isolates of the same origin were indistinguishable from each other.     

Differentiation of Campylobacter Populations as Demonstrated by Flagellin Short Variable Region Sequences

The DNA sequence of the flaA short variable region (SVR) was used to analyze a random population of Campylobacter isolates to investigate the weakly clonal population structure of members of the genus. The SVR sequence from 197 strains of C. jejuni and C. coli isolated from humans, bovine, swine, and chickens identified a group of 43 strains containing disparate short variable region sequences compared to the rest of the population. This group contains both C. jejuni and C. coli strains but disproportionately consisted of bovine isolates. Relative synonymous codon usage analysis of the sequences identified two groups: one group typified C. jejuni, and the second group was characteristic for C. coli and the disparate alleles were not clustered. The data show that there is significant differentiation of Campylobacter populations according to the source of the isolate even without considering the disparate isolates. Even though there is significant differentiation of chicken and bovine isolates, the bovine isolates did not show any difference in ability to colonize chickens. It is possible that disparate sequences were obtained through the lateral transfer of DNA from Campylobacter species other than C. jejuni and C. coli. It is evident that recombination within the flaA SVR occurs rapidly. However, the rate of migration between populations appears to limit the distribution of sequences and results in a weakly clonal population structure.     

Flocculation Effects on Bound Water in Sludges as Measured by Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance relaxation times (T₁ and T₂) were measured for flocculated and unflocculated samples of activated sludge. The weight of water and solids in the sludge samples was found and related to T₁ to find the relative percentage of bound water. The results suggest that the amount of bound water increases as the samples become more unflocculated. The values of T₁ and T₂ also indicate that unflocculated individual particles are characterized by loose packing of shorter molecules and that the addition of larger molecules may induce flocculation.     

Sensitivity of Campylobacter spp. to irradiation in poultry meat

The sensitivity of Campylobacter jejuni (three strains), Camp. coli (three strains), Camp. fetus (one strain) and Camp. lari (one strain) to irradiation in poultry meat was investigated. There was no significant difference in the counts obtained on Blood or Skirrows agar. Preston agar gave a significantly lower recovery of the pathogens after irradiation so these results were not included in calculations of D ₁₀ values. The D ₁₀ values ranged from 0.12 to 0.25 kGy and there was a significant difference in the radiation sensitivity between different Campylobacter spp. and within strains of the same species. These values indicate that Campylobacter spp. are more radiation-sensitive than Salmonella and Listeria monocytogenes irradiated under similar conditions. Therefore irradiation treatments suggested to eliminate the latter from poultry carcasses would also be sufficient to remove Campylobacter.     

Differentiation of the subspecies of Campylobacter fetus by genomic sizing.

Campylobacter fetus subsp. fetus and C. fetus subsp. venerealis are currently differentiated by tolerance to glycine and by their epidemiology. Analysis of C. fetus DNA by pulsed-field gel electrophoresis, after digestion with the restriction endonucleases SmaI and SalI, was used to differentiate between the subspecies. All strains presently identified as C. fetus subsp. fetus had a genomic size of 1.1 Mb, whereas the majority of the C. fetus subsp. venerealis strains had a genomic size of 1.3 Mb. An additional group of strains, which were previously described as C. fetus subsp. venerealis biovar "intermedius" and were able to tolerate higher concentrations of glycine than the rest of the C. fetus subsp. venerealis strains, had an average genome size of 1.5 Mb. We suggest that pulsed-field gel electrophoresis may be useful as an additional aid in the differentiation of C. fetus strains at the subspecies level.     

Sensitivity of the pathogen of Campylobacter infection to various antibiotics]

Antibiotic sensitivity of 21 Campylobacter strains isolated from humans, monkeys and birds with diarrhea was assayed and the findings are presented. It was shown that all the isolates were highly sensitive to ofloxacin, pefloxacin, ciprofloxacin, josamycin, pipemidinic acid and amoxyclav (a combination of amoxycillin and clavulanic acid). The predominating majority of the strains were resistant to rifampicin, amoxycillin and phosphomycin. The majority of the cultures were sensitive to erythromycin and roxithromycin while some of the isolates were resistant to them which could be used as an additional biological characteristic of Campylobacter cultures and considered in choosing agents for antibiotic therapy.     

Variability in Sensitivity to Polyoxin B of Isolates of Alternaria mali and Decreased Fitness of Polyoxin resistant Isolates

The 540 monoconidial isolates of Altemaria mali were obtained in 1983 from apple orchards at Seoul, Suweon, Cheongju, Kochang, Daegu, and Jinju in Korea. The sensitivity of A. mali to polyoxin B greatly varied among isolates and locations. Most of isolates were sensitive to polyoxin B, but 11 isolates showed a high level of resistance, particularly from Kochang and Daegu where polyoxin B had been applied frequently. Conidial germination and mycelial growth of resistant isolates were not inhibited at higher concentrations of polyoxin B compared to the sensitive ones. The polyoxin-resistant isolates were not resistant to the fungicides iprodione and polydong and showed a reduced activity for conidial formation and mycelial growth. The isolates of A. mali with resistance to polyoxin B appear to have decreased fitness in nature.     

Differentiation of Bulgarian Isolates from Cucumber Mosaic Virus by Serological Methods

Cucumber mosaic virus (CMV) is of great importance to the Bulgarian economy and hence a detailed knowledge of its diversity under local geographic and climatic conditions is required. An extended study was carried out on CMV strains the currently occur in Bulgaria. Fifty-one isolates and strains found in different regions and various crops were biologically characterized and serologically differentiated into subgroups I and II using different variants of enzyme-linked immunosorbent assay (ELISA) [double antibody sandwich (DAS)-, antigen-coated plate (ACP)-, triple antibody sandwich (TAS)- with poly and monoclonal antibodies] and immunodiffusion tests. The ELISA modifications with monoclonal antibodies individually (ACP) or in combination with polyclonal antibodies (TAS-ELISA) are suitable for mass screening of CMV isolates. The hyperimmune sera against strains from CMV subgroups I and II were very efficient for use in isolate differentiation via gel double immunodiffusion. The results obtained correlated with the polymerase chain reaction and restriction fragment length polymorphism data reported by other authors. The majority of the isolates belonged to subgroup I, whereas 10, mainly from tomato and pepper, belonged to subgroup II. Most of the subgroup II isolates came from the north of Bulgaria. The results of the present study will help to clarify the virus epidemiology and to develop specific control measures.     

Plant Water Potential Gradients Measured in the Field by Freezing Point

A portable freezing point meter was used in the field to measure the water potential gradients in sunflower (Helianthus annuus), beans (Phaseolus vulgaris), corn (Zea mays), wheat (Triticum aestivum), pumpkin (Cucurbita pepo), potato (Solanum tuberosum), alfalfa (Medicago sativa), and sugarbeets (Beta vulgaris). The measurements were made between daybreak and sunrise, and again during the middle of the afternoon on days when the potential evapotranspiration varied between 6.5 and 8.0 mm of water. The gradients varied from a maximum of 0.2 bar per cm in a wheat, down to an undetectable value for pumpkin. Although most of the soil in the root zone was kept at potentials above –1 bar, the bulk of the root tissue had water potentials of –5 to –10 bars. Differences in water potential between shaded and unshaded leaves, and between leaf tissue and guttation fluid suggested a similar drop of several bars between xylem elements and the surrounding leaf tissue in some plant species. The implications of such drops are discussed with respect to plant water transport equations and pressure cell potential measurements.     

DNA Damage Measured by the Comet Assay in Eight Agronomic Plants

For most crops growing in polluted areas or treated with agricultural chemicals, no genotoxicity assays are available. We have studied the possibility of using the alkaline protocol of the plant-based molecular assay — the Single Cell Gel Electrophoresis (SCGE) assay (also called Comet assay) as a method for detecting induced DNA damage in 8 agronomic important plants (ordered according to the diameter of the nuclei): sugar beet, alfalfa, tobacco, lentil, maize, potato, hard wheat, and bread wheat. The monofunctional alkylating agent ethyl methanesulphonate (EMS) was applied as a model genotoxic agent on young excised leaves of the tested crops for 18 h at 26 °C in the dark. With increasing concentrations of 2 to 10 mM EMS, the DNA damage, expressed by the averaged median tail moment values, significantly increased in nuclei of all crops studied. No correlation between the diameter of nuclei and sensitivity to EMS treatment was observed. The data obtained demonstrate the feasibility of using the Comet assay for detecting induced DNA damage in crops. This revised version was published online in July 2006 with corrections to the Cover Date.     

Novel Clonal Complexes with an Unknown Animal Reservoir Dominate Campylobacter jejuni Isolates from River Water in New Zealand

Campylobacter jejuni is widely distributed in the environment, and river water has been shown to carry high levels of the organism. In this study, 244 C. jejuni isolates from three river catchment areas in New Zealand were characterized using multilocus sequence typing. Forty-nine of the 88 sequence types identified were new. The most common sequence types identified were ST-2381 (30 isolates), ST-45 (25 isolates), and ST-1225 (23 isolates). The majority of the sequence types identified in the river water could be attributed to wild bird fecal contamination. Two novel clonal complexes (CC) were identified, namely, CC ST-2381 (11 sequence types, 46 isolates) and CC ST-3640 (6 sequence types, 12 isolates), in which all of the sequence types were new. CC ST-2381 was the largest complex identified among the isolates and was present in two of the three rivers. None of the sequence types associated with the novel complexes has been identified among human isolates. The ST-2381 complex is not related to complexes associated with cattle, sheep, or poultry. The source of the novel complexes has yet to be identified.Contamination of the environment by bacterial pathogens is a significant health concern, as it provides a continuous source of organisms for the infection and reinfection of humans and animals. Enteric pathogens gain entry into the environment through the discharge of sewage into water and via contamination from animal feces (22). Fecal contamination is responsible for the continued presence and spread of a range of pathogenic organisms, including Campylobacter, norovirus, and Escherichia coli O157. Determining the roles of various environmental sources in human enteric disease requires an understanding of the distribution, survival, population structure, and pathogenic potential of the pathogens in the environment.Campylobacter is the most common cause of gastrointestinal illness in the industrialized world (17), imposing significant economic costs on health systems, and is associated with a number of neurological sequelae (32, 33). The majority of human campylobacter infections are caused by Campylobacter jejuni (90%), with Campylobacter coli mostly responsible for the remainder. Although Campylobacter has been isolated from a wide range of animals (41) and birds (47, 48), contaminated poultry and poultry products remain the most significant sources of human infections (10, 38, 50, 51). Campylobacter is a spiral gram-negative organism that grows best under low-oxygen conditions at 42°C. The organism is unable to grow outside an animal host, and survival in the environment is dependent on ambient temperature, oxygen levels, and sunlight.Studies worldwide examining rivers and waterways show that there is significant contamination by Campylobacter, with the sources being sewage outflow, direct fecal deposition, and pasture runoff (12, 22, 34, 37, 39). Similarly, coastal waters and estuaries can be contaminated by either sewage or bird fecal deposition (23, 35). The inability of Campylobacter to grow in the environment and its sensitivity to sunlight are thought to ensure that the organism is eventually purged from the system. However, the high levels of the organism identified in water systems have been highlighted as a risk for human infection.The characterization of campylobacter populations by multilocus sequence typing (MLST) has shown that the organism is weakly clonal and that certain clonal complexes are associated with particular animals (5, 9, 26). Isolates from human cases of infection show a wide variety of sequence types and many clonal complexes. Source attribution studies using MLST have identified poultry as causing approximately 60% of human infections (14, 38, 50). Cattle have been identified as a potential source of infection due to the high level of similarity between bovine and human strains (18, 19). There remains, however, a significant number of infections for which the source is not certain.New Zealand has one of the highest rates of campylobacteriosis in the developed world. This is due to the significant quantity of fresh chicken consumed coupled with high levels of contamination found in poultry products (1, 10, 51, 52). Campylobacter has been isolated from a range of environmental sources within New Zealand, including its rivers and streams (12, 37). Isolation rates for rivers in New Zealand range from 55 to 90%, comparable to results of studies overseas, and show the same seasonal variation as that seen elsewhere in the world (20). Pulsed-field gel electrophoresis (PFGE) analysis identified indistinguishable macrorestriction profiles for cattle, human, and river isolates, suggesting river water as a potential source of infection (8). In this study, C. jejuni isolates from three rivers in New Zealand, two on the South Island and one on the North Island, were characterized using MLST.     

Diversity of Environmental Mycobacterium Isolates from Hemodialysis Water as Shown by a Multigene Sequencing Approach

Here we used a multigene sequencing approach for the identification and molecular typing of environmental mycobacteria of the fast-growing subgroup. Strains were isolated from hemodialysis water and clinical samples. Eleven type strains of related species of the genus were also included in this study. To gain further insight into the diversity of the environmental mycobacteria, we analyzed several housekeeping genes (16S rRNA, ITS1, gyrB, hsp65, recA, rpoB, and sodA). No individual phylogenetic tree allowed good discrimination of all of the species studied. However, a concatenated and a consensus analysis, combining the genes, allowed better discrimination of each strain to the species level, and the increase in sequence size also led to greater tree robustness. This approach is useful not only for the discrimination and identification of environmental mycobacteria but also for their molecular typing and studies of population genetics. Our results demonstrate high genetic diversity among the isolates obtained, which are probably new species of the genus.     

Induced DNA Damage Measured by the Comet Assay in 10 Weed Species

For most plant species growing in polluted areas no mutagenicity assays are available. We have studied the possibility of using the alkaline protocol of the Comet assay as a method for detecting induced DNA damage in wildly growing weeds. The monofuctional alkylating agent ethyl methanesulphonate (EMS) was applied on leaves of 10 weed species (ordered according to the diameter of the nuclei): Arabidopsis thaliana, Convolvulus arvensis, Bellis perennis, Urtica dioica, Lamium album, Chenopodium rubrum, Plantago media, Poa annua, Taraxacum officinale, and Agropyron repens. With increasing concentrations of EMS (2 to 10 mM) the DNA damage, expressed by the averaged median tail moment values, significantly increased in nuclei of all weeds studied. Using the Head Extent parameter of the Komet version 3.1, we have measured the diameter size of the nuclei of the 10 weed species either immediately after the isolation of the nuclei or after 20 or 45 min of treatment with alkaline buffer (pH > 13). According to the increase of the diameter of the nuclei (including the formed halo) resulting from the to alkaline buffer treatment, electrophoretic conditions (unwinding and electrophoresis time) for the Comet assay can be selected for the individual weed species.     

Antimicrobial Susceptibility Profiles and Molecular Typing of Campylobacter jejuni and Campylobacter coli Isolates from Ducks in South Korea

Campylobacter is a food-borne zoonotic pathogen that causes human gastroenteritis worldwide. Campylobacter bacteria are commensal in the intestines of many food production animals, including ducks and chickens. The objective of the study was to determine the prevalence of Campylobacter species in domestic ducks, and the agar dilution method was used to determine resistance of the isolates to eight antibiotics. In addition, multilocus sequence typing (MLST) was performed to determine the sequence types (STs) of selected Campylobacter isolates. Between May and September 2012, 58 duck farms were analyzed, and 56 (96.6%) were positive for Campylobacter. Among the isolates, 82.1% were Campylobacter jejuni, 16.1% were C. coli, and one was unidentified by PCR. Of the 46 C. jejuni isolates, 87.0%, 10.9%, and 21.7% were resistant to ciprofloxacin, erythromycin, and azithromycin, respectively. Among the C. coli isolates, all 9 strains were resistant to ampicillin, and 77.8% and 33.3% were resistant to ciprofloxacin and azithromycin, respectively. The majority of the Campylobacter isolates were classified as multidrug resistant. Twenty-eight STs were identified, including 20 STs for C. jejuni and 8 STs for C. coli. The most common clonal complexes in C. jejuni were the ST-21 complex and the ST-45 complex, while the ST-828 complex predominated in C. coli. The majority of isolates were of STs noted in ducks and humans from earlier studies, along with seven STs previously associated only with human disease. These STs overlapped between duck and human isolates, indicating that Campylobacter isolates from ducks should be considered potential sources of human infection.     

Antimicrobial Resistance Profiles of Campylobacter jejuni Isolates from Wild Birds in Sweden

In order to determine the occurrence and frequency of resistant strains of the bacterium Campylobacter jejuni and to establish baseline MICs in isolates from an environmental reservoir, the resistance profiles of 10 antimicrobial substances were determined for 137 C. jejuni isolates from wild birds in Sweden. Observed MICs were generally low, with only low to moderate incidence of resistance to the tested compounds. One isolate, however, was resistant to nalidixic acid and ciprofloxacin, indicating that quinolone-resistant genotypes of C. jejuni have the potential to spread to wild bird hosts.     

Detection and Genotyping of Campylobacter jejuni and Campylobacter coli by Use of DNA Oligonucleotide Arrays

Campylobacter have emerged as the most common bacterial food-borne illness in the developed world. The ability to reduce Campylobacter infections in humans is linked to the full comprehension of the principal key aspects of its infection cycle. A microbial diagnostic microarray detecting Campylobacter housekeeping, structural, and virulence associated genes was designed and validated using genomic DNA from reference and field strains of Campylobacter jejuni and coli isolated from human, chicken, and raw milk. This microarray was confirmed to be a powerful diagnostic tool for monitoring emerging Campylobacter pathotypes as well as for epidemiological, environmental, and phylogenetic studies including the evaluation of genome plasticity.     

Calcium Dependency and Tetrodotoxin Sensitivity of Neostriatal Dopamine Release in 5-Day-Old and Adult Rats as Measured by In Vivo Microdialysis

Abstract: The calcium dependency and tetrodotoxin sensitivity of extracellular dopamine levels were assessed by microdialysis in the neostriatum of 5-day-old rat pups and were compared with those obtained in adult rats. The removal of calcium from the dialysate reduced spontaneous levels of extracellular dopamine to 20% of normal in the 5-day-old pups and to 10% of normal in the adults. Calcium-free dialysate also decreased potassium-evoked dopamine release to ～20% of baseline in both ages. Furthermore, the addition of tetrodotoxin to the dialysate decreased spontaneous levels of extracellular dopamine to 10% of baseline in both ages. The effects of calcium removal and the addition of tetrodotoxin on extracellular levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid were less pronounced. The results of this study demonstrate that extracellular levels of dopamine sampled by microdialysis in rats as young as 5 days of age are both calcium dependent and tetrodotoxin sensitive; thus, they are derived from neuronal activity and not from injury caused by acute implantation of the probe. Other age-related differences support the hypothesis that dopamine release and turnover is greater In immature rats and may represent a form of compensation for incomplete dopamine nerve terminal ingrowth.     

Differentiation of Closely Related Carnobacterium Food Isolates Based on 16S-23S Ribosomal DNA Intergenic Spacer Region Polymorphism

A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another. Carnobacterium divergens NCDO 2763^T and C. mobile DSM 4849^T generated one major S-ISR band (ca. 400 bp) and minor M-ISR and L-ISR bands (ca. 500 and ca. 600 bp, respectively). The ISRs amplified from C. gallinarum NCFB 2766^T and C. piscicola NCDO 2762^T were larger (S-ISR, ca. 600 bp; M-ISR, ca. 700 bp; and L-ISR, ca. 800 bp). The L-ISR contained two tDNAs coding for tRNA^Ile and tRNA^Ala genes. The M-ISR included one tRNA^Ala gene, and the S-ISR did not contain a tDNA gene. The RFLP scheme devised involves estimation of variable PCR product sizes together with HinfI, TaqI, and HindIII restriction analysis. Forty-two isolates yielded four unique band patterns that correctly resolved these isolates into four Carnobacterium species. This method is very suitable for rapid, low-cost identification of a wide variety of Carnobacterium species without sequencing.