The goal of the present study was to quantify the levels of HAV viral particles and other indicators of fecal pollution in coastal ocean waters in the vicinity of the U.S.-Mexico border. Relatively little is known regarding the levels of human enteric viruses in southern California coastal waters near the U.S.-Mexico border. In one study, among samples collected from 12 beach locations from Malibu to the border of Mexico, 33% (4 of 12) of marine samples were positive for adenoviruses (12
). MPN concentration estimates indicated that there were 880 to 7,500 adenoviruses per liter of water. These marine sites were located outside of river discharge points, and the authors of that study noted that bacterial indicators did not correlate with the presence of viruses. Using qRT-PCR, Brooks et al. (1
) detected HAV in all eight samples taken during rain events from either the mouth of the Tijuana River (near the U.S.-Mexico border) or the nearby surf zone at IB at levels ranging from 90 to 3,523 copies/liter and 347 to 2,656 copies/liter, respectively. These relatively high levels of HAV measured during wet weather were attributed to the inadequate sewage collection infrastructure in the region of Tijuana, Mexico (1
In the present study, 86 and 100% of wet weather samples collected from the surf zone adjacent to the Tijuana River mouth were positive for HAV and enterovirus, respectively. The concentrations of HAV in these samples ranged from 951 to 30,771 viral particles/liter (geometric mean = 7,569), and the values of enterovirus ranged from 30 to 4,417 viral particles/liter (geometric mean = 327) (Fig. ). Due to the close proximity of this sampling site to the Tijuana River, these levels were anticipated to be higher than levels at the IB pier. The levels of HAV and enterovirus were significantly higher (P < 0.05) for the Tijuana River mouth than for samples collected at the same time from the IB pier (Fig. ).
HAV and enterovirus were detected in 71 and 86%, respectively, of wet-weather IB pier samples. The HAV concentrations in these samples ranged from 105 to 3,445 viral particles/liter (geometric mean = 715), and enterovirus levels ranged from 7 to 375 viral particles/liter (geometric mean = 45) (Fig. ). Concentrations of HAV and enterovirus were below the limit of detection for all six samples collected at Imperial Beach during the dry-weather season (Fig. ). All of the dry-weather samples contained fewer than 7.0 viral particles per liter of HAV and 4.0 viral particles per liter of enterovirus (Fig. ).
qRT-PCR offers improved sensitivity and specificity over traditional viral culture (18
); however, an inherent limitation of qRT-PCR is its inability to discriminate between infectious and noninfectious viral particles (2
). However, Gantzer et al. (9
) noted the instability of infectious enterovirus particles in wastewater and concluded that the presence of enterovirus viral particles (detected by qRT-PCR) can still be valuable as an indicator of recent viral contamination. Furthermore, for viruses such as HAV, which are difficult and time-consuming to culture, qRT-PCR may be an invaluable tool for rapid environmental monitoring. In the present study, we were able to estimate the relationship between the number of viral particles (as indicated by SYBR gold staining) and PFU for our stock control viruses. Using the virus titers provided by the ATCC, we found this relationship to be 78 viral particles/PFU for HAV (strain HM-175) and 48 viral particles/PFU for poliovirus 2 (strain W-2). Similar results for poliovirus were obtained by Donaldson et al. (5
), who found a relationship of 55 viral particles/PFU and Fuhrman et al. (8
), who found a relationship of 66 viral particles/PFU. No comparable results exist in the literature for HAV.
HAV cDNA obtained from the four positive 2003-2004 wet-weather samples was cloned and sequenced, and a BLAST search identified three highly similar HAV strains (Fig. ). All three strains were at least 98% identical. Ticehurst et al. (26
) reported that different human HAV strains of diverse geographic origin were remarkably closely related. The isolates in the present study were significantly similar to isolates from southern Italy (~99% similar to isolate IT-DAL-00, accession number AJ505803), Argentina (~97% similar to isolate Arg873, accession number AF452067), and Japan (~97% similar to isolate FH3, accession number AB020569) (Fig. ). Unlike the present study, in which the virus types were fairly evenly distributed among the samples, a previous study on the Venice Lagoon (23
) found that a single strain of HAV (accession number AY441443) was present in a majority of the samples.
Nine different enterovirus types were isolated from the five positive 2003-2004 wet-weather samples. A neighbor-joining tree grouped the enterovirus isolates into two major clades (Fig. ). One clade contained echoviruses 6, 11, and 30, coxsackievirus A5, and enterovirus B, whereas the second clade contained polioviruses 1, 2, and 3 and enterovirus 90. This is in general agreement with previously published enterovirus phylogeny (20
). While the enterovirus types in the present study were relatively evenly distributed (Fig. ) among the samples, a previous study on the Venice Lagoon (23
) found that a single virus, poliovirus 2, was present in a majority of the samples. Likewise, Donaldson et al. (5
) found that coxsackievirus A9 was the dominant enterovirus type. Since we found poliovirus 2 (the same type of enterovirus as our positive control), one might argue that this resulted from a contamination event in the laboratory. However, this is unlikely because negative controls run in parallel with positive samples were consistently negative by both PCR and sequencing.
Twelve of fourteen wet-weather samples (86%) exceeded the California state water quality standard for one or both of the bacterial indicators, E. coli and enterococci (Fig. ). In contrast, there were no bacterial exceedances for any of the dry-weather samples (Fig. ). Regression analyses of the viral densities (as measured by qRT-PCR) and indicator levels showed a significant correlation between the densities of both bacterial indicators and the levels of HAV (R2 > 0.61, P < 0.0001) and enterovirus (R2 > 0.70, P < 0.0001) (Table ). These results suggest that bacterial indicator levels may be predictive of the levels of viruses at the Tijuana River mouth and IB pier.
A multiple regression analysis showed that HAV and enterovirus levels were directly (positively) related (P < 0.05) to the levels of the fecal indicator bacteria, E. coli and enterococci, one another, river flow rate, and precipitation, and inversely related to water temperature (Table ). There was no correlation between tidal height and the levels of either virus. Collectively, these variables were able to predict HAV levels in a sample 35 to 67% of the time (P ≤ 0.002) and enterovirus levels 19 to 73% of the time (P ≤ 0.025) (Table ).
Although the association (percent concordance) with viral levels was slightly higher for E. coli
than for enterococci, these differences were not significant (P
> 0.05), suggesting that both bacterial indicators were similarly able to predict levels of virus. A series of large-scale epidemiological studies carried out by the U.S. EPA (29
) found that, among the indicator organisms, only two—E. coli
= 0.51) and enterococci (r
= 0.81)—exhibited a strong correlation to swimming-associated gastroenteritis. Based on these findings, the U.S. EPA's draft of the Implementation Guidance for Ambient Water Quality Criteria for Bacteria
) recommended criteria for marine waters solely based on enterococci. Our results suggest that E. coli
might, as well, be a suitable indicator of viral contamination in sewage-contaminated marine waters.
In contrast to our results, a number of recent studies using PCR have shown that viral contamination cannot be well assessed by using bacterial indicators (2
). There may be a number of important reasons for the differing results. All of these studies were conducted on urban rivers where a variety of nonpoint sources of contamination, including animal fecal sources, could have contributed to the lack of a relationship between fecal indicator bacteria and human virus levels. In our study, the existence of a predominant source of human fecal contamination resulting from the inadequate sewage infrastructure in Mexico may have facilitated our finding of statistically significant correlations between bacterial indicator and human virus levels in coastal waters. As such, the present study presents the first quantitative assessment of the statistical relationship between levels of HAV, enterovirus, E. coli
, and enterococci in marine waters.