Mine-derived mercury: effects on lower trophic species in Clear Lake, California.

Considerable ecological research on mercury (Hg) has focused on higher trophic level species (e.g., fishes and birds), but less on lower trophic species. Clear Lake, site of the abandoned Sulphur Bank Mercury Mine, provides a unique opportunity to study a system influenced by mine-derived Hg. An exponentially decreasing gradient of total Hg (TotHg) away from the mine allowed us to evaluate Hg bioaccumulation in planktonic and benthic invertebrates and evaluate population- and community-level parameters that might be influenced by Hg. Studies from 1992-1998 demonstrated that TotHg in lower trophic species typically decreased exponentially away from the mine, similar to trends observed in water and sediments. However, a significant amount of invertebrate TotHg (approximately 60% for sediment-dwelling chironomid insect larvae) likely derives from Hg-laden particles in their guts. Spatially, whole-body methylmercury (MeHg) did not typically exhibit a significant decrease with increasing distance from the mine. Temporally, TotHg concentrations in plankton and chironomids did not exhibit any short-term (seasonal or annual) or long-term (multiyear) trends. Methylmercury, however, was elevated during late summer/fall in both plankton and chironomids, but it exhibited no long-term increase or decrease during this study. Although data from a 50-yr monitoring program for benthic chaoborid and chironomid larvae documented significant population fluctuations, they did not demonstrate population-level trends with respect to Hg concentrations. Littoral invertebrates also exhibited no detectable population- or community-level trends associated with the steep Hg gradient. Although sediment TotHg concentrations (1-1200 mg/kg dry mass) exceed sediment quality guidelines by up to 7000 times, it is notable that no population- or community-level effects were detected for benthic and planktonic taxa. In comparison with other sites worldwide, Clear Lake's lower trophic species typically have significantly higher TotHg concentrations, but comparable or lower MeHg concentrations, which may be responsible for the discrepancy between highly elevated TotHg concentrations and the general lack of observed population- or community-level effects. These data suggest that MeHg, as well as TotHg, should be used when establishing sediment quality guidelines. In addition, site-specific criteria should be established using the observed relationship between MeHg and observed ecological responses.

Time of day that Aedes sierrensis females enter oviposition sites prior to laying eggs.

An ovitrap with a time-segregated entrance was used to separate eggs laid by Aedes sierrensis females according to the time of day that females entered the ovitrap. During a 37-day period in Lake County, CA, females that entered the ovitrap between sunrise and sunset laid 82% of the total number of eggs collected. A daily peak in oviposition (eggs per hour) was produced by females that entered during the 2-h period ending at sunset. Overall, females that oviposited had entered the ovitrap throughout the diel cycle except for a 2-h period ending at sunrise. Those eggs laid by females that entered the ovitrap between 2 h after sunset and 2 h before sunrise provided the 1st evidence that Ae. sierrensis females are capable of locating oviposition sites during the night.

Abundance and Bloodfeeding Patterns of Mosquitoes (Diptera: Culicidae) in an Oak Woodland on the Eastern Slope of the Northern Coast Range of California.

The abundance and bloodfeeding patterns of mosquitoes was studied from 2008 to 2010 at an 18 ha. oak woodland in Lake County, CA. Host-seeking females were collected weekly from sunset to sunrise by paired dry-ice-baited CDC style traps, whereas resting females were aspirated from paired walk-in red boxes. Sequences of the COI gene amplified from bloodmeals from engorged resting females were used to identify the bloodmeal hosts. Aedes sierrensis (Ludlow) and Aedes increpitus Dyar complex mosquitoes were univoltine, although the timing of emergence and abundance varied temporally and seemed weather dependent. Abundance of both Anopheles franciscanus McCracken and Anopheles freeborni Aitken peaked in mid to late summer. Females of both genera bloodfed primarily on mule deer and black-tailed jackrabbits, and few fed on either dogs or humans that were consistently present within the woodland. In contrast, multivoltine Culex tarsalis Coquillett and Culex stigmatosoma Dyar were abundant throughout summer, especially from July to September. Both Culex species bloodfed on a wide variety of avian hosts, with most bloodmeals originating from California scrub-jay, wild turkey, oak titmouse, and house finch. Culex tarsalis fed on proportionately more mammals as summer progressed, peaking at 33% in September.

Identification of Campylobacter spp. and discrimination from Helicobacter and Arcobacter spp. by direct sequencing of PCR-amplified cpn60 sequences and comparison to cpnDB, a chaperonin reference sequence database.

A robust method for the identification of Campylobacter spp. based on direct sequencing of PCR-amplified partial cpn60 sequences and comparison of these to a reference database of cpn60 sequences is reported. A total of 53 Campylobacter isolates, representing 15 species, were identified and distinguished from phenotypically similar Helicobacter and Arcobacter strains. Pairwise cpn60 sequence identities between Campylobacter spp. ranged from 71 to 92 %, with most between 71 and 79 %, making discrimination of these species obvious. The method described overcomes limitations of existing PCR-based methods, which require time-consuming and complex post-amplification steps such as the cloning of amplification products. The results of this study demonstrate the potential for use of the reference chaperonin sequence database, cpnDB, as a tool for identification of bacterial isolates based on cpn60 sequences amplified with universal primers.

Identification and characterization of Shigella boydii 20 serovar nov., a new and emerging Shigella serotype.

Analysis of 163 putative Shigella isolates from Canada and the USA showed biochemical reactions consistent with Shigella species, although none of the isolates reacted with antiserum raised against any of the well-established or provisional Shigella serotypes. All these isolates, provisionally designated serotype SH108, were positive for the ipaH gene and the invasion-associated locus. All fermented mannitol, were serologically indistinguishable from each other and showed no reaction in antisera prepared against Escherichia coli serotypes O1 to O181. PCR-RFLP analysis of the genes involved in O-antigen synthesis revealed a common pattern among these isolates that was distinct from recognized Shigella serotypes and E. coli. Between 1999 and 2003, isolates from across Canada were submitted to the National Laboratory for Enteric Pathogens for antibiotic susceptibility testing, phage typing and PFGE. These assays revealed heterogeneity among the members of this serotype. Antimicrobial susceptibility testing with seven antibiotics identified six profiles, with 90 % (45/50) of the isolates resistant to four or more antibiotics and 72 % (36/50) resistant to five or more. All isolates were typable using a panel of 16 phages, with 11 different phage types (PTs) represented. The most common PTs found were PT 3 (64 %), PT 6 (10 %) and PT 16 (6 %). Analysis of XbaI-restricted genomic DNA revealed 16 highly related patterns that were not readily distinguishable from those obtained for some other Shigella serotypes. The World Health Organization Collaborating Center for Shigella has added serotype SH108 to the Shigella scheme as S. boydii serotype 20 (serovar nov.). Strain SH108 (isolate 99-4528) is the reference strain for this serotype.

Natural variability in the seasonal occurrence and densities of adult populations of Ochlerotatus sierrensis.

This study examined the utility of Fay traps baited with carbon dioxide (Fay/CO2 traps) and ovitraps for monitoring adult Ochlerotatus sierrensis over time and space in the Coast Range of northern California. During a 3-year study in a dense oak woodland, Fay/CO2 traps collected adults from March to November at rates that were correlated with air temperature through peak activity periods ending in late June for males and late July for females. Variability in total annual rainfall did not explain the 3-fold difference in the numbers of females caught among years. Yearly collections were all male-biased, but sex ratios varied to the extent that early season densities of males did not reliably predict subsequent densities of females. Determinations of the seasonal activity periods of females by Fay/CO2 traps and ovitraps were similar except that adults were captured 2-4 wk before oviposition occurred. The availability of natural oviposition sites affected ovipositional totals into the ovitraps, precluding the use of egg counts as a direct measure of population size of females either among or within years. In a subsequent comparison of 3 sites, Fay/CO2 trap captures of females 1st peaked in mid-April at each location, but peak periods of host-seeking activity lasted 1, 7, and 18 wk in woodlands with open, moderate, and closed tree canopies, respectively. Significant differences in the densities of host-seeking females were not detected by the use of ovitraps because egg counts had similar magnitude and peaked between mid-May and mid-August at each location.

Verotoxigenic Escherichia coli (VTEC): a major public health threat in Canada.

BACKGROUND: Verotoxigenic Escherichia coli (VTEC) was first described in Canada during the 1980s as an emerging foodborne disease in association with morbidity and mortality in outbreaks of hemorrhagic colitis caused by E coli O157:H7. OBJECTIVE: To describe the surveillance activities and epidemiological laboratory markers of VTEC that are used at the National Laboratory for Enteric Pathogens (NLEP) to investigate sporadic cases and outbreaks of E coli O157:H7 and non-O157 VTEC in Canada. METHODS: Passive surveillance was conducted by obtaining data on laboratory confirmed cases of VTEC from the Provincial Laboratories of Public Health across Canada. The laboratory epidemiological markers generated for isolates of VTEC included biotyping, serotyping, phage typing, toxin detection and characterization, and molecular typing using pulsed-field gel electrophoresis. RESULTS: Major outbreaks of VTEC O157:H7 disease have been associated with ground beef, unpasteurized apple juice, salami and untreated water. In 1999 and 2000, a total of 46 outbreaks of E coli O157:H7 disease were investigated. Among those, one outbreak was associated with contact at a petting zoo and a second with the consumption of salami. An outbreak in 2000 in Ontario was associated with water and resulted in more than 1000 cases of human illness, with six deaths. The NLEP has also identified more than 100 non-O157 VTEC serotypes from cattle and meat products. At least 23 VTEC serotypes found in humans were also identical to those found in cattle and meat products. CONCLUSIONS: The laboratory-based information that is generated is used to define the incidence, sources of infection, risk factors, trends, distribution and transmission of VTEC to humans from food, water and animal sources. Prevention and control of outbreaks are high-priority health concerns.

Comparison of Campylobacter jejuni lipooligosaccharide biosynthesis loci from a variety of sources.

Campylobacter jejuni strains exhibit significant variation in the genetic content of the lipooligosaccharide (LOS) biosynthesis loci with concomitant differences in LOS structure. The C. jejuni LOS loci have been grouped into six classes based on gene content and organization. Utilizing PCR amplifications of genes from these loci, we were able to classify a majority (80%) of the LOS biosynthesis loci from 123 strains of C. jejuni that included 39 of the Penner serotype reference strains. We found that a particular LOS class was not always associated with a specific Penner serotype, and 14 of 16 Guillain-Barré syndrome-associated isolates tested in this study shared the same LOS class. The remaining isolates that could not be classified were often distinguishable from each other based on the results of gene-specific PCR and the lengths of their LOS biosynthesis loci as determined by long (XL) PCR. Sequence analysis of two of these unique XL PCR products demonstrated two new LOS classes. These results support the hypothesis that the LOS locus is a hot spot for genetic exchange and rearrangements. Analysis of the LOS biosynthesis genes by PCR assays can be used for typing C. jejuni and offers the advantage of inferring potential LOS structures.

Detection and characterization of the hemolysin genes in Aeromonas hydrophila and Aeromonas sobria by multiplex PCR.

A multiplex PCR assay was designed to amplify the Aeromonas hydrophila and A. veronii bv. sobria hemolysin and aerolysin genes. The assay was evaluated by using 121 clinical isolates and 7 reference strains of Aeromonas spp., and these were divided into five genotypes on the basis of the results of the multiplex PCR. The five genotypes were characterized as type 1 for those carrying the ahh1 gene only (36% of isolates), type 2 for those carrying the asa1 gene only (8.5% of isolates), type 3 for those carrying both the ahh1 and the asa1 genes (4% of isolates), type 4 for those carrying the ahh1 gene and the A. hydrophila aerA (aerolysin) gene (37.5% of isolates), and type 5 for those in which no hemolysin genes were detected (14% of isolates). The most common single hemolysin gene carried among all the Aeromonas isolates examined was ahh1, with 99 of 128 (77%) of isolates testing positive for this gene either alone or in combination with other hemolysin genes. Phenotypic expression of toxins was evaluated in a Vero cell culture cytotoxicity assay. These results indicated that there is a statistically significant correlation between the cytotoxin titers and the hemolysin genotype. Isolates belonging to genotype 4 (carrying both the ahh1 gene and the aerolysin and hemolysin aerA genes) expressed higher cytotoxin titers than isolates of the other genotypes (P < 0.001). These isolates were more cytotoxic in cell culture and may have greater clinical significance.

Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus.

A multiplex PCR assay was used to simultaneously detect genes from the five major clinically relevant Campylobacter spp. Those genes selected were hipO and 23S rRNA from Campylobacter jejuni; glyA from each of C. coli, C. lari, and C. upsaliensis; and sapB2 from C. fetus subsp. fetus. The assay was evaluated with 137 clinical and environmental isolates and was found to be rapid and easy to perform and had a high sensitivity and specificity for characterizing isolates, even in mixed cultures.

Escherichia coli ehl1 gene-positive serotype O18ac:H31 associated with an outbreak of diarrhea in a neonatal nursery in Neuquén City, Argentina.

Between 9 October and 12 November 1996, an outbreak of bloody diarrhea occurred in the neonatal nursery ward of the Policlínico Neuquén, in Neuquén, a city in the southwestern region of Argentina. Seven patients of the intermediate care unit were affected. Isolates of Escherichia coli O18ac:H31 that were non-lactose and -sorbitol fermenting were recovered from outbreak cases. Although the strains were negative for a number of virulence factors typically found in diarrheagenic groups of E. coli, all isolates from the present neonatal outbreak possessed the enterohemolysin gene, ehl1. All isolates showed resistance to the antibiotics ampicillin and chloramphenicol. These isolates showed a low adherence property in HeLa cells without any recognizable pattern. In order to evaluate the outbreak dissemination in the neonatology ward, a prevalence study was conducted on 13 November. Stool specimens were obtained from 16 neonates hospitalized in the sector and from 33 medical staff members. E. coli isolates with identical biochemical characteristics of the outbreak strain were recovered from 11 of 16 inpatients and from 4 of 33 staff members during the prevalence study. A total of 15 E. coli strains recovered both from the outbreak and the prevalence study were processed by random amplified polymorphic DNA (RAPD)-PCR and pulsed-field gel electrophoresis (PFGE). By RAPD-PCR 14 of 15 strains showed patterns with 85 to 100% similarity, and by PFGE these strains were identical, each showing a unique pattern with 15 bands between 40 and 400 kb. One strain isolated from a nurse during the prevalence study presented a pattern not related to the others, and this was characterized as E. coli O81:HNM resistant to ampicillin only and negative for all the virulence factors studied. This outbreak occurred despite strict regulations in place to prevent cross-infection in the hospital. Postoutbreak prevalence studies were performed weekly thereafter without detecting new cases.

Campylobacter transmission in a Peruvian shantytown: a longitudinal study using strain typing of campylobacter isolates from chickens and humans in household clusters.

Campylobacter jejuni is a major cause of pediatric diarrhea in developing countries-free-ranging chickens are presumed to be a common source. Campylobacter strains from monthly surveillance and diarrhea cases were compared by means of restriction-fragment length polymorphism (RFLP), rapid amplified polymorphic DNA, and Lior serotyping. RFLP analysis of 156 human and 682 avian strains demonstrated identical strains in chickens and humans in 29 (70.7%) of 41 families, and 35%-39% of human isolates from diarrhea and nondiarrhea cases were identical to a household chicken isolate. Isolation of the same RFLP type from a household chicken and a human within 1 month was highly protective against diarrhea (odds ratio, 0.07; P<.005). Campylobacter strains from symptomatic humans were unlikely to be identical to strains recently carried by household chickens, limiting the potential benefits from household-based control measures.

Risk factors associated with Campylobacter jejuni infections in Curaçao, Netherlands Antilles.

A steady increase in the incidence of Guillain-Barré syndrome (GBS) with a seasonal preponderance, almost exclusively related to Campylobacter jejuni, and a rise in the incidence of laboratory-confirmed Campylobacter enteritis have been reported from Curaçao, Netherlands Antilles. We therefore investigated possible risk factors associated with diarrhea due to epidemic C. jejuni. Typing by pulsed-field gel electrophoresis identified four epidemic clones which accounted for almost 60% of the infections. One hundred six cases were included in a case-control study. Infections with epidemic clones were more frequently observed in specific districts in Willemstad, the capital of Curaçao. One of these clones caused infections during the rainy season only and was associated with the presence of a deep well around the house. Two out of three GBS-related C. jejuni isolates belonged to an epidemic clone. The observations presented point toward water as a possible source of Campylobacter infections.