Health Assessment and Seroepidemiologic Survey of
Potential Pathogens in Wild Antillean Manatees
(Trichechus manatus manatus)
Kathryn Sulzner1, Christine Kreuder Johnson1, Robert K. Bonde2, Nicole Auil Gomez3, James Powell3,
Klaus Nielsen4, M. Page Luttrell5, A. D. M. E. Osterhaus6, A. Alonso Aguirre7,8*
1Wildlife Health Center, School of Veterinary Medicine, University of California Davis, Davis, California, United States of America, 2Sirenia Project, United States Geological
Survey, Gainesville, Florida, United States of America, 3Sea to Shore Alliance, St. Petersburg, Florida, United States of America, 4Canadian Food Inspection Agency,
Nepean, Ontario, Canada, 5Southeastern Cooperative Wildlife Disease Study, Department of Population Health, College of Veterinary Medicine, University of Georgia,
Athens, Georgia, United States of America, 6Erasmus Medical Centre, Rotterdam, The Netherlands, 7Smithsonian-Mason School of Conservation, Front Royal, Virginia,
United States of America, 8Department of Environmental Science and Policy, George Mason University, Fairfax, Virginia, United States of America
The Antillean manatee (Trichechus manatus manatus), a subspecies of the West Indian manatee, inhabits fresh, brackish, and
warm coastal waters distributed along the eastern border of Central America, the northern coast of South America, and
throughout the Wider Caribbean Region. Threatened primarily by human encroachment, poaching, and habitat
degradation, Antillean manatees are listed as endangered by the International Union for the Conservation of Nature.
The impact of disease on population viability remains unknown in spite of concerns surrounding the species’ ability to
rebound from a population crash should an epizootic occur. To gain insight on the baseline health of this subspecies, a total
of 191 blood samples were collected opportunistically from wild Antillean manatees in Belize between 1997 and 2009.
Hematologic and biochemical reference intervals were established, and antibody prevalence to eight pathogens with
zoonotic potential was determined. Age was found to be a significant factor of variation in mean blood values, whereas sex,
capture site, and season contributed less to overall differences in parameter values. Negative antibody titers were reported
for all pathogens surveyed except for Leptospira bratislava, L. canicola, and L. icterohemorrhagiae, Toxoplasma gondii, and
morbillivirus. As part of comprehensive health assessment in manatees from Belize, this study will serve as a benchmark
aiding in early disease detection and in the discernment of important epidemiologic patterns in the manatees of this region.
Additionally, it will provide some of the initial tools to explore the broader application of manatees as sentinel species of
nearshore ecosystem health.
Citation: Sulzner K, Kreuder Johnson C, Bonde RK, Auil Gomez N, Powell J, et al. (2012) Health Assessment and Seroepidemiologic Survey of Potential Pathogens
in Wild Antillean Manatees (Trichechus manatus manatus). PLoS ONE 7(9): e44517. doi:10.1371/journal.pone.0044517
Editor: Niko Speybroeck, Universite ´ Catholique de Louvain, Belgium
Received June 4, 2012; Accepted August 8, 2012; Published September 12, 2012
Copyright: ? 2012 Sulzner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Financial support for many of the serological assays performed in this study was provided in part by the University of California, Davis, CA, Wildlife
Health Center’s Wildlife Fellowship Fund and by a grant from the Preventive Veterinary Department, University of California, Davis, CA. Additional laboratory tests
and services were offered in-kind by Dr. Pat Conrad, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of
California, Davis, CA; Dr. William Reisen, Center for Vectorborne Disease Research, University of California, Davis, CA; Dr. Dave Stallknecht and Page Luttrell,
Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA; Dr. Klaus Nielsen, Canadian Food Inspection Agency, Nepean,
Ontario, Canada; and Dr. A.D.M.E Osterhaus, Department of Virology, Erasmus Medical Centre, Rotterdam, The Netherlands. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
The Antillean manatee (Trichechus manatus manatus), a subspecies
of the West Indian manatee, is an herbivorous, aquatic mammal
restricted to warm coastal waters and inlets along Central
America, the northern coast of South America, and the Caribbean
[1,2,3]. With populations continuing to decline, the Antillean
manatee is currently listed as endangered by the International
Union for the Conservation of Nature (IUCN) . The manatees
inhabiting the waters of Belize are thought to serve as a vital
source population for manatee populations occupying the
neighboring coasts of Mexico, Guatemala, and Honduras ;
thus, ongoing efforts to conserve this population are crucial.
Unfortunately, low recruitment, due in part to a lengthy calving
interval, may not be adequate to maintain population viability in
the face of mounting anthropogenic threats [2,5,6]. Based on
preliminary assessments, two-thirds of all manatee mortalities in
Belize can be traced to habitat loss, perinatal death, and human
activities including poaching, trauma suffered from collisions with
watercraft, and fatalities from fishing equipment .
Current strategies to protect manatees in Belize are focused on
mitigating anthropogenic pressures but do not effectively address
the remaining one-third of manatee deaths resulting from
unspecified causes . In particular, little is known about the
impact of disease at the population level or on the stability of
sirenian populations elsewhere [2,6]. Although manatees are
thought to be fairly resistant to natural disease [5,7,8,9], shifts in
the aquatic environment brought on by climate change, sea level
rise, human encroachment, habitat destruction, and pathogen
PLOS ONE | www.plosone.org1September 2012 | Volume 7 | Issue 9 | e44517
pollution at the land-water interface, may make this nearshore
species increasingly susceptible to infectious agents [10,11,12,13].
Sudden changes in manatee health may signify a larger
environmental disturbance at play, as has been demonstrated
with brevetoxin-related epidemics [14,15]. Manatees may there-
fore be useful sentinels of the surrounding ecosystem [7,10,16],
illustrating the broader application of health research in this
In order to evaluate baseline health in Belize’s manatee
population, a collaborative, multi-agency health assessment
initiative was launched in 1997, complementing objectives
outlined in the Belize’s Manatee Recovery Plan . Between
1997 and 2009, blood samples and other health-related data were
collected from 115 wild Antillean manatees captured and
examined in southern Belize. The present study was integral to
this larger manatee health initiative by fulfilling two primary
objectives: The first was to establish normal hematologic and
biochemical reference intervals for the manatees of this region; the
second was to describe the seroepidemiology of eight potential
pathogens in the wild manatee population of southern Belize. The
pathogens of interest were selected for their zoonotic capabilities
and for their association with morbidity and mortality events in
other marine mammals.
Materials and Methods
Approval for this project was granted to lead scientists from the
following governmental and non-governmental agencies and
programs: The Coastal Zone Management Authority and
Institute’s (CZMAI), Manatee Project, under the jurisdiction of
Belize’s Ministry of Agriculture, Fisheries, and Co-operatives;
Belize’s National Manatee Working Group; Ecohealth Alliance;
and Sea to Shore’s Manatee Conservation Program in Belize. The
Institutional Animal Care and Use Committees of Ecohealth
Alliance and U.S. Geological Survey, Southeast Ecological
Science Center, permitted authorization for research, including
manatee capture and sampling protocols. Data and samples were
collected under research permits issued by the Belize Forest
Department, Ministry of Natural Resources, and the U.S. Fish and
Wildlife Service permit number M79 1721-4 issued to the U.S.
Geological Survey, Sirenia Project.
Manatees and Sample Collection
Between 1997 and 2009, blood samples were collected
opportunistically from 115 apparently healthy, wild Antillean
manatees in Belize. With few exceptions, data collection took place
biannually, typically occurring during alternating wet and dry-
seasons (i.e., May/June – November and December – April/May,
respectfully) [17,18]. Captures occurred in, or around the
periphery of, one of four primary sites: Southern Lagoon in Gales
Point Wildlife Sanctuary (17.20532uN, 88.33643uW); Northern
(16.53184uN, 88.37703uW); and the Drowned Cayes area
(17.48281uN, 88.09765uW) (Fig. 1). For the purposes of this study,
manatees captured in Western, Quashie Trap, Buttonwood, and
Sapodilla lagoons, and those captured along the coast near Mullins
River mouth were included in the Southern Lagoon subpopulation
since travel among these sites is common.
A standard capture technique was employed [19,20] in which
manatees were approached by boat in near shore water of 1–1.5
meters in depth. A large, 13 cm stretch-mesh, nylon net (152 m
long, 6 m deep) was then lowered into the open water surrounding
the target manatee . The net was then collapsed, and
individuals, once restrained, were carefully transported to shore
or brought onto the deck of the capture boat for examination. A
physical exam was performed on each manatee by a trained
veterinarian or biologist under veterinary supervision prior to
sample collection . The sex of the individual was determined
by assessment of dimorphism of the genital-anal distance
[20,22,23]. Age classification was based on standardized total
straight length measurements specific to Antillean manatees :
adult (.225 cm); subadult (176–225 cm); and calf (,176 cm).
Detailed morphometrics were recorded, and subcutaneous fat
thickness measurements were taken with an ultrasound. Vital signs
were recorded throughout the holding time to monitor for signs of
Following the recommended technique for venipuncture in
manatees [20,26], blood samples were obtained from the brachial
vascular bundle, located on the medial aspect of the flipper. Blood
draws were performed using an 18 or 21 gauge, 1.5 inch needle
attached to an extension set equipped with a LuerH adapter in a
VacutainerH collar (Becton-Dickson (BD), Franklin Lakes, New
Jersey, USA). Approximately 20–40 ml of whole blood were
collected from each animal into potassium EDTA anticoagulant
VacutainerH blood tubes (BD, Franklin Lakes, New Jersey, USA)
for complete and differential blood cell counts. An additional 60–
80 ml of whole blood were obtained for serologic and biochemical
analysis; this quantity was divided into plain red top or tiger top
serum separator VacutainerH tubes for serum (BD, Franklin Lakes,
New Jersey, USA) and green top lithium heparinized VacutainerH
tubes for plasma (BD, Franklin Lakes, New Jersey, USA). All blood
samples were placed on ice packs at 4uC immediately after
venipuncture. Samples intended for serology or biochemistry were
centrifuged within eight hours of collection. Following centrifuga-
tion, the serum (supernatant) and plasma from these tubes was
transferred to either one or four ml aliquots depending on total
Once collected, blood samples were brought to the Belize
Medical Associates Laboratory (BMAL) (Belize Medical Associ-
ates, 5791 St. Thomas Street, Belize City, Belize), where they were
either processed for hematology and biochemistry analysis within
12–24 hours of collection or transferred to a 220u freezer. Sera
and plasma to be archived were transported to Florida under the
Convention on International Trade in Endangered Species
(CITES) permit authority and stored in a 280uC freezer at the
U.S. Geological Survey, Southeast Ecological Science Center in
Gainesville, Florida (USGS SESC, Gainesville, FL). Subsamples of
serum and plasma were shipped to the University of Florida,
College of Veterinary Medicine, Clinical Pathology Laboratory
(UFPL) (University of Florida Pathology Laboratories, Gainesville,
FL) for additional biochemistry analysis.
Complete and differential blood cell counts performed at
BMAL included the following parameters: packed cell volume
(PCV); white blood cell count (WBC); red blood cell count (RBC);
hemoglobin (Hb); mean corpuscular volume (MCV); mean
corpuscular hemoglobin (MCH); mean corpuscular hemoglobin
concentration (MCHC); and platelets (PLT). White blood cell
differentials were performed for granulocytes (i.e., neutrophils,
eosinophils, and basophils) and for agranulocytes (i.e., lymphocytes
and monophils). Differentials were converted to absolute cell
counts for statistical analysis.
At the start of the study, complete serum biochemistries were
performed at BMAL. After 2003, additional biochemistry profiles
were performed on plasma samples at UFPL. For quality control
purposes, a blind test was run to compare serum biochemistry
Antillean Manatee Health Assessment and Serosurvey
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results from BMAL with plasma biochemistry results from UFPL
in individuals from which both serum and plasma samples were
obtained. With the exception of minor disparities involving three
analytes: alanine aminotransferase (ALT), aspartate aminotrans-
ferase (AST), and triglycerides, there were no significant differ-
ences in biochemistry results between the two laboratories or
between serum and plasma samples from the same individual. To
maintain consistency in reference interval determination for ALT,
AST, and triglycerides, serum samples continued to be processed
at BMAL for these three analytes.
Plasma biochemistry profiles conducted at UFPL were per-
formed on a Hitachi 911TMChemistry Analyzer (Boerhinger
Mannheim/Roche Applied Science, Indianapolis, Indiana, USA)
using reagents and technique described elsewhere . The
biochemistry profile included the following blood analytes:
glucose; blood urea nitrogen (BUN); creatinine; alkaline phospha-
tase (ALP); gamma-glutamyl transferase (GGT); cholesterol; total
protein (TP); albumin (ALB); potassium; magnesium; chloride;
sodium; calcium; phosphorous; total bilirubin; direct bilirubin;
amylase; lipase; lactate dehydrogenase; creatinine kinase phos-
phokinase; serum amyloid A; and iron.
Pathogens selected for serologic testing included the following
bacterial, parasitic, and viral agents listed respectively: Brucella spp.
and Leptospira interrogans spp.; Neospora caninum, Sarcocystis neurona,
and Toxoplasma gondii; and avian influenza virus type A (AIV),
morbillivirus, St. Louis encephalitis virus (SLEV), Western equine
encephalitis virus (WEEV), and West Nile virus (WNV). None of
the pertinent serologic tests available have been validated in
manatees. In view of this, assays for which positive controls have
already been established in other terrestrial or marine mammals
were chosen as the next best alternative.
A fluorescence polarization assay was performed to test for
antibodies to Brucella spp. (Klaus Nielsen, Canadian Food
Inspection Agency, Nepean, Ontario, Canada) using positive
controls of bovine origin and corresponding methods to identify
seropositive animals described elsewhere . Detection of
antibodies to the following panel of L. interrogans serovars: L.
bratislava; L. canicola; L. gryppotyphosa; L. hardjo; and L. icterohemor-
rhagiae was achieved using a modified aggluntination test (MAT)
(positive titer .1:100), (California Animal Health & Food Safety
Laboratory System (CAHFS) - Davis Laboratory, University of
California, Davis, CA).
An indirect fluorescent antibody test (IFAT) was run for
detection of antibodies to N. caninum, S. neurona, and T. gondii,
(Department of Pathology, Microbiology and Immunology,
School of Veterinary Medicine, University of California, Davis,
CA). In order to perform the IFAT on manatee sera, fluorescein
isothiocyanate (FITC) - labeled goat anti-mouse IgG Fc - specific
secondary antibody against purified manatee IgG was produced at
the University of Florida (Interdisciplinary Center for Biotechnol-
ogy Research Hybridoma Core Laboratory, University of Florida,
Gainesville, FL). Results for T. gondii were further verified with a
latex agglutination test (LAT). In the absence of serological test
validation for T. gondii in manatees, we relied on published studies
and laboratory recommendations as a guide for selecting cut-off
titers for the IFAT ($ 1:320)  and LAT ($ 1:40) [30,31,32].
Serologic testing for morbillivirus was accomplished using a
viral neutralization (VN) assay against canine distemper virus
(CDV) and against porpoise and dolphin morbilliviruses (PMV/
DMV) (A.D.M.E Osterhaus, Department of Virology, Erasmus
Medical Centre, Rotterdam, The Netherlands). A neutralization
antibody titer response $ 10 was considered positive . A
commercial blocking enzyme-linked immunosorbent assay (Flock-
Check AI MultiS-Screen Antibody Test Kit, IDEXX Laborato-
ries, Westbrook, ME), validated with serum samples from ferrets
that were experimentally infected with AIV, was performed to
Figure 1. Seroprevalence (%) of exposure to Leptospira bratislava in the sample population as a whole and by capture site in wild
Antillean manatees (Trichechus manatus manatus) in Belize between 1997 and 2009.
Antillean Manatee Health Assessment and Serosurvey
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assess serum antibodies to AIV type A (Department of Population
Health, College of Veterinary Medicine, University of Georgia,
Athens, GA). Absorbance values were read with a Bio-Rad
Benchmark Microplate Reader (Hercules, California, USA) at
650 nm, and serum sample to negative control (S/N) absorbance
ratios were calculated for each sample. Samples with S/N values
greater or equal to 0.50 were considered negative for the presence
of antibodies to AIV, and samples with S/N values less than 0.50
were considered positive. Manatee sera samples were screened for
SLEV, WEEV, and WNV with an Enzyme immunoassay (EIA)
(Center for Vectorborne Disease Research, University of Califor-
nia, Davis, CA) using biotin labeled goat anti-mouse IgG Fc-
specific secondary antibody against purified manatee IgG
prepared at the University of Florida (Interdisciplinary Center
for Biotechnology Research Hybridoma Core Laboratory, Uni-
versity of Florida, Gainesville, FL). Samples were considered
positive if a ratio of greater than two was observed based on the
average reading of two positive wells/one negative well.
Statistical analyses were performed for hematology, biochem-
istry, and serology data using the statistical software program,
STATA 11 (StataCorp, College Station, Texas). Basic descriptive
measures and graphical summaries including mean, median,
standard deviation, and histograms were used to evaluate the
distribution of each hematologic and biochemical variable.
Normality was assessed with the Shapiro-Wilks W test for
normality , along with standard methods to assess skewness
and kurtosis. The same procedures were performed on the
residuals for each variable . Corresponding plots (e.g. residual
versus fitted, residual versus predictor, kernel density, and
histogram) were also evaluated to further confirm which variables
approximated a Gaussian distribution and verify the decision to
remove certain values designated as outliers for each blood
parameter . Several variables (e.g. RBC, WBC, lymphocyte
count, glucose, BUN, creatinine, and ALT) were log-transformed
to better approximate a Gaussian distribution. The log-trans-
formed variables were then re-examined by the previous methods
to ensure that they satisfied the criteria and met the assumptions of
the statistical analyses chosen.
A two-way analysis of variance (ANOVA) was used to evaluate
sources of variability in blood values in relation to sex and age. In
examining differences in blood parameters by capture site, both
Placencia and Northern lagoons were excluded from the analysis
of all hematologic parameters due to inadequate sample size
(n,5); this was also the case for several biochemical parameters.
Consequently, when comparisons were restricted to two capture
sites (e.g. Southern Lagoon and Drowned Cayes), an unpaired t-
test was used to assess differences in mean parameter values. When
more than two capture sites met the sample size requirements for a
given blood parameter, a one-way ANOVA was performed. An
un-paired t-test was used to assess differences between group
means across wet and dry seasons. For comparisons involving
blood parameters that did not approximate a Gaussian distribu-
tion, even if log-transformed (i.e., monocytes, basophils, and
eosinophils), a Kruskal-Wallis one-way analysis of variance was
conducted. To avoid violating assumptions of independence, only
the first adequate blood sample (i.e., non-hemolyzed, non-lipemic)
obtained from each individual among those captured multiple
times was incorporated into the blood data analyses. Results with a
P-value ,0.05 were considered statistically significant. A Bonfer-
roni adjustment, with an overall significance level of a=0.05, was
used for post hoc comparison of pairs for grouping factors with
three or more levels .
Statistical tests were also used to assess the effect of covariates on
the seroprevalence of pathogen exposure in manatees from Belize.
In order to maintain statistical power, these analyses were limited
to cases where the sample size of seropositive individuals for a
given pathogen exceeded ten manatees in number. Following
these guidelines, a Pearson’s chi-square test of independence or
Fisher’s exact test was used to evaluate significant differences by
sex, age class, season, capture site, and year in relation to
serostatus to L. interrogans spp.
Of the 115 manatees for which blood samples were submitted
for hematology, biochemistry, or serology, males and females
were similarly represented, and age groups were distributed in
the same relative proportions within each sex (Table 1). Some
individuals (n=31) were captured multiple times over the 12-
year period, resulting in multiple blood samples from these
individuals. The majority of manatees were captured in the
Southern Lagoon (n=82), whereas considerably fewer manatees
were captured in the Northern Lagoon (n=8), Placencia
Lagoon (n=6), and Drowned Cayes (n=19). Based on physical
assessment at the time of capture, all individuals included in the
study appeared healthy.
In examining sources of variation in mean hematology and
biochemistry values, (expressed as estimated mean 6 standard
deviation), age class comprised the greatest source of variation.
Significant parameter differences were also observed in relation
to capture location, whereas differences based on sex and season
figured less prominently. Accordingly, reference intervals for the
majority of blood parameters were reported for the population
as a whole (Table 2), with separate reference intervals presented
for those blood parameters associated with significant differences
by age class (Table 3) and by capture location (Table 4).
The mean values of several hematology parameters including
PCV, RBC, PLT, WBC, and lymphocytes differed significantly in
relation to age class (Table 3), while sex had little influence on
hematologic indice values. Important interactions between these
two covariates were not identified. As with comparisons by sex,
mean hematology parameters varied little by capture location and
Table 1. Total number of Antillean manatees (Trichechus
manatus manatus) included in data results for hematology,
biochemistry, and serology in relation to sex and age.ab.
Age GenderHematology BiochemistrySerology
AdultMale 28 41 40
SubadultMale8 10 11
aAge classifications are based on standardized total straight length
measurements in centimeters: adults .255; subadults 176–225; and calves
bThe age classes of 9/112 manatees for which samples were submitted for
serologic testing were not available; this included seven males and two females.
Antillean Manatee Health Assessment and Serosurvey
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season. The only exceptions to this were mean MCV values, which
differed significantly among capture site subpopulations (Table 4),
and eosinophil counts, which were signficantly higher during the
dry season (264.46347.32/UL) than during the wet season
Age class and capture site were both found to be signfiicant
sources of variation in several biochemical parameter values
(Table 3 and Table 4). In contrast, mean biochemical analyte
levels were similar among male and female manatees. With respect
to season, only albumin values differed significantly, with higher
mean levels found during the dry season (4.760.68 g/dL) than
during the rainy season (3.860.83 g/dL, P,0.001).
Seroprevalence of Selected Pathogens
All manatees sampled in Belize had negative antibody titers to
Brucella spp., N. caninum, S. neurona, AIV type A, SLEV, WEEV,
and WNV. Seroprevalence results for Leptospira spp. established
that 23% (26/112) of the sample population was seropositive to at
least one serovar of L. interrogans. Among those manatees that were
seropositive, 14% (16/112) had antibody titers to L. bratislava, 4%
(5/112) to L. canicola, and 4% (5/112) to L. icterohemorrhagiae. None
of the manatees had positive antibody titers to L. interrogans
serovars; grippa, hardjo, and pomona. With regard to serologic
screening for targeted protozoal organisms, one manatee had an
equivocal positive titer to T. gondii following the IFAT, but all other
individuals were seronegative. On the LAT, 7% (8/112) of
manatees had positive antibody titers to T. gondii. Serology results
for morbillivirus revealed low neutralizing antibody titers to CDV
and DMV/PMV in 4% (4/112) of manatees sampled.
In examining serostatus in relation to sex, age class, season, and
capture location, we found a significant association between
seropositivity to L. interrogans and capture site (x2=11.25,
P=0.010). Manatees from the Northern Lagoon had the highest
prevalence of positive antibody titers to L. bratislava (x2=9.99,
P=0.010) (Figure 1). Seropositivity to L. bratislava was also more
prevalent in manatees captured during the dry season (x2=5.36,
P=0.021) than during the rainy season. No significant association
between serostatus and sex or age class was detected.
Table 2. Hematology and biochemistry values for all age
classes in wild Antillean manatees (Trichechus manatus
manatus) in Belize between 1997 and 2009.
AnalyteNMean ± SD MinMax
62 40.063.5928.3 47.3
Neutrophils (/L)811916.66990.48 31.04472.0
Monocytes (/UL)813.8617.210.0 96.0
Eosinophils (/UL) 81 160.86277.250.0 1333.0
Basophils (/UL)82 3.9616.470.0 104.0
Glucose (mg/dL) 7982.1637.9343.1 365.0
69 35.4616.71 7.0 93.0
Triglycerides (mg/dL)104 90.2640.270.0248.0
Albumin (g/dL)80 4.260.892.36.4
Calcium (mg/dL)8210.361.19 6.714.0
Potassium (mmol/L)825.360.70 3.97.1
Sodium (mmol/L) 82151.268.81119.0 171.0
Magnesium (mg/dl)815.261.32 2.59.3
Chloride (mmol/L) 8295.869.03 73.0 118.0
aMCH = mean corpuscular hemoglobin, MCHC = mean corpuscular
hemoglobin concentration, MCV = mean corpuscular volume, BUN = blood
urea nitrogen, AST = aspartate aminotransferase, ALT = alanine
aminotransferase, GGT = c-glutamyltransferase.
Table 3. Hematology and biochemistry values by age class for wild Antillean manatees (Trichechus manatus manatus) in Belize
between 1997 and 2009 with P valuesbindicating significant age differences.
NMean ± SDNMean ± SDNMean ± SD
5710.260.9920 10.860.755 12.162.07P=0.001
53 32.863.32 1935.062.75539.265.90P,0.001
422.560.4018 2.760.224 3.260.44P=0.012
57 4.662.06 206.862.38513.264.40P,0.001
aHb = hemaglobin, PCV = packed cell volume, RBC = red blood cell count, WBC = white blood cell count, Lymphs = lymphocytes, PLT = platelet count, Creat =
creatinine, ALP = alkaline phosphatase, Chol = cholesterol, TP = total protein, Phos = phosphorous.
bP values from F-test with 2 and n–3 df.
Antillean Manatee Health Assessment and Serosurvey
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Governmental and non-governmental agencies in Belize have
made huge strides in manatee conservation over the past two
decades; this has been largely achieved through community
outreach programs, partnerships with neighboring countries, and
dedicated research initiatives. In spite of these steps forward, the
overall health status of manatees in this region and the impact of
infectious disease on population health have not been well
investigated. A few pilot studies have been conducted previously
on Antillean manatees to determine hematology and biochemical
reference intervals, but sample sizes were limited, and one of these
studies was restricted to captive manatees [1,19,38,39]. In the
Florida subspecies, normal blood parameters have been docu-
mented in healthy free-ranging and captive manatees [27,40,41]
along with preliminary seroprevalence data [42,43]. Our study is
the first large-scale effort to establish hematology and biochemistry
reference intervals in healthy, wild Antillean manatees and report
on baseline pathogen exposure in this subspecies.
Hematology and Biochemistry Parameters
Mean blood values for both hematology and biochemistry
parameters differed most significantly in relation to age class.
Many of the age differences detected were either similar to the
findings in the Florida subspecies [27,40], or were typical of age-
related blood parameter variations observed in other mammals
[44,45]. In the present study, we elected to include calves in
comparisons by age in spite of sample size limitations. Establishing
health parameters that are age-specific may enhance our present
understanding of calf health and assist in determining causes
underlying perinatal mortality.
Similar to findings in Florida manatees [40,44], we found that
leukocyte and lymphocyte counts decreased with age, with the
highest counts observed in young calves. One finding unique to
Belizean manatees, regardless of age, was that neutrophils
comprised a much smaller percentage of the total leukocyte count
relative to lymphocytes; prior studies have reported the ratio to be
relatively equal  or often reversed in the case of newborn
manatees [40,44]. In general, leukocyte counts and inflammatory
response differ markedly among marine mammals . Manatees
typically exhibit a subtle or short-lived rise in leukocytes in
response to inflammation, similar to the pattern observed in bovids
[46,47]. Consequently, monitoring relative shifts in manatee
leukocyte counts may be more useful than relying on total counts
for diagnostic purposes . More recently, serum amyloid A and
albumin/globulin ratio have been established as more sensitive
markers of inflammation in manatees and are currently being
incorporated into health profiles [46,48].
Significant age differences were also detected in relation to red
blood cell indices. Specifically, we established that PCV, Hb, and
RBC decreased with increasing age. Overall, this is an unusual
trend in terrestrial mammals  and was not identified as a
significant age-related trend in Florida manatees . Although it
does not account for the discrepancy between manatee subspecies,
it has been suggested in other marine mammals that higher red
blood cell indices in neonates may assist in oxygen regulation as
they learn to hold their breath during dives . As shallow water
mammals, manatee calves do not have to adapt to deep water
dives; however, they may benefit from increased oxygen storage
and carrying capacity while learning to remain submerged for
increased periods of time. An additional explanation for this
finding may be that younger manatees are prone to greater
capture stress than adults and consequently experience an
epinephrine-type response similar to that reported in horses
. Physiologically, a stress reaction of this nature can result in
splenic contraction with swift release of erythrocytes into the
With respect to biochemistry parameters, significant age-related
trends were detected for TP, cholesterol, ALP, phosphorous, and
creatinine. The higher TP levels observed in adult manatees in
Belize relative to subadults and calves may be indicative of low
grade, chronic inflammation , which can be associated with
the normal aging process [50,51]. For all other analytes mentioned
above, the highest mean values were observed in calves, followed
by subadults and adults, respectively. Similar age-related findings
for ALP, cholesterol, and phosphorous were observed in Florida
manatees . Physiologically, rapid bone growth in young
animals causes elevated levels of ALP and phosphorous .
Higher cholesterol levels in calves may be due to nursing and
associated milk composition [27,52] and may also reflect differing
rates of lipid catabolism tied to age-dependent developmental
Table 4. Hematology and biochemistry values by capture location for wild Antillean manatees (Trichechus manatus manatus) in
Belize between 1997 and 2009 with P valuesbcindicating significant capture site differences.
Southern Lagoon Drowned CayesNorthern LagoonPlacencia Lagoon
N Mean ± SDN Mean ± SDNd
Mean ± SDNd
Mean ± SD
19 129.067.44 10121.266.191–2–P=0.003b
58 4.662.84 1310.864.565 4.163.994–P,0.001c
584.360.9213 3.960.665 5.260.674–P=0.035c
7591.29639.0 1985.83640.535 125653.13555.2615.87P=0.048c
52 20.3615.69 1311.766.181–3–P=0.017b
aMCV = mean corpuscular volume, BUN = blood urea nitrogen, ALB = Albumin, K = Potassium, TG = Triglycerides, AST = aspartate aminotransferase, ALT = alanine
transaminase, GGT = gamma-glutamyl transpeptidase.
bP values from t test with n1+ n2–2 df.
cP values from F-test with 2 and n–3 df.
dSample sizes with ,5 observations were not included in the analysis for the blood parameter assessed.
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Capture site was a significant source of variation for MCV,
BUN, ALB, potassium, triglycerides, AST, ALT, and GGT.
However, the biological relevance of these subpopulation differ-
ences is limited since all mean parameter values were clinically
normal relative to reference intervals established previously in
West Indian manateesand
[1,19,27,38,39,40,41,44]. Most of the differences we identified
can probably be explained by subtle variations in diet, water
salinity, and other site-specific environmental conditions that differ
throughout Belize [54,55]. It should also be noted that ALT, AST
and GGT are not liver-specific in manatees [44,56] and changes in
enzyme levels are therefore not a direct indication of hepato-
biliary function. More recent guidelines for diagnosing liver
disease in manatees focus on measuring sorbitol dehydrogenase,
glutamate dehydrogenase, and tracking bilirubin levels [56,57].
In accordancewith prior
[27,38,39,40,41], few significant sex differences were detected
among blood parameters, and those that were identified were
consistent with normal, healthy levels reported in other manatee
populations. Variation in blood parameters by season was also
uncommon, but we found that albumin levels were significantly
lower during the rainy season than during the dry season. This
variation might be related to changes in hydration status stemming
from seasonal alterations in water levels and associated shifts in salt
and fresh water availability . Additionally, we observed higher
eosinophil counts during the dry season compared to the wet
season, which may reflect shifts in parasite load following heavier
periods of rainfall [58,59].
in other marine mammals
Routine screening for common marine mammal pathogens in
manatees from Belize, and more broadly, monitoring for emerging
disease trends across sirenian populations, are essential given the
vulnerability of manatees and dugongs worldwide. In comparing
our findings to seroprevalence results reported in the 1996 Florida
manatee study , several significant differences should be
highlighted. First, 7% of Florida manatees were seropositive to
Brucella spp. , whereas none of the manatees sampled in Belize
had positive antibody titers to this pathogen. Additionally, a single
manatee in the Florida study tested positive for exposure to avian
influenza . Another distinction is that exposure to protozoal
agents, morbillivirus, and WNV  were not assessed in the 1996
survey of the Florida subspecies .
Aside from the aforementioned differences, seroprevalence
findings were relatively similar in both populations. Like manatees
from Belize, all of the Florida manatees had negative titers to
SLEV and WEEV . Additionally, seroprevalence to Leptospira
spp. was found in comparable levels in both subspecies. Overall,
22% of Florida manatees  and 23% of manatees from Belize
had positive antibody titers to one or more serovars of L. interrogans.
A third study investigating leptospirosis exposure in captive
Amazonian manatees (Trichechus inunguis) in Brazil recently
established that 31% of the sample population (n=74) was
seropositive to L. interrogans spp.  and serves as a further
Among those individuals with positive leptospirosis titers in
Belize and Florida, many were seropositive to multiple serovars. In
contrast, this was reported to be the case with only one
leptospirosis-positive manatee in Brazil . In the present study,
to avoid ambiguities resulting from possible cross-reaction among
L. interrogans serovars, only the serovar with the highest titer was
considered positive. This approach, which assumed that an
individual was unlikely to be exposed to more than one serovar
may be overly conservative, as multiple seroprevalences to L.
interrogans serovars have been reported in other marine mammals
. Evaluating studies side-by-side like this has the potential to
offer insight on emerging trends; however, differences in testing
methods, serovar panels, and interpretation of assay results
preclude the ability to make direct comparisons about pathogen
exposure status between sirenian populations. Consequently, those
engaged in manatee disease research should seek to establish
uniform assay and reference laboratories that can apply standard
methodology in diagnostic techniques and interpretation of test
In spite of relatively low seroprevalence of pathogen exposure in
both Belize and Florida manatee populations, disease remains a
threat. Indications of lowered immunity and increased suscepti-
bility to infection in the Florida subspecies became apparent with
the discovery of a novel, manatee-specific papillomavirus in 1997
[43,63,64]. In general, the number of infectious agents discovered
in marine mammals has risen over the past two decades [10,65].
Anthropogenic drivers are likely responsible for many of the
factors contributing this trend. Weakened immunity and/or
increased exposure risk may arise from climatic alterations,
depletion of food sources through over-fishing, and exposure to
chemical byproducts and pathogen waste from terrestrial runoff
[10,12,13,66]. In turn, the risk of interspecies disease transmission
increases as population growth and landscape change push
humans, domestic animals, and wildlife in closer proximity
[11,12,13]. In combination, these stressors place marine species
living close to the land-water interface at heightened risk of
As nearshore species, with regular movement between fresh and
salt water environments [67,68], manatees may be susceptible to
infection through a variety of means including contaminated land-
water runoff, contact with other nearshore species , and
arthropod-borne infections contracted when feeding on shoreline
vegetation . As important pathways of disease transmission are
determined and high risk zones identified, preventive measures
may be developed with added focus given to those pathogens that
were associated with low levels of seropositivity in the manatee
population of Belize. A preliminary discussion of these pathogens
is given in the section that follows.
Maintained in a variety of wild and domestic animal hosts,
leptospirosis poses a serious human health risk worldwide [70,71].
While there have been no case reports of leptospirosis in manatees
around Belize, almost a quarter of the manatees captured in this
study were seropositive to L. bratislava, L. canicola, and/or L.
icterohemorrhagiae. Clinical infection with Leptospira spp. has been
confirmed in several species of pinniped [72,73], but is particularly
well documented in California sea lions . Cyclical epizootics in
this species tend to follow El Nin ˜o years , suggesting that
environmental drivers may influence pathogen or host dynamics
[71,73,74]. Similar epidemiologic and ecological factors may give
rise to outbreaks of L. interrogans spp. in manatees around Belize.
The potential for high die-offs should an epizootic occur warrants
routine serologic screening of this pathogen in manatees of this
region as well as examination for characteristic lesions and
relevant histopathology performed on all carcasses recovered for
A significantly greater proportion of manatees captured in the
Northern Lagoon were seropositive to L. interrogans spp. compared
to the other capture sites; this association was most pronounced for
L. bratislava exposure. Prevalence levels were similar among
manatees from the Southern and Placencia lagoons, whereas all
manatees sampled from the Drowned Cayes were seronegative.
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Potential reasons for the higher exposure levels in the Northern
Lagoon may be due to the fact that it is more land-locked than the
other capture locations (Figure 2). Consequently, it may be subject
to greater water stagnancy, preventing quick removal of the
pathogen from the environment through regular water turnover
. Given its geography, there may also be greater potential for
exposure to infection from terrestrial reservoir hosts via land-water
runoff. A seasonal association with seroprevalence to leptospirosis
was also identified, with a significantly higher percentage of L.
bratislava-positive manatees detected during the dry season than
during the wet season. However, we could not investigate the
effect of season on the incidence of infection since serology only
provides evidence of exposure at some unknown time in the past.
Moreover, wet and dry seasons were too brief in duration to infer
incidence based on changing titer levels of individuals that were
captured multiple times.
Toxoplasma gondii has been documented as a cause of high
morbidity and mortality in a variety of marine mammals
[29,76,77,78,79,80,81,82] and poses a serious human health risk
to immunocompromised individuals and children infected in utero
. Felidae serve as the definitive hosts for T. gondii , and in
southern sea otters, both feral and domestic cats have been
implemented in the transmission pathway by shedding oocysts in
reservoirs and other water sources . Similarly, transport of
oocysts from land-based effluent may be a source of exposure to T.
gondii in manatees , especially given their occasional reliance
on sewage effluents as fresh water drinking sources . Despite
natural behaviors such as this that may increase their exposure risk
to T. gondii, manatees in Belize had a low prevalence of
seropositivity to this zoonotic protozoan. Among the eight
manatees in Belize that were seropositive for T. gondii on the
LAT, none had titers that exceeded 1:320. Four of these
Figure 2. Map of four primary study sites in Belize where capture and release of free-ranging manatees occurred between 1997 and
Antillean Manatee Health Assessment and Serosurvey
PLOS ONE | www.plosone.org8 September 2012 | Volume 7 | Issue 9 | e44517
seropositive manatees were sampled multiple times, but the
magnitude of difference between serial titers was either too small,
or the time elapsed between serial titers too long, to make valid
inferences about the timing of infection or seroreversion.
Serology data on T. gondii in other manatee populations is
limited, but a recent study in captive Amazonian manatees in
Brazil reported a 39.2% seroprevalence to T. gondii . A partial
explanation for the lower exposure levels in manatees from Belize
may be related to the fact that domestic cats are infrequently
observed on shoreline beaches in the region. However, jaguar
tracks are commonly sited at the water’s edge, underscoring the
importance of regular screening and increased focus on identifying
probable sources of contamination. Additional studies of T. gondii
in other manatee populations are currently underway, but to date
there have only been two isolated cases of the protozoan
documented in West Indian manatees; one of these cases was
described in an Antillean manatee from Guyana , a second
case was reported in a Florida manatee that died from
complications of menigoencephalitis . A concerted effort to
collect histologic samples from beached carcasses and obtain serial
titers on suspect cases of T. gondii in captive manatees will assist in
efforts to establish a gold standard and common testing
methodology and will aid in establishing the overall impact of
this protozoan on manatee population health.
In the late 1980s and early 1990s, four novel morbilliviruses
were traced to mass mortalities in several pinniped and cetacean
populations across northwestern Europe, Siberia, and the Med-
iterranean [33,86]. Among the morbilliviruses implicated in the
outbreak, phocine distemper virus (PDV), and the more closely
related porpoise morbillivirus (PMV) and dolphin morbillivirus
(DMV) are believed to be endemic in certain marine mammal
communities . However, it is thought that heavy losses can
occur when these pathogens are introduced into naı ¨ve populations
. As demonstrated in Mediterranean monk seals (Monachus
monachus) , such a scenario has the potential to devastate
endangered species, like manatees, which have low reproductive
rates, heavy maternal investment, and long-life spans .
Although manatees around Belize coexist with several cetacean
species, we found minimal serologic evidence of exposure to
morbillivirus in the manatees we sampled. Of the 112 manatees
tested, only one adult female manatee and three adult males had
low positive antibody titers against CDV and PMV/DMV.
Among the four manatees that were seropositive to morbillivirus,
antibody titers were slightly higher against PMV/DMV than
against CDV, but the low neutralization responses overall (i.e., no
titer .1:20) were not sufficient enough to suggest that a
morbillivirus of cetacean origin was the likely source of infection.
Similar seroprevalence results for morbillivirus were published on
Florida manatees in 1995 in which 4% of the sample population
(n=148) was reported to be seropositive . Neutralization titers
against DMV and PMV were slightly higher in Florida manatees
than those reported in the present study and led researchers to
hypothesize that bottlenose dolphins may have transmitted the
infection . In the same survey, a small number of wild
manatees from Guyana and hand-reared Amazonian manatees
were also screened, and all individuals tested were found to be
Morbillivirus has yet to be isolated from a manatee, and reports
of suspect cases have not been documented in the species .
Investigators from the Florida study speculated that the presence
of low positive titers may indicate that either viral replication is
inadequate to cause clinical signs of infection or that manatees
cohabitate in numbers too small to enable adequate host-host
transmission to elicit an outbreak . However, given the
devastating losses suffered in previously unexposed pinniped and
cetacean populations, and the fragility of manatee populations in
general, regular testing for morbillivirus in health assessments and
continued monitoring for clinical cases is crucial.
Understanding how disease may affect population growth in
Antillean manatees from Belize is especially important at present
since coastal development is increasingly taxing the resilience of
this nearshore mammalian species. Human encroachment has
collectively destroyed important seagrass beds and resulted in the
clearance of vital mangrove areas . Subsequent changes in
water quality and food abundance not only place constraints on
the manatees of this region, but also challenge the robustness of
other organisms that inhabit these waters [2,9]. Antillean
manatees may be well suited as sentinel species in this regard
[7,9,10]. As such, the importance of cause-specific mortality
studies and disease surveillance in healthy populations of this
species must be emphasized.
Although low prevalence of pathogen exposure was detected in
the manatee population from Belize, supplemental mortality data
and improved testing methodologies are needed to gain a better
understanding of the current situation. These next steps are crucial
as a single disease outbreak in an immunologically naı ¨ve,
seronegative population could cause epidemic mortality and
substantially impede recovery [12,88]. Given this possibility,
supporting long-term research that expands on the findings
presented here, may be critical to the success of Belize’s manatee
recovery efforts and assist in safeguarding the health and
conservation of the surrounding ecosystem.
We would like to thank all of the scientists and research assistants at
CZMAI in Belize, who dedicated many hours of work and offered
invaluable input to this study over the years. Further appreciation is given
to Drs. Pat Conrad, William Reisen, and Linda Green as well as laboratory
technicians, Andrea Packham, Ann Melli, and Ying Fang for their
expertise and significant contributions to the laboratory diagnostics.
Any use of trade, product, or firm names is for descriptive purposes only
and does not imply endorsement by the U.S. Government.
Conceived and designed the experiments: RKB JP NAG AAA. Performed
the experiments: RKB JP NAG AAA MPL KN AO. Analyzed the data:
KS CKJ. Contributed reagents/materials/analysis tools: KS CKJ RKB
AAA. Wrote the paper: KS CKJ AAA RKB.
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