Two novel gull-specific quantitative PCR (qPCR) assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR green assay targeting Streptococcus spp. (gull3) and a hydrolysis TaqMan assay targeting Catellicoccus marimammalium (gull4). The objectives of this study were to compare the host specificity of a previous C. marimammalium qPCR assay (gull2) with that of the new markers and to examine the presence of the three gull markers in environmental water samples from different geographic locations. Most of the gull fecal samples tested (n ‫؍‬ 255) generated positive signals with the gull2 and gull4 assays (i.e., >86%), whereas only 28% were positive with gull3. Low prevalence and abundance of tested gull markers (0.6 to 15%) were observed in fecal samples from six nonavian species (n ‫؍‬ 180 fecal samples), whereas the assays cross-reacted to some extent (13 to 31%) with other (nongull) avian fecal samples. The gull3 assay was positive against fecal samples from 11 of 15 avian species, including gull. Of the presumed gull-impacted water samples (n ‫؍‬ 349), 86%, 59%, and 91% were positive with the gull2, the gull3, and the gull4 assays, respectively. Approximately 5% of 239 non-gull-impacted water samples were positive with the gull2 and the gull4 assays, whereas 21% were positive witg the gull3 assay. While the relatively high occurrence of gull2 and gull4 markers in waters impacted by gull feces suggests that these assays could be used in environmental monitoring studies, the data also suggest that multiple avian-specific assays will be needed to accurately assess the contribution of different avian sources in recreational waters.

dNovel markers of fecal pollution in tropical waters are needed since conventional methods recommended for other geographical regions may not apply. To address this, the prevalence of thermotolerant coliforms, enterococci, coliphages, and enterophages was determined by culture methods across a watershed. Additionally, human-, chicken-, and cattle-specific PCR assays were used to identify potential fecal pollution sources in this watershed. An enterococcus quantitative PCR (qPCR) assay was tested and correlated with culture methods at three sites since water quality guidelines could incorporate this technique as a rapid detection method. Various rainfall events reported before sample collection at three sites were considered in the data analyses. Thermotolerant coliforms, enterococci, coliphages, and enterophages were detected across the watershed. Human-specific Bacteroides bacteria, unlike the cattle-and chicken-specific bacteria, were detected mostly at sites with the corresponding fecal impact. Enterococci were detected by qPCR as well, but positive correlations with the culture method were noted at two sites, suggesting that either technique could be used. However, no positive correlations were noted for an inland lake tested, suggesting that qPCR may not be suitable for all water bodies. Concentrations of thermotolerant coliforms and bacteriophages were consistently lower after rainfall events, pointing to a possible dilution effect. Rainfall positively correlated with enterococci detected by culturing and qPCR, but this was not the case for the inland lake. The toolbox of methods and correlations presented here could be potentially applied to assess the microbial quality of various water types.

A study was conducted to determine the primary sources of fecal pollution in a subtropical watershed using host-specific assays developed in temperate regions. Water samples (n ‫؍‬ 534) from 10 different sites along the Rio Grande de Arecibo (RGA) watershed were collected mostly on a weekly basis (54 sampling events) during 13 months. DNA extracts from water samples were used in PCR assays to determine the occurrence of fecal bacteria (Bacteroidales, Clostridium coccoides, and enterococci) and human-, cattle-, swine-, and chicken-specific fecal sources. Feces from 12 different animals (n ‫؍‬ 340) and wastewater treatment samples (n ‫؍‬ 16) were analyzed to determine the specificity and distribution of host-specific assays. The human-specific assay (HF183) was found to be highly specific, as it did not cross-react with nontarget samples. The cattle marker (CF128) cross-reacted to some extent with swine, chicken, and turkeys and was present in 64% of the cattle samples tested. The swine assays showed poor host specificity, while the three chicken assays showed poor host distribution. Differences in the detection of hostspecific markers were noted per site. While human and cattle assays showed moderate average detection rates throughout the watershed, areas impacted by wastewater treatment plants and cattle exhibited the highest prevalence of these markers. When conditional probability for positive signals was determined for each of the markers, the results indicated higher confidence levels for the human assay and lower levels for all the other assays. Overall, the results from this study suggest that additional assays are needed, particularly to track cattle, chicken, and swine fecal pollution sources in the RGA watershed. The results also suggest that the geographic stability of genetic markers needs to be determined prior to conducting applied source tracking studies in tropical settings. C ulturable counts of fecal indicator bacteria (FIB), such as enterococci and fecal coliforms, are used to measure fecal pollution levels in watershed systems. In order to be effective, indicators (i) should not survive for extended periods of time in the environment, (ii) should be exclusively associated with the intestinal tracts of humans and other warm-blooded animals, and (iii) should be associated with the occurrence of human enteric pathogens. However, growing evidence suggests that under some environmental conditions, FIB can survive outside the animal gut, that they are associated with a wide array of nonmammalian vertebrates and invertebrates, and that their correlation with pathogens varies significantly. Some of the evidence has been obtained from studies conducted in tropical regions (1, 2). For instance, currently used FIB can occur naturally in water accumulated in tropical epiphytic plants (3), can survive in tropical marine waters in the presence of nutrients (4), and can proliferate in tropical soils (5, 6). Studies in Mediterranean coastal areas have shown that Escherichia coli densities are often high in th...