Global model fitting to compare survival curves for faecal indicator bacteria and ruminant-associated genetic markers.

AIMS: To compare decay profiles of ruminant- and cattle-associated molecular markers for faecal contamination and Escherichia coli, facilitating their correct application in water quality studies. METHODS AND RESULTS: We generated decay profiles for cultivable E. coli, a general Bacteroidales genetic marker (GenBac3), ruminant markers (CF128, Rum2Bac) and cattle markers (CowM2, CowM3) using faeces-seeded mesocosms, and selected best fitting models for each decay profile. Global model fitting tested for differences between decay profiles. After normalizing for initial concentration, decay curves differed significantly between E. coli and all genetic markers except CowM3. Decay curves for CF128 differed from GenBac3 and Rum2Bac, but Rum2Bac and GenBac3 decay profiles did not differ. Despite similar survival profiles for some markers, highly varied initial concentrations affected time to nondetection. CONCLUSIONS: Decay curves and time until nondetection differed among markers from the same host. However, the Rum2Bac and GenBac3 markers had similar decay profiles and could potentially be investigated further for source allocation using the ratio method. SIGNIFICANCE AND IMPACT OF THE STUDY: As the use of genetic markers for microbial source tracking becomes increasingly common, caution is necessary. Both the shape of decay curves and time to nondetect may differ depending on the marker selected, resulting in possible misinterpretation of results and precluding application of a 'ratio method' of source allocation.

Molecular phylogeny of the animal kingdom.

A rapid sequencing method for ribosomal RNA was applied to the resolution of evolutionary relationships among Metazoa. Representatives of 22 classes in 10 animal phyla were used to infer phylogenetic relationships, based on evolutionary distances determined from pairwise comparisons of the 18S ribosomal RNA sequences. The classical Eumetazoa are divided into two groups. Cnidarians arose from a protist ancestry different from the second group, the Bilateria. Within the Bilateria, an early split gave rise to Platyhelminthes (flatworms) and the coelomate lineage. Coelomates are thus monophyletic, and they radiated rapidly into four groups: chordates, echinoderms, arthropods, and eucoelomate protostomes.

Sequence of the Octopus dofleini hemocyanin subunit: structural and evolutionary implications.

Sequencing of the subunit of the hemocyanin of Octopus dofleini has been completed from a cDNA library. This represents the first molluscan hemocyanin to be completely sequenced. The sequence determined is for one of the two distinguishable cDNAs which have been recognized for this protein. The protein subunit has 2896 amino acids and contains seven functional units, each carrying two sets of three invariant histidine residues constituting the binding sites (A and B) for two copper atoms. The accompanying paper identifies this site in the C-terminal functional unit (Odg). Differences in sequence for the two cDNAs, for the region in which both are available, are concentrated in the "linker regions" between functional units. The sequences of the seven units exhibit high similarity, averaging about 40% identity, with a concentration of conserved sequences in the region surrounding the copper binding sites. The sequences around the B-site show significant homology to the sequences of arthropod hemocyanins. Comparison of the functional unit sequences in terms of hydrophobicity and surface exposure profiles, as well as regions of probable secondary structure, indicate that all functional units probably have a common tertiary folding; the protein subunit is a string of similarly folded beads. A number of putative N-linked carbohydrate binding sites can be recognized in the sequence; one of these corresponds to the carbohydrate observed in the X-ray diffraction study of functional unit Odg as disclosed in the accompying paper. Phylogenetic analysis of the sequences of the O. dofleini functional units, and comparison with other available molluscan sequences indicates that the multi-domain subunit structure must have arisen over a relatively brief period, preceeding the differentiation of major molluscan types.

Characterization of murine bronchoalveolar macrophage respiratory burst: comparison of soluble and particulate stimuli.

Stimulation of the respiratory burst of murine bronchoalveolar macrophages obtained by lung lavage was studied using four different stimuli and different assay conditions. One soluble stimulus, phorbol myristate acetate (PMA), two intracellular particles, zymosan and Blastomyces dermatitidis conida, and one extracellular particle, B. dermatitidis yeast, were incubated with either freshly obtained macrophages in suspension or 2- and 48-hour macrophage monolayers. Suspension cultures were incubated with stimuli for 90 minutes and monolayers for 10 minutes before O2- was assayed. PMA did not elicit O2- production in macrophage suspensions or 2-hour macrophage monolayers, but 48-hour macrophage monolayers exhibited a 13-fold increase above control values (P = .0001). On the other hand, zymosan elicited an increase in O2- production in both suspensions and monolayers, although monolayers incubated for 48 hours produced almost fourfold more O2- than the other systems (P = .025). Opsonization had no effect on the ability of zymosan to elicit respiratory burst. B. dermatitidis conidia resulted in a two- to threefold increase in O2- production in macrophage suspensions and a five- to eightfold increase in 48-hour monolayers, representing significantly less respiratory burst stimulation than with either zymosan or PMA. Similarly, B. dermatitidis yeasts demonstrated similar submaximal stimulation of O2-, 3-4 times that over control values, and again this was less than zymosan and PMA. We conclude that 1) freshly obtained murine bronchoalveolar macrophages do not respond to PMA with an increase in O2- production, but that responsiveness is evident after 48 hours of incubation in monolayers; 2) B. dermatitidis conidia and yeasts do not stimulate respiratory burst activity to the same degree as zymosan or PMA; and 3) opsonization of zymosan is not necessary for stimulation of the murine bronchoalveolar macrophage oxidative burst, confirming previous data that functional complement receptors are not present on these cells.

18S rRNA sequences of Leishmania enriettii promastigote and amastigote.

Dideoxy sequencing with reverse transcriptase and universal primers was used to obtain partial sequences of the 18S rRNAs from the promastigote and amastigote life-cycle stages of L. enriettii. Approximately 1400 nucleotides of sequence from the two stages were compared. Unlike Plasmodium berghei, in which 18S rRNAs from the mosquito stage and the mammalian stage of the life cycle are only 96.5% similar, the amastigote and promastigote rRNAs of L. enriettii are identical. In addition, a comparison of 1425 bases of the L. enriettii promastigote sequence with the published sequence of L. donovani revealed only four differences; the two sequences are 99.8% similar. A likely explanation for this high similarity, considering the 97% similarity between L. donovani and the related genus Crithidia fasciculata, is that the two species are closely related and of comparatively recent origin. The low diversity between the 18S rRNA sequences of Leishmania species is similar to that reported for 13 Tetrahymena species, where similarities ranged from 98.1 to 99.9%, but different from the pattern reported in the genus Naegleria, where divergence was greater.

A phylogenetic comparison of the 16S rRNA sequence of the fish pathogen, Renibacterium salmoninarum, to gram-positive bacteria.

The 16S rRNA of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonids, was sequenced by reverse transcriptase to produce a nearly complete sequence (97%) of 1475 nucleotides. Phylogenetic comparisons to seventeen genera and signature sequence analysis indicated that R. salmoninarum was a member of the high G + C Gram-positive eubacterial subdivision although the reported G + C value is only 53%. A phylogenetic tree details the relationship of R. salmoninarum to ten actinomycetes from diverse environments.

Identification of bacteria crucial to histamine accumulation in pacific mackerel during storage.

Bacterial growth and histamine formation in Pacific mackerel during storage at 0, 4, 15, and 25 degrees C were monitored. To identify bacterial species contributing to histamine formation, several groups of bacteria were isolated by using selective media under temperatures corresponding to the various storage conditions. Initially, low counts of bacteria were found in the gill, skin, and intestine of fresh fish, and only weak histamine formers were found in the gill. Histamine was found in the muscle when fish were stored above 4 degrees C, and aerobic plate counts reached 10(6) CFU/g. When fish became unsuitable for human consumption by abusive storage, toxicological levels of histamine were always found. The highest level of histamine formed was 283 mg/100 g in 2 days. The optimum temperature for supporting growth of prolific histamine formers was 25 degrees C. The most prolific and prevalent histamine former was Morganella morganii, followed by Proteus vulgaris, both of which were isolated on violet red bile glucose (VRBG) agar. At 15 degrees C, a significant level of histamine was still produced in fish muscle, although prolific histamine formers were less frequently detected than at 25 degrees C. The isolates on thiosulfate citrate bile salts sucrose (TCBS) agar were weak histamine formers and identified as Vibrio parahaemolyticus and Vibrio alginolyticus. At 4 degrees C, less than 57.4 mg/100 g of histamine was found in fish stored for 14 days. Most isolates were natural bacterial flora in the marine environment and identified as weak histamine formers. At 0 degrees C, neither histamine former nor histamine production was detected up to 14 days of storage.

Source and identification of histamine-producing bacteria from fresh and temperature-abused albacore.

Histamine-producing bacteria were isolated from fresh and temperature-abused albacore using two different isolation procedures. Typically, the bacterial isolates on Niven's or modified Niven's medium produced negligible or low levels of histamine (<300 ppm) in histamine enumeration broth. The most frequently found species using this approach was Hafnia alvei. By prescreening on selective media (eosin methylene blue [EMB] agar for enteric bacteria; deMan Rogosa Sharpe agar for lactic acid bacteria: KF streptococcus agar for streptococci; pseudomonas isolation [PI] agar for pseudomonads; and staphylococcus medium 110 agar for staphylococci) prior to plating on histidine decarboxylase differential media, detection rate of true histamine formers increased. Prolific histamine producers capable of forming >1,000 ppm histamine in culture broth were isolated when PI and EMB agars were used for prescreening. Among the selective media tested, EMB agar was most effective in selecting high histamine producers, as demonstrated by the highest rate of true positives based on histamine analysis. Histamine-producing isolates were mostly enteric bacteria, including Morganella morganii, H. alvei, Klebsiella spp., Citrobacter freundii, Enterobacter spp., and Serratia spp. M. morganii isolated on PI agar from temperature-abused albacore muscle was found to be the highest histamine former. This species was not isolated from fresh albacore. while other enteric bacteria were frequently detected on the gills. However, only a few species isolated from both fresh and temperature-abused muscles were identified as high histamine formers.

A PCR assay To discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA.

Our purpose was to develop a rapid, inexpensive method of diagnosing the source of fecal pollution in water. In previous research, we identified Bacteroides-Prevotella ribosomal DNA (rDNA) PCR markers based on analysis. These markers length heterogeneity PCR and terminal restriction fragment length polymorphism distinguish cow from human feces. Here, we recovered 16S rDNA clones from natural waters that were close phylogenetic relatives of the markers. From the sequence data, we designed specific PCR primers that discriminate human and ruminant sources of fecal contamination.

Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes.

We describe a new PCR-based method for distinguishing human and cow fecal contamination in coastal waters without culturing indicator organisms, and we show that the method can be used to track bacterial marker sequences in complex environments. We identified two human-specific genetic markers and five cow-specific genetic markers in fecal samples by amplifying 16S ribosomal DNA (rDNA) fragments from members of the genus Bifidobacterium and the Bacteroides-Prevotella group and performing length heterogeneity PCR and terminal restriction fragment length polymorphism analyses. Host-specific patterns suggested that there are species composition differences in the Bifidobacterium and Bacteroides-Prevotella populations of human and cow feces. The patterns were highly reproducible among different hosts belonging to the same species. Additionally, all host-specific genetic markers were detected in water samples collected from areas frequently contaminated with fecal pollution. Ease of detection and longer survival in water made Bacteroides-Prevotella indicators better than Bifidobacterium indicators. Fecal 16S rDNA sequences corresponding to our Bacteroides-Prevotella markers comprised closely related gene clusters, none of which exactly matched previously published Bacteroides or Prevotella sequences. Our method detected host-specific markers in water at pollutant concentrations of 2.8 x 10(-5) to 2.8 x 10(-7) g (dry weight) of feces/liter and 6.8 x 10(-7) g (dry weight) of sewage/liter. Although our aim was to identify nonpoint sources of fecal contamination, the method described here should be widely applicable for monitoring spatial and temporal fluctuations in specific bacterial groups in natural environments.

Characteristics of the pulmonary cellular immune response to two strains of Blastomyces dermatitidis in the mouse.

In order to assess the cellular responses in the lung in murine pulmonary blastomycosis, serial lung lavages were performed in normal BALB/cByJ mice that had received an intranasal inoculation with 1 of 2 strains of Blastomyces dermatitidis of opposite virulence. The virulent strain, ATCC 26199, induced an increasing number of neutrophils recovered from the lung lavages. An early peak of incoming neutrophils was seen at 1 to 2 days, followed by a more rapid accumulation of intraalveolar neutrophils, until by the ninth day after infection, 80% of the cells recovered from the mice were neutrophils. In contrast, the avirulent strain, ATCC 26197, induced the same degree (20%) of neutrophil influx on Days 1 and 2. Thereafter, the percentage of neutrophils consistently declined, so that by Day 6 after inoculation, the differential counts were normal. The total number of cells recovered from the mice challenged with the virulent strain did not increase significantly until Day 4; then the total number of cells consistently increased until the experiment was terminated on Day 14. The total number of cells obtained from the mice challenged with the avirulent organism never exceeded the normal range. To assess the effects of nonviable inert particles, mice were given Sephadex G-25 beads by the intranasal route, and the total number of cells recovered from the lung lavage fluid and the differential counts were determined. The peak neutrophil influx occurred 6 h after aspiration of the beads, compared with 24 to 48 h with the fungi.(ABSTRACT TRUNCATED AT 250 WORDS)

Phylogenetic position of phylum Nemertini, inferred from 18S rRNA sequences: molecular data as a test of morphological character homology.

Partial 18S rRNA sequence of the nemertine Cerebratulus lacteus was obtained and compared with those of coelomate metazoans and acoelomate platyhelminths to test whether nemertines share a most recent common ancestor with the platyhelminths, as traditionally has been implied, or whether nemertines lie within a protostome coelomate clade, as suggested by more recent morphological analyses. Maximum-parsimony analysis supports the inclusion of the nemertine within a protostome-coelomate clade that falls within a more inclusive coelomate clade. Bootstrap analysis indicates strong support for a monophyletic Coelomata composed of a deuterostome and protostome-coelomate clade. Support for a monophyletic protostome Coelomata is weak. Inference by distance analysis is consistent with that of maximum parsimony. Analysis of down-weighted paired sites by maximum parsimony reveals variation in topology only within the protostome-coelomate clade. The relationships among the protostome coelomates cannot be reliably inferred from the partial sequences, suggesting that coelomate protostomes diversified rapidly. Results with evolutionary parsimony are consistent with the inclusion of the nemertine in a coelomate clade. The molecular inference corroborates recent morphological character analyses that reveal no synapomorphies of nemertines and flatworms but instead suggest that the circulatory system and rhynchocoel of nemertines are homologous to coelomic cavities of protostome coelomates, thus supporting the corresponding hypothesis that nemertines belong within a protostome-coelomate clade. The sequence data provide an independent test of morphological character homology.

The phylogenetic status of arthropods, as inferred from 18S rRNA sequences.

Partial 18S rRNA sequences of five chelicerate arthropods plus a crustacean, myriapod, insect, chordate, echinoderm, annelid, and platyhelminth were compared. The sequence data were used to infer phylogeny by using a maximum-parsimony method, an evolutionary-distance method, and the evolutionary-parsimony method. The phylogenetic inferences generated by maximum-parsimony and distance methods support both monophyly of the Arthropoda and monophyly of the Chelicerata within the Arthropoda. These results are congruent with phylogenies based on rigorous cladistic analyses of morphological characters. Results support the inclusion of the Arthropoda within a spiralian or protostome coelomate clade that is the sister group of a deuterostome clade, refuting the hypothesis that the arthropods represent the "primitive" sister group of a protostome coelomate clade. Bootstrap analyses and consideration of all trees within 1% of the length of the most parsimonious tree suggest that relationships between the nonchelicerate arthropods and relationships within the chelicerate clade cannot be reliably inferred with the partial 18S rRNA sequence data. With the evolutionary-parsimony method, support for monophyly of the Arthropoda is found in the majority of the combinations analyzed if the coelomates are used as "outgroups." Monophyly of the Chelicerata is supported in most combinations assessed. Our analyses also indicate that the evolutionary-parsimony method, like distance and parsimony, may be biased by taxa with long branches. We suggest that a previous study's inference of the Arthropoda as paraphyletic may be the result of (a) having two few arthropod taxa available for analysis and (b) including long-branched taxa.

Genetic diversity in Sargasso Sea bacterioplankton.

Bacterioplankton are recognized as important agents of biogeochemical change in marine ecosystems, yet relatively little is known about the species that make up these communities. Uncertainties about the genetic structure and diversity of natural bacterioplankton populations stem from the traditional difficulties associated with microbial cultivation techniques. Discrepancies between direct counts and plate counts are typically several orders of magnitude, raising doubts as to whether cultivated marine bacteria are actually representative of dominant planktonic species. We have phylogenetically analysed clone libraries of eubacterial 16S ribosomal RNA genes amplified from natural populations of Sargasso Sea picoplankton by the polymerase chain reaction. The analysis indicates the presence of a novel microbial group, the SAR11 cluster, which appears to be a significant component of this oligotrophic bacterioplankton community. A second cluster of lineages related to the oxygenic phototrophs--cyanobacteria, prochlorophytes and chloroplasts--was also observed. However, none of the genes matched the small subunit rRNA sequences of cultivated marine cyanobacteria from similar habitats. The diversity of 16S rRNA genes observed within the clusters suggests that these bacterioplankton may be consortia of independent lineages sharing surprisingly distant common ancestors.

Synergistic inhibition of HIV-1 by CD4 binding domain reagents and V3-directed monoclonal antibodies.

Human immunodeficiency virus (HIV) binds to the surface of CD4 positive lymphocytes and monocyte/macrophages via a high affinity interaction between CD4 and the HIV envelope glycoprotein gp120 and is internalized by fusion of the virus and the cell membrane. The third variable (V3) domain of gp120 also plays a central role in the fusion and viral entry process. Reagents that neutralize HIV by binding to the CD4 binding domain or V3 domain of gp120 have been proposed as therapeutics in the post-HIV exposure and perinatal setting. However, the neutralization potency of these proposed reagents, sCD4, V3 directed and CD4 binding domain directed monoclonal antibodies (MAbs), is intermediate at best and may be of limited use in a clinical setting. We have demonstrated that the combination of reagents to these two primary targets for neutralization resulted in synergy with 10- to 1000-fold increase in virus neutralization. The addition of these combined reagents a second time 3 and 5 days postinfection resulted in an additional 10-fold increase in neutralization suggesting a block in HIV spread from cell to cell. These data suggest that a combination of CD4 binding domain reagents and V3 antibody infused at intervals may significantly reduce the viral load in AIDS patients.

Diversity and depth-specific distribution of SAR11 cluster rRNA genes from marine planktonic bacteria.

Small-subunit (SSU) ribosomal DNA (rDNA) gene clusters are phylogenetically related sets of SSU rRNA genes, commonly encountered in genes amplified from natural populations. Genetic variability in gene clusters could result from artifacts (polymerase error or PCR chimera formation), microevolution (variation among rrn copies within strains), or macroevolution (genetic divergence correlated with long-term evolutionary divergence). To better understand gene clusters this study assessed genetic diversity and distribution of a single environmental SSU rDNA gene cluster, the SAR11 cluster. SAR11 cluster genes, from an uncultured group of the alpha subclass of the class Proteobacteria, have been recovered from coastal and midoceanic waters of the North Atlantic and Pacific. We cloned and bidirectionally sequenced 23 new SAR11 cluster 16S rRNA genes, from 80 and 250 m in the Sargasso Sea and from surface coastal waters of the Atlantic and Pacific, and analyzed them with previously published sequences. Two SAR11 genes were obviously PCR chimeras, but the biological (nonchimeric) origins of most subgroups within the cluster were confirmed by independent recovery from separate gene libraries. Using group-specific oligonucleotide probes, we analyzed depth profiles of nucleic acids, targeting both amplified rDNAs and bulk RNAs. Two subgroups within the SAR11 cluster showed different highly depth-specific distributions. We conclude that some of the genetic diversity within the SAR11 gene cluster represents macroevolutionary divergence correlated with niche specialization. Furthermore, we demonstrate the utility for marine microbial ecology of oligonucleotide probes based on gene sequences amplified from natural populations and show that a detailed knowledge of sequence variability may be needed to effectively design these probes.