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Occurrence of Human Pathogenic Bacteria and Toxoplasma gondii in Cetaceans Stranded in the Philippines: Providing Clues on Ocean Health Status

Authors:
  • Institute of Biology, University of the Philippines
  • De La Salle University-Dasmariñas, Dasmarinas City, Cavite, Philippines

Abstract

The general consensus of a rapidly changing ocean ecosystem being affected by anthropogenic activities needs to be understood in relation to both wildlife and human health. The risks and challenges for the Philippines include lack of scientific information on waterborne diseases that are potentially zoonotic. The present study fills in this knowledge gap by detecting the occurrence of bacteria, Giardia, and Toxoplasma gondii in locally found cetacean species. Cetaceans (n = 30) that stranded from January 2012 through March 2013 were appropriately responded to, and biological materials were taken whenever applicable. A total of 25 bacteria were isolated from nine stranders. Phenotypic and genotypic methods of isolate identification yielded 12 consensus genera: Acinetobacter, Aeromonas, Burkholderia, Enterococcus, Moraxella, Proteus, Providencia, Rhizobium, Serratia, Sphingomonas, Staphylococcus, and Vibrio. No screened strander was positive for Giardia. Serological assay detected antibodies for T. gondii in five stranders, while nested polymerase chain reaction positively amplified the B1 gene of the parasite in two stranders. This study provides the first report on bacteria and T. gondii in cetaceans found in the Philippines. Since the detected microorganisms include species recognized to cause new infections in marine mammals worldwide, the findings of the study underscore the potential of stranded cetaceans to serve as sentinels for studying the movement of emerging pathogens in marine habitats, provide clues on the health status of their free-ranging populations, and present the health risks available to humans who share the same water resource with them.
Aquatic Mammals 2015, 41(2), 149-166, DOI 10.1578/AM.41.2.2015.149
Occurrence of Human Pathogenic Bacteria and
Toxoplasma gondii in Cetaceans Stranded in the Philippines:
Providing Clues on Ocean Health Status
Marie Christine M. Obusan,
1
Lemnuel V. Aragones,
1, 2
Cristina C. Salibay,
3
Maria Auxilia T. Siringan,
2
and Windell L. Rivera
2, 4
1
Institute of Environmental Science and Meteorology, College of Science,
University of the Philippines, Diliman, Quezon City, 1101
E-mail: christine.obusan@gmail.com
2
Natural Sciences Research Institute, University of the Philippines, Diliman, Quezon City, 1101
3
Biological Sciences Department, College of Science and Computer Studies,
De La Salle University–Dasmariñas, Dasmariñas, Cavite
4
Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City, 1101
Abstract
The general consensus of a rapidly changing ocean
ecosystem being affected by anthropogenic activi-
ties needs to be understood in relation to both wild-
life and human health. The risks and challenges for
the Philippines include lack of scientific informa-
tion on waterborne diseases that are potentially zoo-
notic. The present study fills in this knowledge gap
by detecting the occurrence of bacteria, Giardia,
and Toxoplasma gondii in locally found cetacean
species. Cetaceans (n = 30) that stranded from
January 2012 through March 2013 were appropri-
ately responded to, and biological materials were
taken whenever applicable. A total of 25 bacteria
were isolated from nine stranders. Phenotypic and
genotypic methods of isolate identification yielded
12 consensus genera: Acinetobacter, Aeromonas,
Burkholderia, Enterococcus, Moraxella, Proteus,
Providencia, Rhizobium, Serratia, Sphingomonas,
Staphylococcus, and Vibrio. No screened strander
was positive for Giardia. Serological assay detected
antibodies for T. gondii in five stranders, while
nested polymerase chain reaction positively ampli-
fied the B1 gene of the parasite in two stranders.
This study provides the first report on bacteria and
T. gondii in cetaceans found in the Philippines. Since
the detected microorganisms include species recog-
nized to cause new infections in marine mammals
worldwide, the findings of the study underscore the
potential of stranded cetaceans to serve as sentinels
for studying the movement of emerging pathogens
in marine habitats, provide clues on the health status
of their free-ranging populations, and present the
health risks available to humans who share the same
water resource with them.
Key Words: cetaceans, strandings, bacteria,
Giardia, Toxoplasma gondii, sentinels
Introduction
Marine mammals face the challenges of a chang-
ing environment primarily affected by human
activities. Thus, they are in a good position to
serve as sentinels of their habitat conditions
(Bossart, 2011). Moore (2008) suggested that the
utility of these animals to serve as sentinels stems
from their ecological diversity as well as inherent
variability in marine ecosystems. Furthermore,
Stewart et al. (2008) provided that information
on their emerging or recurring health problems
can be used as a measure of ocean health and
indicator of impending human health issues.
Assessments of health status among marine mam-
mals usually involve general pathogen screenings
in stranded and free-ranging populations (Gerber
et al., 1993; Calle et al., 2002; Duignan, 2003;
Hanni et al., 2003; Zarnke et al., 2006; Aguirre
et al., 2007; Greig et al., 2007; Bogomolni et al.,
2008; Alekseev et al., 2009; Zuerner et al., 2009;
Brownstein et al., 2011). Outbreaks of infectious
diseases, sometimes characterized by high mor-
bidity and mortality, were documented in several
marine mammal populations worldwide, posing
threats to public health (Lipscomb et al., 1996;
Nielsen et al., 2001; Van Bressem et al., 2009).
While it seems necessary to investigate pathogens
in cases of mass mortalities or large-scale disease
epidemics, knowledge of their occurrence in the
absence of these occasions is urgently needed,
particularly in the case of Philippine marine mam-
mals which do not have baseline data yet.
150 Obusan et al.
There may already be clinical signs of infec-
tious diseases in some marine mammal popula-
tions in the Philippines, but the lack of research
attention being given to them makes them unrec-
ognized, much less documented. Except for the
reported case of aspergillosis in a melon-headed
whale (Peponocephala electra) calf that stranded
and died in Bataan (Torno et al., 2008), the occur-
rence of other bacterial, protozoan, or fungal
pathogens causing either overt or hidden manifes-
tations of infection is not known in any marine
mammal species found in the country. The pres-
ent study fills in the current knowledge gap on the
occurrence of pathogenic agents among locally
found cetacean species and the potential of these
animals to serve as sentinels of emerging and zoo-
notic diseases. For such purpose, the researchers
maximized the sampling opportunity provided by
local stranding events in the country, observed
to have an increasing trend in recent years. Of
the 30 identified marine mammal species in the
Philippines, 28 are cetaceans and 22 of these are
reported to strand (Aragones et al., 2010), provid-
ing the much needed chance to study these diffi-
cult-to-observe albeit charismatic animals. While
it is not the primary aim of the study to investigate
the role of pathogen or disease occurrence in sam-
pled stranding events, the involvement of such
may be suggested as done elsewhere (Lopez et al.,
2002; Kreuder et al., 2003; Gonzalez-Solis et al.,
2006; Stoddard et al., 2008; Fauquier et al., 2009;
Forman et al., 2009; Colegrove et al., 2010). This
study generally aimed to detect the occurrence of
bacteria, Giardia, and Toxoplasma gondii in ceta-
ceans stranded in the Philippines.
Methods
Stranded Cetacean Samples
Cetaceans stranded in Philippine waters from
January 2012 through March 2013 were opportu-
nistically sampled. Live or dead individuals were
characterized in terms of species, sex (i.e., based
on genital and/or mammary slits), length (i.e.,
tip of the snout to the tip of the tail or notch of
the flukes), age class (inferred from length based
on species), and type of stranding (e.g., single or
mass; live or dead). The chance of responding to a
stranded cetacean is affected by the proximity of the
stranding site, and so the researchers collaborated
with some stakeholders (i.e., Philippine Marine
Mammal Stranding Network [PMMSN]; Bureau
of Fisheries and Aquatic Resources [BFAR]) for
monitoring stranding events all over the country.
The Philippines is an archipelagic country with
three major islands: (1) Luzon, (2) Visayas, and
(3) Mindanao. To respond to strandings reported
within Luzon, the researchers travelled by land
(i.e., at least 5 h and at most 12 h) or water (i.e.,
6 h) immediately after a report was made. On the
other hand, Visayas and Mindanao can be reached
by plane or ship. Biological materials were col-
lected from stranded cetaceans considering the
(1) animal disposition (e.g., if it was a live or dead
strander); (2) physical preservation based on the
expanded version (Geraci & Lounsbury, 2005) of
the code system established by the Smithsonian
Institution’s Marine Mammal Events Program
(e.g., bacteriological samples were only collected
from live or freshly dead animals in order to mini-
mize, if not avoid, contamination); and (3) sam-
pling conditions (e.g., handling limitations result-
ing to contamination of body parts to be sampled).
Detection of Pathogens
The detection of potentially emerging and infec-
tious zoonotic pathogens was limited to bacteria
(with Vibrio spp. as preliminary targets) and proto-
zoa (T. gondii and Giardia spp.). Screening of ceta-
ceans for these microorganisms proceeded depend-
ing on the type of biological material(s) obtained
from each stranded sample (see Table 1). The labo-
ratory work was performed at the Microbiological
Research and Services Laboratory (MRSL) and
the Molecular Protozoology Laboratory (MPL) of
the Natural Sciences Research Institute (NSRI),
University of the Philippines, Diliman as well as
at the Biological Sciences Department, College
of Science and Computer Studies, De La Salle
University, Dasmariῆas, Cavite, Philippines.
Phenotypic Methods for Bacteria Screening
Specimens were obtained from routine and non-
routine sites using sterile rayon swabs with trans-
port medium (i.e., Stuart). For routine sites, swab
samples were collected from the blowhole and anus
of cetaceans (Buck et al., 1991, 2006; Miller et al.,
2010; Morris et al., 2011). For the blowhole area,
swabs were inserted into the hole during a breath,
gently moved along the wall, and removed during
the next breath in live stranders. Another method
is by lowering a sterile Petri dish directly over the
blowhole to collect the exhaled breath condensate
(blow) and then swabbing afterwards. Anal swabs
were collected by inserting rayon swabs into the
anal orifice and gently swabbing the area. For
freshly dead individuals, swab samples were also
taken from blowhole and anal areas. Considering
the physical condition and disposition of sampled
cetaceans, the actual number of swab samples col-
lected from the blowhole and anus varied, and
so these samples were pooled for each body site
and stranded individual. Whenever applicable,
swab samples from nonroutine sites (e.g., lesions,
organs, and abdominal or thoracic fluid) were
also obtained from both live and dead animals
(Bogomolni et al., 2008), especially in relation to
151
Occurrence of Human Pathogenic Bacteria and Toxoplasma in Cetaceans Stranded in the Philippines
Biological materials for
Specimen
for protozoa
detection
Feces
Blood
Feces
Blood
Serum
pathogen screening
Feces
Blood
Swab specimen for
bacteria isolation
Anus
Abdominal fluid
d
Blowhole
Genital area
Blowhole
Anus
Lung
d
Abdominal fluid
Skin lesion
Blowhole
Anus
Liver
Blowhole
Anus
Condition
code at the
sampling
b
time of
2
3
3
1
2
3
1
3
c
1
Sex/
age class
Male/
adult
Male/
subadult
Male/
subadult
Female/
subadult
Male/
adult
Female/
subadult
Male/
subadult
Female/
adult
Female/
subadult
2012
2012
Stranded cetaceans responded for sampling from January 2012 through March 2013
Stranding date
7 February 2012
9 February 2012
9 March 2012
16 March 2012
9 April
29 April
7 June 2012
11 June 2012
27 July 2012
Stranding location
omas,
La Union
Bolinao,
Pangasinan
,
, Pier
Legaspi City
Claveria, Cagayan
Alaminos, Pangasinan
alisay
Sto. T
Lubang Island,
Occidental Mindoro
Cabangan, Zambales
Bigaa,
Legazpi City
T
Camarines Norte
hynchus
Pantropical spotted dolphin
hynchus
Cetacean species
Dwarf sperm whale
Stenella attenuata
ostris
Kogia sima
or
Short-finned pilot whale
Globicephala macror
Stenella longir
Spinner dolphin
Globicephala macr
Short-finned pilot whale
Stenella attenuata
Pantropical spotted dolphin
edanensisSteno br
Rough-toothed dolphin
Tursiops aduncus
Indo-Pacific bottlenose dolphin
ursiops truncatusT
Common bottlenose dolphin
able 1.
Strander
code and
number
S1
S2
S3
S4
S5
S6
S7
S8
S9
T
152 Obusan et al.
Blood
Blood
Feces
Feces
Blood
Serum
Feces
Feces
Blood
Serum
Feces
Blood
Serum
Blood
Blood
Serum
Blood
Feces
Blood
Serum
Blowhole
Anus
Blowhole
Anus
Blowhole
Anus
Heart
d
Lung
d
Liver
d
Blowhole
Anus
1
1
3
2
1
1
1
1
2
1
2
3
2
Female/
adult
/
a
ND
calf
Female/
adult
Male/
subadult
Female/
adult
/
a
ND
calf
Male/
adult
Male/
adult
Female/
subadult
Male/
subadult
Male/
adult
Male/
subadult
Female/
adult
28 July 2012
28 July 2012
2012
2012
8 August
30 August
12 September 2012
12 September 2012
16 September 2012
17 September 2012
5 November 2012
7 November 2012
16 November 2012
30 December 2012
4 January 2013
Cruz, Davao City
Cruz, Davao City
Camarines Norte
Bacarra, Ilocos Norte
Cagayan
Cagayan
Zambales
Capalonga,
La Union
iwi, Albay
Santa Ana,
Santa Ana,
San Fernando,
San Jose,
Camarines Sur
San Antonio,
, Sorsogon
San Antonio,
T
Palauig, Zambales
Lupi, Prieto Diaz,
Sorsogon
Pilar
Stenella attenuata
Pantropical spotted dolphin
Stenella attenuata
Pantropical spotted dolphin
evicepsKogia br
Pygmy sperm whale
Stenella attenuata
Pantropical spotted dolphin
Pygmy sperm whale
ostris
eviceps
eviceps
eviceps
eviceps
ocephalus
Kogia br
Pygmy sperm whale
Kogia br
Kogia br
Pygmy sperm whale
Kogia br
Pygmy sperm whale
ostris
sp.
Stenella longir
Spinner dolphin
Stenella attenuata
Pantropical spotted dolphin
Stenella longir
Spinner dolphin
Physeter macr
Giant sperm whale
Mesoplodon
Beaked whale
S10
11S
S12
S13
S14
S15
S16
S17
S18
S19
S20
S21
S22
153
Occurrence of Human Pathogenic Bacteria and Toxoplasma in Cetaceans Stranded in the Philippines
3 – fair/
Feces
Blood
Serum
Feces
Blood
Serum
Blood
the results of raction,
Blowhole
Anus
to human intein relation
1
2
3
c
1
2
3
3
3
Male/
adult
Male/
adult
Male/
adult
Female/
subadult
Female/
Male/
Male/
subadult
Male/
, 2005): 1 – live; 2 – fresh/carcass in good condition;
adult
adult
adult
were thoroughly investigated
14 January 2013
25 January 2013
14 February 2013
21 February 2013
24 February 2013
25 February 2013
5 March 2013
2 March 2013
stress; cetaceans
Candelaria, Zambales
Pasuquin, Ilocos Norte
Zambales
, Cagayan
Pamplona,
San Antonio,
Cagayan
Morong, Bataan
Buguey
Bauang, La Union
Anda, Pangasinan
of response due to animal
Rough-toothed dolphin
hynchus
was not done at the time
Short-finned pilot whale
Pygmy sperm whale
Lagenodelphis hosei
Indo-Pacific bottlenose dolphin
hynchus
edanensis
eviceps
or
Steno br
Globicephala macror
Kogia br
s dolphinFraser
ursiops aduncusT
Globicephala macr
Short finned pilot whale
edanensisSteno br
Rough-toothed dolphin
ursiops truncatusT
Common bottlenose dolphin
gans basically intact; 4 – poor/advanced decomposition; and 5 – mummified/skeletal remains
collection for biological
S23
S24
S25
S26
S27
S28
S29
S30
ND = not determined
Physical condition at the time of sampling based on Smithsonian Institution Condition Codes (Geraci & Lounsbury
decomposed but or
Sampling
which are discussed in another report
Additional swab samples taken right after the strander died
a
b
c
d
154 Obusan et al.
suspected infection. The swab samples were stored
in coolers (about 7° to 10° C) and transported
to the laboratory within 24 h for processing.
Those inoculated to media upon collection were
directly incubated 18 to 24 h at 35 + 2° C. For the
enrichment of target Vibrio spp., swabs were inoc-
ulated immediately into APW (alkaline peptone
water) with 2 to 3% sodium chloride (NaCl) broth
or streaked onto TCBS (Thiosulfate Citrate Bile
Salts Sucrose) or TSA (Trypticase Soy Agar with
2 to 3% NaCl) plates when transit time is < 8 h
(Elliot et al., 1998). Swabs from transport medium
were placed in APW and incubated 12 to 24 h at
35 + 2° C prior to subculture on TCBS plates.
The plates were incubated 18 to 24 h at 35 + 2°C.
Suspect isolates were confirmed for Gram and
cytochrome oxidase reactions, microscopically
observed for cellular morphology, and identified
using the rapid diagnostic kit Analytical Profile
Index system (API 20 NE). Distinctive colonies
primarily characterized by color and identified by
the API system were streaked and cultured on new
TCBS or TSA (with 2 to 3% NaCl) plates several
times to purify bacterial isolates until pure cultures
were obtained. Presumptive non-vibrios were fur-
ther examined for other phenotypic characteristics
before confirming identification using API 20 NE
or VITEK 2 systems (BioMerieux SA, France).
Serological Method for Toxoplasma Screening
Blood extraction proceeded from the fluke vascula-
ture of live animals or vena cavae from freshly dead
stranders (Geraci & Lounsbury, 2005). To recover
serum, whole blood was either placed in serum
separator tubes or kept warm for 30 min until clot-
ted and centrifuged at 1,500 rpm for 1 min. Sera
were stored at 4° to 8° C and processed within 48 h.
Antibodies against T. gondii were detected using
Toxocell Latex Agglutination Test (LAT: BIOKIT
Manufacturing Company, Barcelona, Spain). Serum
recovery depended on the amount and quality of
blood obtained.
Molecular Methods for Giardia, Toxoplasma,
and Bacteria Screening—For Toxoplasma screen-
ing, blood was obtained as described above. For
Giardia screening, fecal samples were collected
from live cetaceans by enema with 50 to 100 ml
sterile saline (R. Fayer, pers. comm., 6 May 2011).
The liquid feces was placed in sterile plastic cups
for > 7 d (Fayer et al., 2008) or fixed in 10%
formalin or 70% ethanol until processing. When
available, swabbed fresh feces from the rectal
area of live individuals (Buck et al., 2006) and
solid feces (5 to 10 g) from necropsied individuals
(Geraci & Lounsbury, 2005) were also collected
and suspended in a buffer (e.g., PBS) or fixated
and then resuspended prior to DNA extraction. All
samples were held at 4° to 5° C until processing.
Bacteria that were isolated and identified through
phenotypic methods were pretreated for DNA
extraction. Pellets (maximum 2 × 10
9
cells) from
18 to 24 h pure cultures were harvested by cen-
trifugation at 5,000 ×
g (7,500 rpm) for 10 m and
then resuspended in 180 μl tissue lysis buffer.
Extraction of DNA was done using the Quick-
gDNA
TM
Blood MiniPrep kit (Zymo Research) for
blood samples, Fecal DNA Miniprep
TM
kit (Zymo
Research) for fecal samples, and DNeasy
®
Blood
& Tissue kit (Qiagen) for bacteria cultures follow-
ing manufacturers instructions.
Procedures for polymerase chain reaction
(PCR) were performed for amplifications of target
pathogen gene fragments. For Giardia, 18S-rDNA
at fragments ~130 bp (base pair) or 170 bp was
the target size using the primer pairs RH11 and
RH4 for first step PCR, and GiarF and GiarR for
second step (McGlade et al., 2003; Szénási et al.,
2007; Lim et al., 2009). The thermocycler condi-
tions were three cycles of 94° C for 2 min, 53° C
for 1 min, 72° C for 2 min followed by 50 cycles
of 94° C for 30 s, 53° C for 20 s, 72° C for 30 s,
and an extension of 72° C for 7 min (McGlade
et al., 2003).
For Toxoplasma, amplification yielding a final
nested product of 96 bp directed against the B1
gene was carried out using the primer pairs Outer
Sense strand and Outer Nonsense strand for
first step PCR, and Inner Sense strand and Inner
Nonsense strand for second step. The thermocy-
cler conditions were (1) for first step, denaturing
at 94° C for 2 min followed by 40 cycles of 93
° C
for 10 s, 57° C for 10 s, 72° C for 30 s, and an
extension of 72° C for 30 s; and (2) for second
step, denaturing at 94° C for 2 min followed by 40
cycles of 93° C for 10 s, 62.5° C for 10 s, 72° C
for 15 s, and an extension of 72° C for 15 s (Jones
et al., 2000; Pretti et al., 2010).
For bacteria, the conserved regions 8 and 10 of
the 16S rDNA were amplified by forward primer
1169U20 and reverse primer 1521L19 (Jin et al.,
2005). The reactions were carried out with initial
denaturation at 95° C for 5 min, followed by 35
cycles of 94° C for 25 s, 55° C for 30 s, and 72° C
for 25 s as modified from Jin et al. (2005).
Reactions were performed in 25 μl volume
with the following final concentrations of compo-
nents: 1X PCR Master Mix (Promega, USA), 0.1
to 1.0 μM assigned upstream primer (AITbiotech,
Singapore), 0.1 to 1.0 μM assigned downstream
primer (AITbiotech, Singapore), < 250 ng DNA
template, and nuclease-free water adjusted accord-
ingly. Negative controls consisted of PCR reagents
excluding DNA template. Positive controls included
DNA samples of Giardia sp. (Lim, Department of
Parasitology, University of Malaya, Malaysia),
T. gondii (Ishida, University of Tokyo, Japan; Lim,
Department of Parasitology, University of Malaya,
155
Occurrence of Human Pathogenic Bacteria and Toxoplasma in Cetaceans Stranded in the Philippines
Malaysia), Vibrio parahaemolyticus (Philippine
National Collection of Microorganisms [PNCM],
National Institute of Molecular Biology and
Applied Microbiology [BIOTECH], University of
the Philippines, Los Baῆos), and Escherichia coli
(Microbiological Research and Services Institute
[MRSL], Natural Sciences Research Institute
[NSRI], University of the Philippines, Diliman).
Electrophoresis of PCR products in TAE (Tris-
acetate-EDTA) buffer was performed on 1 to
2% agarose gel at 100 V with ethidium bromide
(0.5 mg/ml) staining. A 100 bp molecular mass
ladder (Vivantis) was included in each gel. For
bacteria, PCR-positive samples were processed for
purification, DNA quantification, and sequencing
(1st Base, Malaysia). Molecular analyses were per-
formed using software programs Bioedit, Version
7.0.5.3 (Hall, 1999) for editing and aligning of
sequences, and MEGA 5 for phylogenetic analyses
(Tamura et al., 2011). Sequence homologies were
determined based on parameters suggested by Hall
(2011).
Results
Stranded Cetaceans
A total of 30 cetaceans that stranded in the
Philippines were responded to from January 2012
through March 2013. Cetacean samples were con-
firmed to be from 11 species: Globicephala mac-
rorhynchus (short-finned pilot whale), Kogia sima
(dwarf sperm whale), Kogia breviceps (pygmy
sperm whale), Lagenodelphis hosei (Frasers dol-
phin), Mesoplodon sp. (unidentified beaked whale),
Physeter macrocephalus (giant sperm whale),
Stenella attenuata (pantropical spotted dolphin),
Stenella longirostris (spinner dolphin), Steno bre-
danensis (rough-toothed dolphin), Tursiops adun-
cus (Indo-Pacific bottlenose dolphin), and Tursiops
truncatus (common bottlenose dolphin). As to age
class, 53% of the stranders were adults (n = 16),
40% were subadults (n = 12), and only 7% were
calves (n = 2). The sex of these calves was not
determined, whereas 57% of the remaining sam-
ples were males (n = 17), and 37% were females
(n = 11).
All of the strandings were from single events.
Most of the responded cetaceans came from
Luzon Island, where a relatively higher number
of strandings were reported during the duration
of the study (see Figure 1). There were more live
(n = 18) than dead stranders (n = 12). Among live
stranders, the majority of cetaceans (n = 16) either
died while being responded to or re-stranded dead
after they were released back into the wild. Only
two were released and did not re-strand.
Swab samples, blood, sera, and feces were
appropriately collected from cetaceans based on
animal disposition, physical preservation, and
sampling conditions. Depending on the type of
biological material(s) obtained, the stranders were
screened for target pathogens: nine individuals
(30% of all cetacean samples) with swab samples
for the presence of bacteria; 15 individuals (50%
of all cetacean samples) and 8 individuals (27% of
all cetacean samples) with blood and serum sam-
ples, respectively, for the presence of T. gondii;
and 10 individuals (33% of all cetacean samples)
with fecal samples for the presence of Giardia
spp. (see Table 2).
Bacteria in Stranded Cetaceans
Twenty-five bacteria isolates were obtained from
the following cetacean species: two short-finned
pilot whales, one dwarf sperm whale, three pygmy
sperm whales, one spinner dolphin, and two
rough-toothed dolphins. Phenotypic and geno-
typic methods of isolate identification yielded
12 consensus genera: Acinetobacter, Aeromonas,
Burkholderia, Enterococcus, Moraxella, Proteus,
Providencia, Rhizobium, Serratia, Sphingomonas,
Staphylococcus, and Vibrio. However, there were
differences in species-level identifications between
the two methods used. In particular, 23 (92%) out
of 25 isolates subjected to phenotypic methods
were successfully identified to the species, while
19 (90%) out of 21 isolates (with available nucleic
acid samples) processed for 16S rDNA sequenc-
ing had highest matches with similar sequences
in the Genbank database at the species level.
Species-level agreements between phenotypic
and genotypic identifications were established
for the following isolates: Burkholderia cepacia,
Staphylococcus epidermidis, Serratia marcescens,
Proteus mirabilis, and Providencia stuartii.
Toxoplasma in Stranded Cetaceans
The following cetaceans were screened for the
presence of T. gondii: three short-finned pilot
whales, three pygmy sperm whales, one beaked
whale, three pantropical spotted dolphins, two
spinner dolphins, two rough-toothed dolphins,
and one Indo-Pacific bottlenose dolphin. All these
cetaceans had qualified blood samples for the
amplification of the target parasite’s B1 gene by
nested PCR, while only eight had serum samples
available for Toxocell LAT. Two individuals were
PCR-positive, while five were serologically posi-
tive for the protozoan parasite.
Giardia in Stranded Cetaceans
Giardia was not detected in the following ceta-
ceans: three short-finned pilot whales, three
pygmy sperm whales, one spinner dolphin, two
rough-toothed dolphins, and one beaked whale.
156 Obusan et al.
Figure 1. Stranding sites of responded cetaceans (samples S1 to S30) in the Philippines from January 2012 through March 2013
157
Occurrence of Human Pathogenic Bacteria and Toxoplasma in Cetaceans Stranded in the Philippines
Detection of protozoa
b
TPCR negative
GPCR negative
Consensus taxon
Moraxella
omonas
ibrio
Aer
V
ganisms in stranded cetaceans sampled in the Philippines from January 2012 through March 2013
Burkholderia cepacia
Identification of isolated bacteria
in GenBank based on
1)
highest
sequence match
(16S rDNA
% bp/bp similarity)
Moraxella lincolnii
omogenes
(FJ22711
Genotypic method
(HF558363)
(83%)
omonas salmonicida
Y910844)
(86%)
(96%)
(EU734821)
(97%)
. achrsubsp
(A
Aer
Burkholderia cepacia
Vibrio harveyi
(96.9%)
Phenotypic method
(API/VITEK 2
% probability)
sp.
a
Moraxella
a
(82.2%)
a
ibrio
a
obactrum
V
(87.9%)
Moraxella
(82.2%)
omonas
sobria
Aer
(98.7%)
omonas
ophila
(83.8%)
ibrio
Aer
hydr
V
(96.2%)
Burkholderia
cepacia
(99.4%)
Ochr
parahaemolyticus
Dwarf sperm whale
Pantropical spotted dolphin
hynchus
ostris
Cetacean species
Kogia sima
Stenella attenuata
Stenella longir
Spinner dolphin
or
Short-finned pilot whale
get microor
Globicephala macr
Occurrence of tar
Strander code
and number
S1
S2
S3
S4
able 2.T
158 Obusan et al.
TPCR negative
GPCR negative
TPCR negative
T negativeTLA
GPCR negative
TPCR negative
TPCR negative
GPCR negative
TPCR negative
positiveTTLA
GPCR negative
omonas
Rhizobium
Sphingomonas
Staphylococcus
cescens
ococcus
epidermidis
Serratia mar
ibrio
Aer
Enter
V
sp.
ococcus casseliflavus
cescens
omonas
(AF367977)
(100%)
Aer
(HM161724)
(81%)
10) (99%)
sp.Rhizobium
(FM173817)
Sphingomonas melonis
1)
(93%)
(JF343163)
(88%)
Staphylococcus epidermidis
(98%)
ibrio harveyi
1
(98%)
(CP003959)
V
(FJ2271
(KC4431
Serratia mar
Enter
cescens
omonas
ophila
ococcus
(99.7%)
faecium
(91%)
Rhizobium
radiobacter
(90%)
Sphingomonas paucimobilis
(88%)
Staphylococcus epidermidis
(99%)
Serratia mar
(91%)
ibrio alginolyticus
(95%)
Aer
hydr
Enter
V
hynchusor
Short-finned pilot whale
Stenella attenuata
Pantropical spotted dolphin
edanensis Steno br
Rough-toothed dolphin
ursiops aduncus
Globicephala macr
T
Indo-Pacific bottlenose dolphin
Tursiops truncatus
Common bottlenose dolphin
Stenella attenuata
Pantropical spotted dolphin
Stenella attenuata
Pantropical spotted dolphin
eviceps
eviceps
Kogia br
Pygmy sperm whale
Stenella attenuata
Pantropical spotted dolphin
Kogia br
Pygmy sperm whale
S10
11
S5
S6
S7
S8
S9
S
S12
S13
S14
159
Occurrence of Human Pathogenic Bacteria and Toxoplasma in Cetaceans Stranded in the Philippines
GPCR negative
TPCR negative
T positive
positiveT
T
TLA
GPCR negative
TPCR positive
TPCR negative
positiveTLA
TPCR negative
TPCR negative
T positive
TLA
TPCR negative
TLA
GPCR negative
ibrio
ibrio
V
V
Proteus mirabilis
Acinetobacter
Acinetobacter
ibrio
ibrio
V
V
ibrio harveyi
ibrio harveyi
oteus mirabilis
Acinetobacter baumannii
Acinetobacter baumanni
1)
1)
1)
1)
(FJ22711
(98%)
(FJ22711
(98%)
V
(NR074898)
(100%)
(JF513192)
(98%)
ibrio harveyi
1
(97%)
1
(97%)
V
(JF513192)
(95%)
Pr
V
(FJ2271
Vibrio harveyi
(FJ2271
ibrio alginolyticus
(96%)
ibrio alginolyticus
(99%)
oteus mirabilis
(99%)
Acinetobacter haemolyticus
(96%)
Acinetobacter haemolyticus
(96%)
(90%)
(91%)
Pr
V
V
ibrio alginolyticusV
ibrio alginolyticusV
eviceps
Pygmy sperm whale
eviceps
ostris
eviceps
Kogia br
Kogia br
Pygmy sperm whale
Kogia br
Pygmy sperm whale
Stenella longir
Spinner dolphin
Stenella attenuata
Pantropical spotted dolphin
ostris
Stenella longir
Spinner dolphin
ocephalusPhyseter macr
Giant sperm whale
sp.Mesoplodon
Beaked whale
S15
S16
S17
S18
S19
S20
S21
S22
160 Obusan et al.
TPCR negative
negativeT
GPCR negative
TPCR negative
negativeT
TLA
TLA
GPCR negative
TPCR positive
Providencia stuartii
ovidencia stuartiiPr
)
ovidencia stuartii
dia
(CP003488)
Giar
(98%)
Providencia stuartii
(CP003488)
(97%)
Providencia stuartii
(99%)
ovidencia stuartii
(97%)
); and GPCR (PCR screening for T. gondii
Pr
Pr
T screening for (LA
hynchus
T
edanensis
Rough-toothed dolphin
eviceps
Steno br
Globicephala macror
Short-finned pilot whale
s dolphin
Kogia br
Pygmy sperm whale
Lagenodelphis hosei
hynchusor
Fraser
ursiops aduncus
Indo-Pacific bottlenose dolphin
Globicephala macr
Short-finned pilot whale
edanensis
T
Steno br
Rough-toothed dolphin
Tursiops truncatus
Common bottlenose dolphin
TLA);
Toxoplasma gondii
S23
S24
S25
S26
S27
S28
S29
S30
No genotypic identification
TPCR (PCR screening for
a
b
161
Occurrence of Human Pathogenic Bacteria and Toxoplasma in Cetaceans Stranded in the Philippines
These were the stranders with fecal material quali-
fied for screening.
Discussion
Screening of sampled cetacean stranders for bac-
teria revealed that a comparatively high propor-
tion (28%) of isolates were comprised of species
of Vibrio. Vibrio spp. appear to be the most com-
monly isolated bacteria in cetaceans (Buck et al.,
2006; Morris et al., 2011). Although Vibrio spp.
are natural inhabitants of the marine environ-
ment, many of its species are known to associate
with animals and are pathogenic (Hervio-Heath
et al., 2002; Panicker et al., 2004; Thompson
et al., 2007). Some species (e.g., V. cholerae,
V. vulnificus, V. parahaemolyticus, V. mimicus,
and V. fluvialis) were reported as serious human
pathogens (Vora et al., 2005; Saha et al., 2006;
Izumiya et al., 2011). In the Philippines, studies
on Vibrio involved isolations of pathogenic and
non-pathogenic strains from shrimps and sclerac-
tinian corals (Monsalud et al., 2003; Arboleda &
Reichardt, 2008; Caipang & Aguana, 2011).
Other isolated bacteria species are either pre-
viously known or emerging agents of primary
or nosocomial infections in humans and other
animals: Burkholderia cepacia; Ochrobactrum
anthropi; Serratia marcescens; Proteus mirabi-
lis; Providencia stuartii; Staphylococcus epider-
midis; and members of the genus Sphingomonas,
Acinetobacter, and Aeromonas (Daily et al.,
1981; Parke & Sherman, 2001; Salles et al.,
2002; Tumbarello et al., 2004; Guillou, 2005;
Teyssier et al., 2005; Grimont & Grimont, 2006;
Fernandez-Delgado et al., 2007; Ryan & Adley,
2010; Abulreesh, 2011). It was previously recog-
nized that bacterial pathogens are routinely recov-
ered even from healthy captive marine mammals
(Dunn et al., 2001). However, more isolates are
being found in stranders than in free-living or
captive cetaceans, implying the probability of
finding more opportunistic bacteria in stranded
animals which are usually debilitated or physi-
cally compromised (Buck et al., 1991; Chan et al.,
2001; Rose et al., 2009). Among stranded marine
mammal populations, bacterial infections were
found to comprise a considerable proportion of
infectious mortality, accounting for 31% prob-
able cause of death in bottlenose dolphins from
the coastal region of South Carolina (McFee &
Lipscomb, 2009), 14% of dead strandings along
the Oregon Coast (Stroud & Roffe, 1979), and
in 29% of mortality cases classified as human-
induced types (i.e., fishery-related and traumatic
injury) in Hong Kong (Parsons & Jefferson, 2000).
The non-concurrence between genotypic and
phenotypic species-level identifications of most
isolates (71%) may have resulted from the limita-
tions of the methods used. The study did not aim
to compare the taxonomic discriminations of the
methods used; rather, 16S rDNA sequencing was
done to complement the phenotypic identification
technique. This is due to the efficiency of com-
mercial systems used being limited by the need
to perform additional tests to definitively identify
strains at the species level (Adderson et al., 2008).
The identified bacteria isolates are data relevant
to the medical management of cetaceans that
locally strand in the Philippines. Many strand-
ing events in the country are being responded to
by trained members of stranding networks (e.g.,
PMMSN) or by a concerned government agency
(i.e., BFAR), and some of these cases necessi-
tate cetacean rehabilitation involving antibiotic
treatments. Thus, sensitivity tests should be part
of any monitoring work on cetacean health. As
suggested earlier (Bogomolni et al., 2008; Rose
et al., 2009; Wallace et al., 2013), the exposure of
marine mammals to antibiotic resistances must be
considered given the ability of these animals not
only to serve as vectors of resistant bacteria but
also to indicate the geographical extent of bacte-
rial resistance development.
As for Giardia detection, the negative results
parallel the findings of Rengifo-Herrera et al.
(2011) in seals sampled from the Antarctic as well
as that of Fayer et al. (2008) in bottlenose dol-
phins sampled from South Carolina and Florida.
It should be considered that the stranded cetaceans
screened in this study only represent their coun-
terparts in the wild, and so there would always
be the possibility of Giardia occurrence in their
populations.
As for Toxoplasma, the amplification of T. gondii
B1 gene from the blood samples of Indo-Pacific
bottlenose dolphin and pygmy sperm whale sug-
gests acute infection of these animals. However,
the presence of antibodies directed against T. gondii
cannot conclusively establish whether the other
serologically positive cetaceans are suffering from
chronic or acute infection though it does confirm
their exposure to the parasite. This is because body
tissues (e.g., skeletal and smooth muscles of the
brain, liver, etc.) that may be harbouring differ-
ent stages of the parasite (i.e., latent bradyzoites
in tissue cysts or active tachyzoites and bradyzo-
ites) were not examined histopathologically or
otherwise. Aside from the serologic and molecu-
lar evidence, other pathological findings clinically
associated with toxoplasmosis in marine mammals
were not available in all samples due to limitations
in stranding response. Other documented cases of
toxoplasmosis in the country involved terrestrial
mammals such as rats (Salibay & Claveria, 2006)
and cats (Advincula et al., 2010).
162 Obusan et al.
As it is in other marine mammals, the occur-
rence of T. gondii in cetaceans tested in the pres-
ent study (i.e., kogiids, spinner dolphin, bottlenose
dolphin, and ziphiid) is intriguing given that the
route for postnatal infection includes the ingestion
of either (1) oocysts from contaminated food and
water or (2) latent bradyzoite-infected tissue by
carnivorism (Dubey et al., 2003, 2008). One inter-
esting case is that of infected Antillean manatees
(T. manatus manatus) in Puerto Rico; these animals
are exclusively herbivorous and, thus, ingestion of
infected meat or animal tissue is unlikely (Bossart
et al., 2012). In the southern sea otter (E. lutris
nereis) California population that experienced sig-
nificant mortality due to toxoplasmic meningoen-
cephalitis (Conrad et al., 2005), a source of infec-
tion was hypothesized to be through the predation
of filter-feeding marine bivalve shellfish (Mytilus
galloprovincialis) found to assimilate and concen-
trate infective oocysts from contaminated marine
water (Arkush et al., 2003). Dietary information on
pygmy sperm whales supports cephalopods (most
commonly members of the families Cranchiidae,
Enoploteuthidae, Histioteuthidae, Lycoteuthidae,
and Ommastrephidae) as the staple diet as well
as consumption of deep-sea shrimps and, rarely,
mesopelagic fishes (Bloodworth & Odell, 2008).
On the other hand, mesopelagic fishes, particularly
myctophids (mainly Ceratoscopelus warmingi,
Diaphus spp., and Myctophum asperum) were
found to be the consistent portion of the spinner
dolphins’ diet inhabiting the Sulu Sea, Philippines
(Dolar et al., 2003). Likewise, cephalopods and
fishes were found to comprise the diets of beaked
whales (MacLeod et al., 2003) as well as Indo-
Pacific bottlenose dolphins (Amir et al., 2005).
Such cold-blooded preys are not hosts to T. gondii,
and it is not known whether sporozoite excysts if
they do ingest oocysts (Jones & Dubey, 2010).
The presence of T. gondii in some stranded ceta-
ceans in the Philippines offers the possibility of
toxoplasmosis in their free-ranging populations.
This is especially important in the case of kogi-
ids for which not much is known about owing to
their cryptic and solitary behavior, difficulty in
taxonomic identification, and generally deepwa-
ter distribution (Bloodworth & Odell, 2008). It is
important to note that among the five serologically
positive samples, three are pygmy sperm whales
(kogiids) that separately beached in the northern
part of the country (i.e., North Luzon) within 6 d.
Similarly, information regarding beaked whales is
said to be so sparse that even the most basic aspects
of their biology are poorly known for some species
(MacLeod et al., 2006). A 12-y marine mammal
stranding database (1998-2009) in the Philippines
documented only two single strandings of pygmy
sperm whales and three single strandings of beaked
whales involving two Blainville’s beaked whale
(Mesoplodon densirostris) (Aragones et al., 2010)
and one Longman’s beaked whale (Indopacetus
pacificus) (Acebes et al., 2005).
The high proportion of live responded ceta-
cean stranders herein (i.e., 60%) is consistent
with the reported percentage (i.e., 65%) for
marine mammal strandings in the Philippines by
Aragones et al. (2010). In their paper, the authors
hypothesized three possible reasons for the rela-
tively high number of live stranding events in the
country: (1) acoustic trauma, (2) gear entangle-
ment, and (3) biotoxins coupled in their prey
items. While physical damages from fishing gears
were observed in some of the sampled cetaceans
herein, it is also possible that infections or diseases
contribute to live stranding frequencies given the
determined occurrences of the target pathogens.
This deserves further investigation in the future.
Until this study, there has been no information
on the presence of the detected microorganisms
in cetacean species found in the Philippines. This
study provides the first report on bacteria isolated
in local cetaceans. Likewise, this is the first effort
to document T. gondii occurrence from a marine
environment and among marine mammals in the
country. The current findings provide clues to the
health status of free-ranging cetacean populations
and underscore the potential of stranded represen-
tatives to serve as sentinels for indicating the con-
ditions of their habitats. As the detected microor-
ganisms include known and emerging pathogens
of humans and other terrestrial animals, such may
have implications on potential contamination
of marine habitats by land-based disease agents
or the unrecognized existence of these agents in
marine environments. Knowledge on the kinds
of pathogens found in cetaceans may be treated
as feedback information on how anthropogenic
activities (e.g., discharge of untreated effluents to
oceans) are affecting the ecology of these species.
Likewise, it presents the health risks available to
humans who share the same water resource and
interact with these animals through responding to
stranding events, doing research work, or involve-
ment in other activities. As the proponents of the
“one health” concept suggest, the health status
of marine mammals provides one of the links
between ocean and human health. Therefore, it is
high time to consider the health conditions of the
diverse cetacean assemblage in the Philippines. As
an offshoot of this study, a local stranding event
response protocol involving organized specimen
collection and improved necropsy work for inves-
tigating emerging diseases in stranded cetaceans
is in the planning stages.
163
Occurrence of Human Pathogenic Bacteria and Toxoplasma in Cetaceans Stranded in the Philippines
Acknowledgments
We thank the Philippine Marine Mammal Stranding
Network (PMMSN), the Bureau of Fisheries and
Aquatic Resources (BFAR) Regional Offices, Ocean
Adventure, and Bryan Clark Hernandez for assistance
provided. This study was funded by the Philippine
Council for Health Research and Development–
Department of Science and Technology (PCHRD-
DOST) through the Accelerated Science and
Technology Human Resource Development Program
(ASTHRDP) scholarship grant for MCMO.
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... Amazon River dolphins (Inia geoffrensis) in Brazil (Santos et al., 2011;Marigo et al., 2013), Mediterranean fin whale (Balaenoptera physalus) and striped dolphins (Stenella coeruleoalba) in Italian coasts (Guardo et al., 2010;Profeta et al., 2015), and common bottlenose dolphins (Tursiops truncatus) in the Eastern Mediterranean Sea (Bigal et al., 2018), among others. It has also been detected in several cetaceans (Stenella attenuata, Tursiops truncatus, T. aduncus, Kogia breviceps, Grampus griseus, Lagenodelphis hosei, S. longirostris, Globicephala macrorhynchus, S. coeruleoalba, and Mesoplodon sp.) in the Philippines (Obusan et al., 2015;Obusan et al., 2019). Toxoplasmosis in cetaceans has often been considered a secondary disease, usually associated with immunosuppression, encephalitis, and abortion in stranded individuals (Grattarola et al., 2016;Mazzariol et al., 2012;Resendes et al., 2002). ...
... Overall, T. gondii was detected in 21% of 19 select cetaceans that stranded during the year 2019. Previous studies reported the local detection of the parasite in 71% of 28 cetaceans that stranded from 2016-2018 (Obusan et al., 2019) and in 3% of 23 cetaceans that stranded from 2012-2013 (Obusan et al, 2015). The differences in the prevalence could be due to the detection ...
... Pantropical spotted dolphins and pygmy sperm whales were reported in previous studies (Obusan et al., 2015;Obusan et al., 2019) as among cetacean species in the Philippines found to have T. gondii (Table 3). To the best knowledge of the authors, the present study is the first to report the detection of the parasite in a Cuvier's beaked whale (Ziphius cavirostris). ...
Article
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Toxoplasma gondii infections affect marine mammal species worldwide. Investigating the presence of the protozoan parasite in marine mammals is crucial to understanding land-sea connection in relation to the movement of pathogenic and potentially pathogenic microorganisms in the marine environment. The main objective of this study was to detect T. gondii, through nested PCR targeting the RE gene of the parasite, in select cetaceans (n=19) that stranded in different parts of the Philippines from January to December 2019. T. gondii was detected in four cetaceans, specifically, in the brain tissue of a pantropical spotted dolphin (Stenella attenuata), brain and stomach tissues of a Cuvier’s beaked whale (Ziphius cavirostris), brain and skeletal tissues of a pygmy sperm whale (Kogia breviceps), and lung tissue of another pantropical spotted dolphin. No statistically significant association was established between the stranding parameters and presence of T. gondii DNA in tissues of cetaceans. To the best knowledge of the authors, this study is the first to report the presence of T. gondii in a Cuvier’s beaked whale (Ziphius cavirostris). The detection of T. gondii in deep dwelling cetacean species supports the claim that toxoplasmosis may have extended beyond coastlines where pathogen run-off is likely. T. gondii prevalence among cetaceans in the Philippines has received attention for the past five years, and there is a need to continue the surveillance of T. gondii among local cetacean populations given its implications in the conservation and management of these marine mammals.
... Previous knowledge considers T. gondii as a land-based parasite, until the importance of its transmission by water [32] was implicated by waterborne outbreaks [33] and reports of infections or prevalence in marine mammals including cetaceans [34][35][36][37][38][39][40][41][42][43], fissipeds [44,45], pinnipeds [46-49, 21, 36], and sirenian [50]. In the Philippines, Obusan et al. (2015) reported the occurrence of T. gondii in cetacean species [51]. This body of evidence suggests waterborne aspects of toxoplasmosis as a zoonotic disease as well as the utility of marine mammals to serve as surrogates for studying its emergence in the marine environment [36]. ...
... Previous knowledge considers T. gondii as a land-based parasite, until the importance of its transmission by water [32] was implicated by waterborne outbreaks [33] and reports of infections or prevalence in marine mammals including cetaceans [34][35][36][37][38][39][40][41][42][43], fissipeds [44,45], pinnipeds [46-49, 21, 36], and sirenian [50]. In the Philippines, Obusan et al. (2015) reported the occurrence of T. gondii in cetacean species [51]. This body of evidence suggests waterborne aspects of toxoplasmosis as a zoonotic disease as well as the utility of marine mammals to serve as surrogates for studying its emergence in the marine environment [36]. ...
... T. gondii in cetaceans found in the Philippines was first reported by Obusan et al. (2015) [51]. Since then, the protozoan parasite has been included as one of the target pathogens for the screening of cetaceans that strand in the country. ...
Article
Full-text available
Background: The stranding events of cetaceans in the Philippines provide opportunities for gathering biological information and specimens, especially from the pelagic forms. As part of an effort to monitor the health of wild cetaceans, this study detected Leptospira spp. and Toxoplasma gondii, causative agents of the emerging zoonotic diseases leptospirosis and toxoplasmosis respectively, in their stranded representatives. From October 2016-August 2018, 40 cetaceans (representing 14 species) that stranded nationwide were sampled for brain, cardiac muscle, skeletal muscle, kidney, and blood tissues, urine, and sera. These were subjected to molecular, serological, culture, and histopathological analyses to detect the target pathogens. Results: T. gondii was detected in 20 (71%) of the 28 cetaceans with biological samples subjected to either molecular detection through RE gene amplification or IgG antibodies detection through agglutination-based serological assay. On the other hand, Leptospira was detected in 18 (64%) of 28 cetaceans with biological samples subjected to bacterial culture, molecular detection through 16S rDNA amplification, or IgM antibodies detection through ELISA-based serological assay. Conclusions: There is the plausibility of toxoplasmosis and leptospirosis in cetacean populations found in the Philippines, however, acute or chronic phases of infections in sampled stranded individuals cannot be confirmed in the absence of supporting pathological observations and corroborating detection tests. Further studies should look for more evidences of pathogenicity, and explore the specific mechanisms by which pelagic cetacean species become infected by Leptospira spp. and T. gondii. As there is growing evidence on the role of cetaceans as sentinels of land-sea movement of emerging pathogens and the diseases they cause, any opportunity, such as their stranding events, should be maximized to investigate the health of their populations. Moreover, the role of leptospirosis or toxoplasmosis in these stranding events must be considered.
... It also contributes to health risk analyses available to humans and domestic species who share the same water resources. Finally, zoo staff, government institutions, and researchers working with this population of animals must take biosecurity measures (Obusan et al. 2015). ...
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Marine mammals, regarded as sentinels of aquatic ecosystem health, are exposed to different pathogens and parasites under natural conditions. We surveyed live South American fur seals Arctocephalus australis and South American sea lions Otaria flavescens in Uruguay for Leoptospira spp., canine distemper virus (CDV), Mycobacterium spp., Toxoplasma gondii and Neospora caninum. Samples were collected from 2007 to 2013. The seroprevalence of Leptospira spp. was, for A. australis (n = 61) 37.6% positive, 50.9% negative and 11.5% suspect, while for O. flavescens (n = 12) it was 67% positive, 25% negative and 8% suspect. CDV RNA was not detected in any of the analyzed samples. Most animals tested as seropositive to tuberculosis antigens by WiZo ELISA (A. australis: 29/30; O. flavescens: 20/20); reactivity varied with a novel ELISA test (MPB70-MPB83-ESAT6-MPB59 antigens). Seroprevalence against N. caninum and T. gondii was 6.7% and 13.3% positive for O. flavescens, and 0% and 2.2% positive for A. australis respectivelly. To evaluate possible sources of infection for pinnipeds, wild rats Rattus rattus and semi-feral cats Felis catus were additionally tested for Leptospira spp. and T. gondii respectively. Water samples tested for Leptospira revealed saprofitic L. bioflexa. Pathogenic Leptospira were detected in kidneys of 2 rats, and cats tested positivie for T. gondii (100%). These results represent a substantial contribution to the study of the health status in wild pinnipeds in Uruguay.
... Marine invertebrates can trap and retain T. gondii oocysts via filtering seawater or ingesting seaweed (Mazzillo et al., 2013) and migratory filter-feeding fish can retain infective oocysts in their digestive tract (Massie et al., 2010). These filter feeders are common food sources of marine mammals (Cabezón et al., 2011;Obusan et al., 2015) and thus represent an important source of infection by T. gondii oocysts (Mazzillo et al., 2013;Poulle et al., 2021). T. gondii-infected marine mammals manifest as meningoencephalitis, multiple organ necrosis and haemorrhage (Lapointe et al., 1998;Roe et al., 2013). ...
Article
Toxoplasma gondii infection in wild marine mammals is a growing problem and is associated with adverse impacts on marine animal health and public health. This systematic review, meta‐analysis and meta‐regression estimates the global prevalence of T. gondii infection in wild marine mammals and analyzes the association between T. gondii infection and epidemiological variables. PubMed, Web of Science, Science Direct, China National Knowledge Infrastructure, and Wanfang Data databases were searched until 30 May 2021. Eighty‐four studies (n = 14,931 wild marine mammals from 15 families) were identified from literature. The overall pooled prevalence of T. gondii infection was 22.44% (3,848/14,931; 95% confidence interval (CI): 17.29% – 8.04%). The prevalence in adult animals 21.88% (798/3119; 95% CI: 13.40 –31.59) was higher than in the younger age groups. North America had a higher prevalence 29.92% (2756/9243; 95% CI: 21.77 – 38.77) compared with other continents. At the country level, the highest prevalence was found in Spain 44.26% (19/88; 95%CI: 5.21 – 88.54). Regarding climatic variables, the highest prevalence was found in areas with a mean annual temperature >20°C 36.28% (171/562; 95% CI: 6.36 – 73.61) and areas with an annual precipitation >800 mm 26.92% (1341/5042; 95% CI: 18.20 – 36.59). The subgroup and meta‐regression analyses showed that study‐level covariates, including age, country, continent, and mean temperature, partly explained the between‐study heterogeneity. Further studies are needed to investigate the source of terrestrial to aquatic dissemination of T. gondii oocysts, the fate of this parasite in marine habitat and its effects on wild marine mammals. This article is protected by copyright. All rights reserved
... detection using serological and molecular methods [51,64]. However, as mentioned, we did not perform confirmatory methods for the identification of the cysts, and so we refer to them as either "unidentified" or "putative". ...
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The relatively high frequency of marine mammal stranding events in the Philippines provide many research opportunities. A select set of stranders (n = 21) from 2017 to 2018 were sampled for bacteriology and histopathology. Pertinent tissues and bacteria were collected from individuals representing eight cetacean species (i.e. Feresa attenuata, Kogia breviceps, Globicephala macrorhynchus, Grampus griseus, Lagenodelphis hosei, Peponocephala electra, Stenella attenuata and Stenella longirostris) and were subjected to histopathological examination and antibiotic resistance screening, respectively. The antibiotic resistance profiles of 24 bacteria (belonging to genera Escherichia, Enterobacter, Klebsiella, Proteus, and Shigella) that were isolated from four cetaceans were determined using 18 antibiotics. All 24 isolates were resistant to at least one antibiotic class, and 79.17% were classified as multiple antibiotic resistant (MAR). The MAR index values of isolates ranged from 0.06 to 0.39 with all the isolates resistant to erythromycin (100%; n = 24) and susceptible to imipenem, doripenem, ciprofloxacin, chloramphenicol, and gentamicin (100%; n = 24). The resistance profiles of these bacteria show the extent of antimicrobial resistance in the marine environment, and may inform medical management decisions during rehabilitation of stranded cetaceans. Due to inadequate gross descriptions and limited data gathered by the responders during the stranding events, the significance of histopathological lesions in association with disease diagnosis in each cetacean stranding or mortality remained inconclusive; however, these histopathological findings may be indicative or contributory to the resulting debility and stress during their strandings. The findings of the study demonstrate the challenges faced by cetacean species in the wild, such as but not limited to, biological pollution through land-sea movement of effluents, fisheries interactions, and anthropogenic activities.
... The parasite Toxoplasma gondii and bacteria of the genus Giardia were found in some samples collected from single stranding events of 30 individuals representing 11 species that were documented from January 2012 to March 2013 (Obusan et al., 2015). From these same stranding events, nine individuals that underwent rehabilitation were screened to test for bacterial susceptibility patterns to antibiotics (Obusan et al., 2018); these individuals represented five species including the roughtoothed dolphin, which exhibited development of some antibiotic resistance. ...
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Global marine mammal research is disproportionately lacking compared to terrestrial mammal research and is strongly biased toward populations in Europe, North America, New Zealand, and Australia. With high extinction risks facing marine mammals in the tropics, we sought to identify potential drivers of research effort and extinction risk evaluations for marine mammals in the Philippines as a model for tropical island nations with limited resources and research capacity. Using a bibliographic approach, we compiled all materials on marine mammal research in the Philippines from 1991 to 2020, which we categorized into eight thematic areas of research focus. We reviewed all materials based on their research focus to assess the current scientific knowledge of local marine mammal populations. Using a simple metric to calculate research effort allocation, we found that all marine mammal species in the Philippines receive inadequate research attention. Using generalized linear models, we analyzed the relationship of potential factors that drive research effort. The model with the lowest Akaike Information Criterion value suggests that frequency of marine mammal stranding incidents may influence an increase in research effort on marine mammals by providing access to biological specimens that would normally be difficult to obtain. Strandings are unfortunate events with often unclear causes, but they provide an opportunity to collect data from behaviorally cryptic animals in areas where financial constraints often hamper scientific progress. We also determined that a national Red List evaluation was predicted by increased research effort. Maximizing local research using all materials from strandings and building research capacity may be an alternative to expensive field-based methods to increase knowledge on local marine mammal populations.
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Analyses of the spatial and temporal patterns of 26 years of stranding events (1995–2011 and 2012–2021, n = 568) in Indonesia were conducted to improve the country’s stranding response. The Emerging Hot Spot Analysis was used to obtain the spatial and temporal hotspot patterns. A total of 92.4% events were single stranding, while the remaining were of mass stranding events. More stranding events were recorded between 2012 and 2021 in more dispersed locations compared to the previous period. Within the constraints of our sampling limitations, East Kalimantan and Bali were single stranding hotspots and consecutive hotspots. East Java and Sabu-Raijua in East Nusa Tenggara were mass stranding hotspots. Temporally, Raja Ampat (West Papua) experienced a significant increase in case numbers. The presence of active NGOs, individuals or government agencies in some locations might have inflated the numbers of reported cases compared to areas with less active institutions and/or individuals. However, our results still give a good understanding of the progression of Indonesia’s stranding responses and good guidance of resource allocation for the stranding network. Several locations in Indonesia that need more efforts (e.g., more training workshops on rescue and necropsies) have been identified in this paper. Suggestions to improve data collection (including georeferencing tips) have also been included.
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Stranding of marine mammals is complex and understanding this phenomenon requires continuous surveillance, monitoring, data collection and research. The Philippine Marine Mammal Stranding Network (PMMSN) has collected 1178 records of stranding events nationwide from 2005 to 2020. This Technical Report is a follow-up to the second Report (i.e., Aragones and Laggui 2019). As stated in the second Technical Report the consequent series of Reports will cover two-year periods only. Thus, this third Report covers the stranding dataset from 2019 to 2020. However, as in the first (Aragones et al. 2017) and second Reports, updates on the general trends for the larger data set (2005 to 2020) will also be provided. This Report showcases analyses of the stranding records from 2019 to 2020 (n=220) for trends in stranding frequency by year, region, season, monsoon, species, sex, age class, original disposition, release and rehabilitation success. The spatial coverage presented in this report was specific to regions and provinces primarily for administrative purposes. Identification of more specific or smaller spatial areas (i.e., by municipality/city) for potential stranding hotspots was assessed using Fishnet Tools (using 15 x 15 km grids). Furthermore, seasonality of stranding events was categorized according to the prevailing monsoons. The Northeast (NE) monsoon months are November to February (NDJF), Southwest (SW monsoon) monsoon months are June to September (JJAS), and Spring Inter-monsoon (Spring IM) in October (or Lull before NE monsoon) and the Winter Inter-monsoon (Winter IM) from March to May (MAM, or Lull before SW monsoon). The stranding data was also presented in the more classic seasonal context of DJF, MAM, JJA, SON. As data analytics advances, future reports will be improved further.
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The occurrence of protozoan parasite, bacterial communities, organic pollutants and heavy metals was investigated in free-ranging species of fin (Balaenoptera physalus, n. 2) and sperm (Physeter macrocephalus, n. 2) whales from the Pelagos Sanctuary, Corsican-Ligurian Provencal Basin (Northern-Western Mediterranean Sea). Out of four faecal samples investigated, two from fin whales and one from sperm whale were found positive to Blastocystis sp. A higher number of sequences related to Synergistetes and Spirochaetae were found in sperm whales if compared with fin whales. Moreover, As, Co and Hg were found exclusively in sperm whale faecal samples, while Pb was found only in fin whale faecal samples. The concentration of both PAH and PCB was always below the limit of detection. This is the first report in which the presence of these opportunistic pathogens, bacteria and chemical pollutants have been investigated in faecal samples of free-ranging whale species and the first record of Blastocystis in fin and sperm whales. Thus, this study may provide baseline data on new anthropozoonotic parasite, bacterial records and heavy metals in free-ranging fin and sperm whales, probably as a result of an increasing anthropogenic activity. This survey calls for more integrated research to perform regular monitoring programs supported by national and/or international authorities responsible for preservation of these still vulnerable and threatened whale species in the Mediterranean Sea.
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Stranding of marine mammals is complex and understanding it requires more data and studies. The Philippine Marine Mammal Stranding Network (PMMSN) has collected 952 records of stranding events nationwide from 2005 to 2018. This Technical Report is a follow-up to the first Report (i.e. Aragones et al. 2017), which analyzed strandings data from 2005 to 2016, and this second series covered two years (2017-2018). The next series of Reports will similarly cover two-year periods only. As in the first Report, this second Report will initially give the general trends for the larger data set (2005 to 2018). The bulk of this Report is about the analyses of the stranding records from 2017-2018 (n= 229) for trends in stranding frequency by year, region, season, species, gender, age class, original disposition, release and rehabilitation success. The spatial coverage presented in this report was specific to regions and provinces primarily for administrative purposes. Identification of more specific or smaller spatial areas (i.e. by municipality/ city) for potential stranding hotspots was assessed using Fishnet Tools (using 15 x 15 km grids). Furthermore, seasonality of stranding events was categorized according to the prevailing monsoons. The Northeast (NE) monsoon months are November to February (NDJF), Southwest (SW monsoon) monsoon months are June to September (JJAS), and Spring Inter-monsoon (Spring IM) in October (or Lull before NE monsoon) and the Winter Inter-monsoon (Winter IM) from March to May (MAM, or Lull before SW monsoon). As data analytics advances, future reports will be improved further.
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Shrimp culture is considered as one of the major industries in most aquaculture-producing countries in Asia. This industry has grown steadily over the years, but its gradual decline is due to several disease outbreaks of either bacterial or viral in origin. Luminous vibrios is, which is caused mainly by Vibrio harveyi has been implicated for mass mortalities in shrimp aquaculture in the Philippines. In addition, another pathogen, V. cambellii also causes vibrios is in shrimp, but this bacterium does not exhibit luminescence. The use of molecular methods particularly the polymerase chain reaction (PCR), facilitated early detection of the causative agent; thus, control measures have been undertaken to ensure efficient management strategies, thus preventing massive mortalities of the cultured stock. This paper reviews the different PCR protocols that have been developed for early detection of pathogenic Vibrio spp. in Philippine shrimp aquaculture. These PCR protocols were developed by targeting specific genes of the pathogen in order to ensure accurate diagnosis of the disease. In addition, the use of direct colony PCR, multiplex PCR and co-amplification together with another viral pathogen is also discussed.
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AIMS: This study focused on the serologic detection of Toxoplasma gondii infection in two groups of cats: stray and household groups. In addition, hematologic assessment of seropositive (sero+) and seronegative (sero-) cats was done. METHODS: Sixty cats were serologically tested for anti-Toxoplasma gondii antibodies using the latex agglutination test. Six collection sites for each group of cats were identified in the urban communities of Sta Rosa and San Pedro, Laguna, Philippines. The 60 cats collected were divided into 30 stray and 30 household cats. RESULTS: Results revealed that 28 (46.67%) of the 60 cats were sero+. There were more household cats (28.33%) which showed seropositivity compared to stray cats (18.33%), however the difference was statistically insignificant (p>0.05) . Hematologic tests through complete blood count showed significantly (p<0.05) higher number of sero+ cats with abnormalities on hemoglobin level, red blood cell count, segmenter (neutrophil) and monocyte counts compared to the control. Other parameters such as percent packed cell volume, white blood cell count, eosinophil and lymphocyte counts showed insignificant (p>0.05) results across sero+ cats and the control. Blood chemistry analysis showed significantly higher (p<0.05) potassium level irregularities in sero+ cats relative to the sero- cats. Other parameters such as amylase , blood sugar, blood uric acid, creatinine and blood urea nitrogen were statistically insignificant (p>0.05). CONCLUSIONS: Although Toxoplasma gondii infection suggests possible cause of hematologic abnormalities, it is recommended that further studies on this aspect be done to provide more basic and clinical research information that would improve cat health management.
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The earth's climate is changing, possibly at an unprecedented rate. Overall, the planet is warming, sea ice and glaciers are in retreat, sea level is rising, and pollutants are accumulating in the environment and within organisms. These clear physical changes undoubtedly affect marine ecosystems. Species dependent on sea ice, such as the polar bear (Ursus maritimus) and the ringed seal (Phoca hispida), provide the clearest examples of sensitivity to climate change. Responses of cetaceans to climate change are more difficult to discern, but in the eastern North Pacific evidence is emerging that gray whales (Eschrichtius robustus) are delaying their southbound migration, expanding their feeding range along the migration route and northward to Arctic waters, and even remaining in polar waters over winter—all indications that North Pacific and Arctic ecosystems are in transition. To use marine mammals as sentinels of ecosystem change, we must expand our existing research strategies to encompass the decadal and ocean-basin temporal and spatial scales consistent with their natural histories.
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The stomach contents of 26 Indo-Pacific bottlenose dolphins (Tursiops aduncus) incidentally caught in gillnet fisheries around Unguja Island (Zanzibar) between February 2000 and August 2002 were examined. The relative importance of each prey species was assessed through indices of relative importance. In total, 1403 prey items comprising 50 species of bony fish and three species of squid were identified from food remains. Five species of fish, Uroconger lepturus, Synaphobranchus kaupii, Apogon apogonides, Lethrinus crocineus, Lutjanus fulvus, and three species of squid, Sepioteuthis lessoniana, Sepia latimanus and Loligo duvauceli, were the most important prey species. Based on an index that included frequency of occurrence, percentage by number and by weight, Uroconger lepturus proved to be the most important prey species of mature dolphins whereas Apogon apogonides was the preferred prey of immature dolphins. These results indicate that Indo-Pacific bottlenose dolphins off the coast of Zanzibar forage on a relatively large number of prey species, but that only a few small- and medium-sized neritic fish and cephalopods contribute substantially to the diet. Further, the ecology and behavior of the preferred fish prey species indicate that the dolphins forage over reef or soft bottom substrata and near the shore.