Tracking the Primary Sources of Fecal Pollution in a Tropical Watershed in a One-Year Study

Abstract

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...

Evaluation of five microbial and four mitochondrial DNA markers for tracking human and pig fecal pollution in freshwater

“…Knowledge regarding the geographical stability of a DNA marker, mainly its sensitivity and specificity, is critical for widespread application of the marker in FST 17 26 . In our study, only human mtDNA marker H-ND5 and pig-associated microbial DNA marker Pig-2-Bac appeared to be geographically stable, as demonstrated by that their performances in this study were in agreement with those seen in the previous studies conducted in the West.…”

“…It is not unusual that the performance of a FST marker varies from location to location, demonstrating a geographical instability 17 . Most of FST markers were developed in the West, including America and France.…”

Coherence among Different Microbial Source Tracking Markers in a Small Agricultural Stream with or without Livestock Exclusion Practices

“…watershed systems where multiple sources of fecal inputs occur (22,54,55). While the overall spatial trends described above made sense from a biophysical standpoint, there was no significant systematic sample-by-sample-or temporal window-based coherence among similar source tracking endpoints, nor were there any strong associated links with pathogens.…”

Effects of urban stream burial on organic matter dynamics and reach scale nitrate retention

“…The qPCR assay was performed in 25 lL reaction volumes containing 19 TaqMan universal PCR master mix with AmpErase uracil-N-glycosylase (Applied Biosystems, Foster City, CA, USA), 0.2 lg lL -1 bovine serum albumin, the corresponding primers at 1 lM and the TaqMan reporter probe at 0.5 lM. Ten-and 50-fold dilutions of each DNA extract were used to test for PCR inhibition (Ryu et al 2011;Toledo-Hernandez et al 2013). The amplification protocol involved an initial incubation at 50°C for 2 min to activate uracil-N-glycosylase, a 10 min incubation at 95°C, followed by 40 cycles of 95°C for 15 s and 56°C for 1 min, and a 10 min incubation at 72°C.…”