Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 200467699706Original ArticleVibrio cholerae in Peruvian watersA. I. Gil et al.
Environmental Microbiology (2004) 6(7), 699–706
doi:10.1111/j.1462-2920.2004.00601.x
Occurrence and distribution of Vibrio cholerae in
the coastal environment of Peru
Ana I. Gil,1 Valérie R. Louis,2 Irma N. G. Rivera,2†
Erin Lipp,2‡ Anwar Huq,2 Claudio F. Lanata,1
David N. Taylor,3§ Estelle Russek-Cohen,4
Nipa Choopun,2 R. Bradley Sack5 and
Rita R. Colwell2,6*
1
Instituto de Investigación Nutricional (IIN), Av. La
Universidad 685, La Molina, Lima 18, Peru.
2
Center of Marine Biotechnology, University of Maryland
Biotechnology Institute, 701 E. Pratt Street, Suite 236,
Baltimore, MD 21202, USA.
3
NAMRID, Naval Medical Research Center Detachment
Unit 3800, Centro Medico Naval, Lima, Peru.
4
Department of Animal Science, University of Maryland,
College Park, MD 20742, USA.
5
Johns Hopkins University, Department of International
Health, Bloomberg School of Public Health, Baltimore, MD
21205, USA.
6
Institute for Advanced Computer Sciences, University of
Maryland, College Park, MD 20742, USA.
Summary
The occurrence and distribution of Vibrio cholerae in
sea water and plankton along the coast of Peru were
studied from October 1997 to June 2000, and included
the 1997–98 El Niño event. Samples were collected at
four sites in coastal waters off Peru at monthly intervals. Of 178 samples collected and tested, V. cholerae
O1 was cultured from 10 (5.6%) samples, and V. cholerae O1 was detected by direct fluorescent antibody
assay in 26 out of 159 samples tested (16.4%). Based
on the number of cholera cases reported in Peru from
1997 to 2000, a significant correlation was observed
between cholera incidence and elevated sea surface
temperature (SST) along the coast of Peru (P < 0.001).
From the results of this study, coastal sea water and
zooplankton are concluded to be a reservoir for V.
Received 27 August, 2003; revised 12 January, 2004; accepted 12
January, 2004. *For correspondence at the Center of Marine Biotechnology. E-mail colwell@umbi.umd.edu; Tel. (+1) 301 405 9550; Fax
(+1) 301 314 6654. Present addresses: †Microbiology Department,
ICB-USP, 1374, Lineu Prestes Avenue – Edif. ICB II, University of
São Paulo, CEP 05508-900, São Paulo, SP, Brazil. ‡Department of
Environmental Health Science, University of Georgia, Athens, GA
30602, USA. §Vaccine Testing Unit, Department of International
Health, Johns Hopkins University, Bloomberg School of Public Health,
Baltimore, MD 21205, USA.
© 2004 Blackwell Publishing Ltd
cholerae in Peru. The climate–cholera relationship
observed for the 1997–98 El Niño year suggests that
an early warning system for cholera risk can be established for Peru and neighbouring Latin American
countries.
Introduction
Cholera is an ancient disease and remains endemic in
many parts of the developing world, continuing to cause
significant morbidity and mortality annually (Barua, 1992).
Since 1990, many more cases of cholera have been
reported to the World Health Organization (WHO, 1991;
2000) than during the decades before 1990. In late January 1991, cholera reappeared in Latin America and initially in Peru, after an absence of more than 100 years
(Tauxe et al., 1994). During 1991, cholera cases occurred
simultaneously at different sites along the coast, over a
distance of more than 1200 km (Seas et al., 2000). However, it was observed (Seas et al., 2000) that clinical cholera was actually beginning to appear in locations along
the coast north of Lima in 1990, before the major epidemic
in Lima. Subsequently, the epidemic spread to include the
rest of South and Central America, from Mexico to Argentina (Tauxe et al., 1994). The source of the epidemic
remains unknown; however, the three major routes of
transmission were identified as water (untreated contaminated water), food (poor sanitary handling of contaminated food) and seafood (Tauxe et al., 1995).
Between 1991 and 2000, cholera has been reported in
Peru in a recurring seasonal pattern, with the largest
number of cases occurring in the summer (January to
March) in Lima and in other major cities along the coast
of Peru. The initial major outbreak and subsequent cases
of cholera caused serious public health and socioeconomic problems for Peru and its neighbouring Latin
American countries. During 1993–95, in Lima, Franco
et al. (1997) detected Vibrio cholerae in the environment
of heavy sewage-contaminated areas before cholera
cases occurred in the community.
An ability to predict the appearance of cholera in a
specific region would be of great public health value. To
be able to make such predictions, it is vital to understand
the ecology of the causative agent, V. cholerae. The
results of many studies carried out over the past 30 years
have confirmed the hypothesis of Colwell et al. (1977) that
700 A. I. Gil et al.
the aquatic environment is the primary reservoir for V.
cholerae (Kaper et al., 1979; Colwell and Huq, 2001).
Furthermore, it has been clearly demonstrated (Huq et al.,
1983) that V. cholerae is associated with zooplankton,
especially copepods. A study in Bangladeshi villages
where cholera was endemic showed that a simple filtration
method, removing most plankton >20 mm from drinking
water, significantly reduced the risk of cholera (Colwell
et al., 2003). V. cholerae is also capable of entering into
a viable but non-culturable (VBNC) state under conditions
adverse to active metabolism and cell division (Roszak
and Colwell, 1987). It has been shown that, in geographical areas where cholera is endemic, VBNC V. cholerae
can be detected in riverine, estuarine and coastal waters
year round, whether or not there are cases of cholera in
the resident populations (Khan et al., 1984; Colwell,
1996). In the temperate estuary of Chesapeake Bay in the
United States, the abundance of V. cholerae in the environment is seasonal (Heidelberg et al., 2002a). Furthermore, it was shown (Heidelberg et al., 2002b) that the
relative proportion of V. cholerae, compared with other
bacteria such as g-proteobacteria and Vibrio/Photobacterium, was higher for V. cholerae associated with zooplankton (especially zooplankton >202 mm) than in the water
column. The results suggest that V. cholerae outcompetes
other bacterial taxa associated with zooplankton and is
commensal with copepods.
The objective of this study was twofold: to determine
the occurrence and distribution of V. cholerae in sea water
and plankton in Peruvian coastal areas and to monitor the
occurrence of cholera cases in nearby cities to identify the
start of an epidemic and relate it to climate and environmental conditions, such as sea surface temperature
(SST), salinity and species composition of plankton. The
presence of culturable and non-culturable V. cholerae was
therefore determined to establish the aquatic environment
along the coast of Peru as a reservoir of the causative
agent of cholera. In addition, a focus of current research
in our laboratory is to develop a predictive model for cholera, as the ability to predict cholera in cholera endemic
areas can be a powerful tool for prevention by public
health workers.
Results and discussion
A total of 178 samples of water and plankton were collected from the four sampling sites between October 1997
and June 2000. Although the incidence of cholera in Lima
and in the nearby port of Callao was our primary interest,
two other sites were included in the study located north
and south of Lima in order to obtain an indication of the
range of distribution of V. cholerae in the coastal waters
of Peru (see Fig. 1). The greater Lima (including Callao)
area is a metropolis of nearly eight million inhabitants
located on the coast, while Trujillo and Arequipa are
smaller localities, with 620 000 and 750 000 inhabitants,
respectively, situated some distance from the coast.
Based on results obtained from the fluorescent antibody
assay (DFA), V. cholerae O1 could be detected in sea
water and plankton collected at all stations located along
the coast of Peru (Table 1). However, V. cholerae O1 was
isolated by culture only at the Lima site. For either method,
Fig. 1. Study sampling sites in Peru. Reprinted
from Lipp et al. 2003, with permission from the
American Society for Microbiology.
© 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 699–706
Vibrio cholerae in Peruvian waters 701
Table 1. Presence of V. cholerae O1 at sampling sites included in
this study, three fractions combined.
% positive (no of positive samples/total
samples analysed)
Sampling sites
Culturable V. cholerae O1
DFA O1
Trujillo
Callao
Lima
Arequipa
Total
0% (0/35)
0% (0/36)
14.7% (10/68)
0% (0/39)
5.6 % (10/178)
8.3%
13.8%
16.9%
25.7%
16.4%
(3/36)
(4/29)
(10/59)
(9/35)
(26/159)
The variable denominator results from occasional missing values.
DFA, direct fluorescent antibody assay.
sea water and both plankton fractions (i.e. P64 and P202,
as described in Experimental procedures) accounted for
positive samples and, on such occurrences, one, two or
all three fractions were positive. V. cholerae strains tested
by polymerase chain reaction (PCR) revealed the presence of the toxR gene, a global regulatory gene found in
V. cholerae and other vibrios. Further molecular genetic
characterization, using direct DNA extraction from sea
water and plankton samples (results reported elsewhere;
Lipp et al., 2003), confirmed the presence of V. cholerae
O1. Furthermore, the cholera toxin gene (ctxA) was also
present in 25% of these samples. V. cholerae O139 was
not detected, although weak agglutination with V. cholerae
O139 antiserum occurred with strains isolated from water
and plankton samples from the Lima site. The presumptive V. cholerae O139 strains were sent to the Center of
Marine Biotechnology, Baltimore, MD, USA, where they
were analysed further by PCR (Rivera et al., 2003). It was
concluded that these strains were not V. cholerae O139
but rather V. cholerae O22, a cross-reacting serogroup
that has been suggested to be the source of exogenous
DNA resulting in the emergence of the serogroup O139
(Dumontier and Berche, 1998). Such cross-reactivity has
been reported previously (Shimada et al., 1994; Isshiki
et al., 1996), and Dalsgaard et al. (2002) showed recently
that diagnostic O139 antiserum cross-reacted with V.
cholerae O22 and O155 serogroups.
Environmental data collected at the sampling sites are
summarized in Table 2. Water temperature and total bacterial counts did not differ significantly among sampling
sites (values statistically different from one another are
indicated by a different letter, a, b or c). On average, air
temperature was lower in Trujillo than at the other sites,
and salinity was higher at this coastal site. Chlorophyll a
concentrations were highest in Trujillo and lowest in Arequipa. Within the zooplankton populations analysed, two
copepod species typical of coastal waters, Acartia tonsa
and Paracalanus parvus, were overwhelmingly dominant
with respect to relative abundance. In over 20% of cases
for each, they constituted more than half, and up to 90%,
of the zooplankton. P. parvus was the most highly prevalent species and was present in 71% of samples in Trujillo,
82% in Callao, 95% in Lima and 74% in Arequipa. A. tonsa
was less prevalent in Trujillo (45% of samples) than in
Arequipa (84% of samples), while it was present in 91%
of samples in Lima and Callao. However, no pattern could
be clearly established between V. cholerae O1 and the
presence of a specific zooplankton species in the larger
plankton fraction (P202). In the smaller fraction (P64),
nauplii were positively associated with V. cholerae O1
detected by DFA (P = 0.0375), whereas chlorophyll a did
not correlate with V. cholerae O1 (P = 0.2246). One limitation of this study was the inability to address patchiness
of chlorophyll a, phytoplankton and zooplankton. Higher
temporal and spatial resolution may help to answer this
question. In this regard, the use of remote sensing to
detect chlorophyll a should help resolve the problem.
Figure 2 provides a comparison of the percentage of
water and plankton samples positive for V. cholerae O1.
The plankton samples were split, based on two sizes, i.e.
between 64 mm and 202 mm and >202 mm. No difference
was detected among the three fractions in the case of
culturable V. cholerae O1. The analysis was limited, however, because the number of positives was low (n = 10).
By DFA, V. cholerae O1 was detected least frequently in
the larger plankton (> 202 mm). This was also the case
when results were examined site by site. Overall, V. chol-
Table 2. Environmental data collected at the study sampling sites with minimum, maximum (upper line) and mean ± standard deviation (lower
line) provided.
Sampling
sites
n
Trujillo
36
Callao
36
Lima
68
Arequipa
39
Air temperature
(∞C)
Water temperature
(∞C)
Salinity
(p.p.t.)
Chlorophyll a
(mg m-3)
Total bacterial countsa
(105 cell ml-1)
16.0 –22.0
19.6 ± 2.3 a
22.0–26.0
24.4 ± 1.7 b
16.0–30.3
22.8 ± 3.6 b
15.0–30.0
23.8 ± 3.7 b
12.0–29.0
17.5 ± 4.0 a
14.1–24.3
17.8 ± 2.7 a
15.0–25.1
18.4 ± 2.5 a
12.0–23.5
16.7 ± 2.9 a
35.0–37.0
35.6 ± 0.7 a
34.0–36.0
35.0 ± 0.4 b
34.0–37.0
35.1 ± 0.6 b
30.0–37.0
35.1 ± 1.2 b
1.5–150.9
35.8 ± 48.8 a
0.0–122.6
18.6 ± 32.3 b
0.0–136.2
13.2 ± 28.6 b
0.0–16.6
2.3 ± 3.2 c
0.02–11.6
1.9 ± 2.5 a
0.4–18.0
4.1 ± 5.3 a
0.01–14.1
2.5 ± 3.1 a
0.003–10.8
1.9 ± 2.5 a
For each column, means that are statistically different are indicated by different letters (a, b or c); P < 0.05. n = number of sampling events per site.
a. DAPI counts (see Experimental procedures).
© 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 699–706
702 A. I. Gil et al.
Fig. 2. Percentage of samples positive for V. cholerae O1. N, total
samples analysed; W, sea water; P64, plankton ≥64 mm but <202 mm;
P202, plankton ≥202 mm in size. DFA, direct fluorescent antibody
assay. Q statistic (Fleiss, 1981) was used to compare the proportion
of V. cholerae in each fraction; values statistically different from one
another are indicated by a different letter (a, b or c); P < 0.05.
erae O1, detected by DFA, occurred more frequently in
sea water than in plankton samples, but the difference
was significant only at P < 0.1. This trend was similar to
that observed for a subset of the same samples that were
analysed using PCR (Lipp et al., 2003) and to results of
a similar study carried out in Chesapeake Bay, MD, USA
(Louis et al., 2003). It is also in accordance with the observations of Heidelberg et al. (2002b), who reported that
only a small fraction of the total water column bacteria is
associated with zooplankton. Although V. cholerae may
have a competitive advantage, compared with other bacteria, when associated with zooplankton, the majority of
bacteria may be free-floating in the water column. It
should be noted that, at peak periods of zooplankton
blooms, bacteria are released into the surrounding sea
water as the bloom ‘crashes’, i.e. when zooplankton die
and their skeletal structures disintegrate (Gooday, 1990;
Flach et al., 1992).
Our current knowledge of V. cholerae suggests that its
presence in coastal water, as estimated by the method of
detection, is dependent on various environmental parameters and complex ecological interplay. Higher water temperatures typically increase the abundance of V. cholerae
in natural waters (Lipp et al., 2002). In the temperate
estuary of Chesapeake Bay, it was shown (Louis et al.,
2003) that increased influx of freshwater increased the
detection frequency of V. cholerae O1. Salinity was concluded to be a statistically significant parameter associated with increased numbers of V. cholerae. However, as
the increase in freshwater inflow was simultaneously
associated with lower salinity, increased nutrient load and
phytoplankton blooms, a combination of these factors is
most likely to be responsible for increased frequency of V.
cholerae. A similar phenomenon appears to occur along
the Peruvian coast, where run-off alters salinity and nutrient loads locally.
Zooplankton blooms typically feed on phytoplankton
blooms, and V. cholerae, with the ability to degrade chitin
and being associated with zooplankton, especially copepods (Huq et al., 1983), benefits from such events. In this
study, the smaller size plankton fraction (P64) included
large phytoplankton and microzooplankton, while the
larger size plankton fraction (P202) principally included
mesozooplankton. The overall apparent lack of association observed here between V. cholerae and plankton may
result, in part, from the complexity of the plankton fractions analysed. The power of discrimination may not have
been high enough to detect the signal associated with the
presence V. cholerae O1. Nonetheless, nauplii contribute
significantly to the distribution of V. cholerae O1 along this
coastal environment. Experiments are in progress to
determine whether V. cholerae is preferentially associated
with certain types or species of plankton.
The number of cholera cases occurring in the population of Peru in proximity to the coastal study sites is shown
in Fig. 3. Seasonal changes in water temperature (SST)
were observed at all sites in the study, i.e. SST increased
in the spring and reached a peak during the summer
(January–March). For all sites included in this study, the
highest SST was observed during the summer of 1998,
as was expected given that it occurred during the forecasted 1997–98 El Niño event. Statistical analysis with
linear regression showed that cholera outbreaks occurring
in the summer of 1998 were correlated with the SST peak
(P < 0.001). When it was predicted that an El Niño event
would occur in 1997–98, we were able to plan a sampling
strategy to determine the presence of V. cholerae in sea
water and plankton before the event and to analyse the
relationship between SST and cholera, as had been
observed in the 1991–92 El Niño event. When SST
decreased, very few or no cases of cholera were reported.
Furthermore, analysis of sewage samples by Speelmon
et al. (2000) led to the conclusion that proliferation of V.
cholerae occurred during the period of warmer temperatures linked with El Niño.
Significant evidence has been accumulated showing
that, in Bangladesh, SST is a prime environmental factor
associated with cholera cases (Lobitz et al., 2000; Pascual et al., 2000; Rodo et al., 2002). In this area of endemicity, cholera is characterized by two annual peaks, with
a large peak in the autumn and a smaller one in the spring
(Faruque et al., 1998). Lobitz et al. (2000) found a significant correlation between cholera incidence and SST. That
is, when SST decreases in the summer during the monsoon, the number of cases declines. When cholera was
prevalent throughout the Indian subcontinent, it was recognized that the pattern of cholera outbreaks was seasonal, but that the timing of epidemics varied from one
area to the next (Pollitzer, 1959). Furthermore, climatic
conditions, such as temperature, precipitation and humidity, were identified as influencing the severity of cholera
© 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 699–706
Vibrio cholerae in Peruvian waters 703
Fig. 3. Seasonal data of cholera incidence rate and sea surface temperature (SST) in Peru during the time of this study. (TR, Trujillo; LI, Lima;
CA, Callao; AQP, Arequipa).
outbreaks (Pollitzer, 1959). Therefore, it is expected that
cholera outbreaks will be dependent on the local ecology
and timing of meteorological events. Unlike the Bay of
Bengal, where the SST is usually above 24∞C year
around, with an average of ª27∞C (Lobitz et al., 2000), the
coast of Peru experiences cold upwelling. In Callao, the
SST averages 17∞C and ranges from 14∞C to 20∞C during
a non-El Niño year (NOAA, 2003). During the 1997–98 El
Niño event, the SST was above normal for ª13 months
(Sanchez et al., 2000) and increased by up to 4∞C above
the seasonal average. This El Niño event had profound
ecological repercussions (Chavez et al., 1999), and the
sustained above-normal temperatures are concluded to
have played a role in the increased number of cholera
cases. In the unique ecological conditions of the Peruvian
coast, characterized by recurring El Niño climatic events,
SST should be considered as an important predictive
parameter for cholera.
In this study, we have demonstrated that V. cholerae O1
is, indeed, present in sea water and plankton along the
Peruvian coast, with culturable V. cholerae O1 isolated
from samples collected off the Lima coast, and the overall
results were corroborated by PCR (Lipp et al., 2003).
Lima, a large coastal urban area, provides suitable conditions for growth of V. cholerae via run-off, ocean pollution
and increased nutrients, resulting in culturable V. cholerae
being detected at this site. However, failure to obtain culturable V. cholerae O1 at sites other than Lima during the
1997–98 cholera outbreak is typical of the difficulty in
© 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 699–706
isolating V. cholerae from unpolluted environmental samples despite its presence (Pollitzer, 1959; Roszak and
Colwell, 1987; Lipp et al., 2002). Also, it was not possible
to collect samples immediately before the epidemic in
Arequipa (November and December 1997) and during the
epidemic in Trujillo (February and March 1998) because
severe weather and unusually high tides at those particular sampling times made it impossible for small craft to
reach the sampling sites. Nevertheless, V. cholerae O1
was detected at all sites by DFA (this study) and PCR
(Lipp et al., 2003) throughout the entire study. The
fluorescent monoclonal antibody and PCR methods
allowed detection of V. cholerae O1 whether cells were
culturable or not. Thus, the DFA and PCR methods offer
useful and reliable approaches for environmental studies
of V. cholerae and related bacteria and, for these reasons,
are preferable to culture (Huq et al., 1990; Lipp et al.,
2003).
As SST can be monitored by satellite (Lobitz et al.,
2000), it can be a useful parameter for continuous monitoring. Although a direct association with chlorophyll a
could not be established in this study, measurements
obtained by remote sensing may provide the increased
temporal and spatial resolution needed for predicting the
increased presence of V. cholerae in the coastal environment of Peru. Further studies on a larger scale are being
conducted to expand the findings of this study for other
Latin American countries where cholera is an ongoing
public health problem.
704 A. I. Gil et al.
Experimental procedures
Study sites and survey parameters
This study included four sampling sites along a 1600 km
stretch of the Peruvian coast. At each site, a station was
located 0.5–1.0 km offshore, using a global positioning system (GPS 12, Garmin, Olathe). Station 1 was established at
Trujillo, in the port of Salaverry, 700 km north of Lima (S
8∞13¢, W 78∞59¢). Station 2 was at the port of Callao, 35 km
west of Lima (S 12∞03¢, W 77∞09¢). Station 3 was located at
Lima, in the port of Chorrillos (S 12∞09¢, W 77∞02¢), and
station 4 was established at Arequipa, in the port of Matarani,
900 km south of Lima (S 16∞59¢, W 72∞06¢) (Fig. 1). The
period encompassing the study was from October 1997 to
June 2000. Sea water and plankton samples were collected
monthly over approximately three annual seasons. Before the
summer season, i.e. during October to December, samples
were collected twice monthly. Also, at stations 2 and 3, samples were collected twice monthly in July 1997 (winter) in
order to standardize the collection methods used in this
study.
Sample collection
Samples were usually collected before noon, using a small
craft from the local port to reach the sampling site. GPS was
used to identify the sampling position. Surface water was
collected using two 2.5 l sterilized polypropylene flasks, and
samples were stored in an insulated box fitted with cold packs
to maintain the temperature between 2∞C and 10∞C. Plankton
samples were collected using 64 mm and 202 mm mesh-size
plankton nets towed 1 m below the surface. The larger meshsize net was placed inside the smaller mesh-size net to
obtain two plankton fractions. The smaller fraction (referred
to as P64) included large phytoplankton and microzooplankton ranging from 64 mm to 202 mm in size, while the larger
fraction (referred to as P202) included mesozooplankton
>202 mm in size. Each fraction (ª 50 ml) was transferred to
a sterilized flask. Samples were maintained at temperatures
between 2∞C and 10∞C, transported to the laboratory and
processed within 4 h of collection.
Isolation of culturable V. cholerae
Standard microbiological methods were used to culture V.
cholerae from sea water and plankton, as described elsewhere (American Public Health Association, 1989). Briefly, 3 l
of water was filtered using 0.2 mm polycarbonate membranes. Membranes were placed in 100 ml of alkaline peptone water (APW; 1% peptone, 1% NaCl, pH 8.4), incubated
at ª25∞C for 18 h, and a loopful was streaked on thiosulphate–citrate–bile salts (TCBS) agar. Five sucrose-positive
colonies were randomly picked from TCBS agar. Presumptive
V. cholerae colonies were identified using triple sugar iron
agar (Difco Laboratories), lysine iron agar (Difco Laboratories), indole and oxidase reactions. Approximately 20 ml of
the zooplankton and phytoplankton samples was filtered onto
0.2 mm polycarbonate membranes. The filters were pro-
cessed following the same procedures as described above
for water samples.
Serology and PCR
Presumptive V. cholerae isolates were tested using antisera
to identify V. cholerae O1 and O139 serotypes by agglutination, as described by Kay et al. (1994). V. cholerae O1 was
identified by agglutination with Inaba-Ogawa polyvalent antiserum (Difco Laboratories). V. cholerae O139 was identified
with monoclonal antiserum O139 [Centre for Health and Population Research (ICDDR,B) Dhaka, Bangladesh]. PCR primers used to identify V. cholerae O1, V. cholerae O139, V.
cholerae O22, the ToxR gene (toxR) and the cholera toxin
gene (ctxA) are described by Rivera et al. (2001).
Direct fluorescent antibody assay and direct viable count
for V. cholerae O1 (DFA)
Vibrio cholerae O1 was detected in water and plankton samples using a direct fluorescent antibody/direct viable count
assay (DFA/DVC) (Hasan et al., 1994). Briefly, 1 l of water
and 20 ml of plankton were filtered using 0.2 mm polycarbonate filters. The filters were rinsed with 1 ml of filter-sterilized
sea water. Concentrated samples were mixed with 0.025%
yeast extract and 0.002% nalidixic acid and incubated at
25∞C in the dark for 18 h (Kogure et al., 1979). After incubation, 10 ml of each sample was placed on a glass slide and
allowed to air dry. Specific fluorescent monoclonal antibody
against V. cholerae O1 (New Horizon Diagnostics) was
added, and the reaction was allowed to proceed for 15 min
at 35∞C in a moist dark chamber. The slides were washed
with distilled water and air dried. Slides were examined with
an epifluorescent microscope (Axiolab; Carl Zeiss), using BP
450–490 nm and LP 520 nm filters, and the presence of
elongated fluorescent cells was recorded. Positive and negative controls from the kit were included on each slide.
Environmental parameters
Sea surface temperature (SST) was measured during sampling using a certified thermometer 0∞C to 50∞C (Sama Precision, Ertco) placed 1 m below the sea surface and allowed
to calibrate for 15 min. Air temperature (AT) was recorded
using a digital thermometer (High Precision, Ertco) at the
beginning and end of each sampling. Salinity was measured
using a hand-held salinometer (Vista A366ATC).
Chlorophyll a determination
Samples were processed by methanol extraction as
described elsewhere (Louis et al., 2003). Briefly, 25 ml of
each water sample (in duplicate) was filtered using a glass
microfibre (GF/F) filter (Whatman). Filters were wrapped in
aluminium foil and frozen immediately at -80∞C until
processed.
© 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 699–706
Vibrio cholerae in Peruvian waters 705
Total bacterial direct count
Water samples (10 ml), fixed immediately after collection with
a final concentration of 2% formaldehyde (Fisher Scientific),
were incubated in the dark at room temperature with a final
concentration of 10 mM 4¢,6-diamino-2-phenylindole (DAPI;
Sigma), for 2 h. After staining, 1–3 ml of sample was filtered
through a 25-mm-diameter, 0.2 mm pore size black polycarbonate membrane (K02BP02500; Osmonics), in duplicate.
Filters were mounted on microscope slides using low-fluorescence immersion oil (type A or FF) and examined at 1000¥
magnification with an epifluorescent microscope (Axiolab;
Carl Zeiss), using an LP 420 nm UV filter.
Plankton identification
Phytoplankton and zooplankton analyses were carried out by
the Instituto del Mar del Perú (IMARPE), Callao, Peru, following IMARPE standard methodology (Schiller, 1933–37; Cupp,
1943; Einarsson and Rojas de Mendiola, 1963; Boltovskoy,
1999).
Briefly, 2 ml samples of 3% formalin-fixed phytoplankton
(P64 fraction) were gently homogenized, and 1 ml was pipetted on to a clean slide and observed at 16 ¥ 12.5 (200¥) and
40 ¥ 2.5 (500¥) using a Leitz, model Dialux 20 compound
microscope.
A total of 2 ml of 3% formalin-fixed zooplankton samples
(P202 fraction) was inoculated on to Bogorov plates for
counting, and the plates were read at 10¥ and 40¥ using a
Nikon model SMZ stereoscopic microscope.
Cholera cases
The number of suspected and confirmed cholera cases
was obtained for each site where environmental samples were collected, as well as for the entire country.
The data were provided by the Office of Epidemiology,
Peruvian Ministry of Health (personal communication,
http://www.minsa.gob.pe/cedco/).
Data analysis
The statistical software package SAS, version 8.2 (SAS Institute) was used to perform statistical analyses. Cochran’s Q
statistic (Fleiss, 1981) was used to test for significant difference among proportions in the three sampling fractions. For
the rest of the analyses, sea water and plankton fractions
were combined, i.e. the combined fraction sample was considered positive when any of the three fractions, sea water,
small plankton or large plankton, was positive. Throughout
the analysis, significance was declared if P < 0.05.
Acknowledgements
We are grateful to Dr Hernan Miranda, UNT, Trujillo, and to
Dr Walter Medina, UNSA, Arequipa, and their laboratory personnel for their co-operation in carrying out the sampling and
laboratory facilities. We are grateful to Mrs Rita Orozco from
IMARPE for collaboration in sampling at Callao station. We
thank Mrs Carmen Barreno and Mr William Lopez for excellent laboratory support. This research was supported in part
© 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 699–706
by NIH grants 1R0A13912901 and R01 NRO 4527 01A1, US
Environmental Protection Agency STAR grant R824995 to
Johns Hopkins University, and NASA grant no. NAG 2-1195
to the University of Maryland Biotechnology Institute. The
authors acknowledge the excellent co-operation of Dr
Jonathan Patz in carrying out the research.
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