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RESEARCH ARTICLE
Bacteriological and histopathological findings
in cetaceans that stranded in the Philippines
from 2017 to 2018
Marie Christine M. ObusanID
1,2☯
*, Jamaica Ann A. Caras
1,3
, Lara Sabrina L. LumangID
1
,
Erika Joyce S. CalderonID
1
, Ren Mark D. Villanueva
1
, Cristina C. Salibay
4
, Maria Auxilia
T. Siringan
2
, Windell L. RiveraID
5
, Joseph S. Masangkay
6
, Lemnuel V. Aragones
3☯
1Microbial Ecology of Terrestrial and Aquatic Systems, Institute of Biology, College of Science,University of the
Philippines Diliman, Quezon City, Philippines, 2Natural Sciences Research Institute, College of Science,
University of the Philippines Diliman, Quezon City, Philippines, 3Marine Mammal Research Stranding Laboratory,
Institute of Environmental Science and Meteorology, College of Science, University of the Philippines Diliman,
Quezon City, Philippines, 4College of Science and Computer Studies, De La Salle University-Dasmariñas, City of
Dasmariñas Cavite, Philippines, 5Pathogen-Host-Environment Interactions Research Laboratory, Institute of
Biology, College of Science, University of the Philippines Diliman, Quezon City, Philippines, 6College of
Veterinary Medicine, University of the Philippines Los Baños, College, Los Baños, Laguna, Philippines
☯These authors contributed equally to this work.
*mmobusan@up.edu.ph
Abstract
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 sam-
pled 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 pro-
files 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 multi-
ple 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 environ-
ment, and may inform medical management decisions during rehabilitation of stranded ceta-
ceans. 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.
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OPEN ACCESS
Citation: Obusan MCM, Caras JAA, Lumang LSL,
Calderon EJS, Villanueva RMD, Salibay CC, et al.
(2021) Bacteriological and histopathological
findings in cetaceans that stranded in the
Philippines from 2017 to 2018. PLoS ONE 16(11):
e0243691. https://doi.org/10.1371/journal.
pone.0243691
Editor: Ulrike Gertrud Munderloh, University of
Minnesota, UNITED STATES
Received: November 27, 2020
Accepted: October 22, 2021
Published: November 11, 2021
Copyright: ©2021 Obusan 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.
Data Availability Statement: All relevant data are
within the manuscript.
Funding: MCMO and LVA received funding for the
study through project grants BIO-19-1-05 (Natural
Sciences Research Institute, University of the
Philippines Diliman) for the conduct of
methodology and 171704SOS (Office of the Vice
Chancellor for Research and Development,
University of the Philippines Diliman) for sample
collection respectively. The funders had no role in
Introduction
The surveillance of wildlife health is part of an early warning system for detecting the emer-
gence or resurgence of disease threats. In the case of cetacean populations in the Philippines,
perhaps the most practical way of investigating their health is through their stranding events.
A marine mammal is considered stranded when it runs aground, or in a helpless position such
as when it is ill, weak, or simply lost [1]. While the event itself deserves attention, as it is not
normal for any marine mammal to strand for no apparent reason, each stranded individual
can give information on the abundance, distribution, health, and other ecological characteris-
tics of its free-living counterparts [2], as well as threats faced by its population [3]. It is impor-
tant that stranding events be responded to as quickly as possible, since some stranded animals
may quickly die depending on the size of the animal and extent of human intervention [4].
Biases exist in investigating the factors involved in cetacean strandings; easy-to-detect cir-
cumstances such as obvious injuries (especially those intentionally inflicted by humans) are
likely to be more reported, whereas the role of diseases or parasites may be underestimated.
The capacity to detect the presence of pathogens or parasites of stranded cetaceans depends on
resources, such as the presence of a stranding network with the capability to respond to strand-
ing events as well as availability of expertise for conducting necropsy and other protocols for
case investigation. Nonetheless, whether or not a pathological condition is the underlying
cause of a stranding, stranded animals are good representatives for monitoring wildlife health.
Also, while live strandings provide good biological samples for laboratory analyses, a dead or
decomposing carcass on the beach is just as useful in providing specimens and other informa-
tion as demonstrated in previous studies.
The available literature on bacteria that were isolated from marine mammals worldwide
support the significance of investigating Gram-negative species and their antibiotic resistance
or susceptibility. Antibiotic susceptibility patterns have been described for populations and
individuals of Atlantic bottlenose dolphins, Pacific bottlenose dolphins, Risso’s dolphins, Cali-
fornia sea lions, beluga whale, sea otters and pinnipeds [5–8]. Strains of zoonotic bacteria resis-
tant to multiple antibiotics used for human and animal treatments were isolated from these
animals, and some of those bacteria were recognized by the American Biological Safety Associ-
ation (ABSA) as human pathogens. Associations between increased prevalence of antibiotic
resistant bacteria in marine mammals and proximity to human activities were strongly sug-
gested [5,7,9–11]. The antibiotic susceptibility profiles of bacteria isolated from cetaceans
found in the Philippines where previously reported, wherein more than half of the bacteria
(n = 14) had single or multiple resistances to a selection of antibiotics [12].
On the other hand, histopathological assessments proved to be useful in determining prob-
able causes of death or debility of stranded cetaceans worldwide [13–17]. Tissue lesions help
confirm parasitic and bacterial infections, co-morbidities, physical injuries (e.g., brought
about by fisheries or human interactions) and bioaccumulation of chemical compounds (e.g.,
persistent organic pollutants) in cetaceans [16,18–22]. Histopathological assessment is a prac-
tical and informative tool that provides pathological evidence and reinforces the necropsy con-
ducted in dead cetaceans as part of the stranding response.
In this study, swab and tissue samples collected from cetaceans that stranded locally from
February 2017-April 2018 were subjected to bacterial isolation (with subsequent antibiotic
resistance screening) and histopathological assessment. Data on antibiotic resistant bacteria,
parasites, and tissue lesions in cetaceans are valuable in evaluating the factors that may be asso-
ciated with their local stranding events, observed to have increased in recent years [23,24]. Of
the 29 confirmed species in the country, 28 were reported to have stranded from 2005–2018
[24]. A yearly average of 105 cetacean strandings occurred in the country from 2014 to 2018
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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.
[24]; 229 events were recorded by the Philippine Marine Mammal Stranding Network
(PMMSN) in collaboration with the Bureau of Fisheries and Aquatic Resources (BFAR) from
2017 (n = 121) to 2018 (n = 108) involving 118 dead and 108 live (n = 3 unknown) stranders.
Materials and methods
All biological samples were collected in coordination with PMMSN and the Marine Mammal
Research and Stranding Laboratory (MMRSL) of the Institute of Environmental Science and
Meteorology (IESM), University of the Philippines, Diliman (UPD). The marine mammal
stranding response and tissue collection is a nationwide effort which is part of the Memoran-
dum of Agreement (MOA) between PMMSN and BFAR. Laboratory work was done at Micro-
bial Ecology of Terrestrial and Aquatic Systems Laboratory (METAS), Institute of Biology,
UPD.
Sample collection
Cetaceans that stranded in the Philippines from February 2017 to April 2018 were opportunis-
tically sampled for tissues and swabs by veterinarians, prosectors, or biologists who were
trained by PMMSN in collaboration with BFAR. Swabs were collected from routine and non-
routine sites depending on animal disposition and physical preservation, i.e., based on the
expanded version of the Code system established by the Smithsonian Institution’s Marine
Mammal Events Program [1]. For routine sites, swab samples were collected from the blow-
hole and anus of live cetaceans. For 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.
Whenever possible, exhaled breath condensate (blow) was collected by lowering a sterile petri
dish directly over the blowhole and the dish was swabbed afterwards. Anal swabs were col-
lected by inserting rayon swabs into the anal orifice, and gently swabbing the area. Swab sam-
ples were also taken from blowhole and anal areas of freshly dead individuals. Swab samples
from non-routine sites (e.g., lesions, organs, and abdominal or thoracic fluid) were also
obtained from both live and dead animals especially in relation to suspected infection. Tissues
were obtained during necropsy following the procedures of Pugliares et al., 2007 [25]. Stranded
cetaceans were characterized in terms of species, sex, age class, stranding type, stranding site,
and stranding season. Data gathered from the stranding and necropsy reports were include in
the analysis.
Histopathological assessment
Tissue samples (<1 cm
3
each) were preserved in 10% neutral buffered formalin, processed by
paraffin-embedded technique, sectioned at 5 μm, and subjected to hematoxylin and eosin
(H&E) staining. Tissue sectioning and H&E staining technique were performed at Providence
Hospital, Quezon City where tissue sections were stained with hematoxylin in water, dehy-
drated using a series of increasing concentrations of alcohol, and applied with eosin as a coun-
terstain. Stained specimens were passed through xylol and toluol before mounting [26]. Using
light microscopy, stained tissue samples were observed for the following: inflammation; fibro-
sis; granuloma lesions; edema; presence of cysts; endothelial damage (including endothelial
deposits); presence of macrophages; granules; microthrombi formation; and hemorrhage.
Bacterial isolation and antibiotic resistance screening
Swab samples in transport media (e.g., Amies) were stored at 4˚C and were sent to the labora-
tory within 18–24 h. Swabs were then enriched in Tryptic Soy Broth (TSB) for 18–24 h at
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37˚C. From the enriched media, inocula were streaked on MacConkey Agar (MCA) plates.
Morphologically distinct Gram-negative colonies were sub-cultured and purified. Bacterial
smears of pure cultures were Gram-stained according to Brown and Smith (2015) [27]. Gram-
negative bacterial isolates were subjected to 16S rRNA gene sequencing-based identification
and antibiotic resistance screening.
Pure bacterial isolates were identified using 16S rRNA gene amplification. Bacterial DNA
was extracted from the purified isolates using either the GF-1 Bacterial DNA Extraction Kit
(Vivantis Technologies) following manufacturer’s instructions, or the Boil Lysis Method fol-
lowing Ahmed and Dablool (2017) [28]. The universal 16S rRNA bacterial gene was amplified
from the DNA of isolates through polymerase chain reaction (PCR). The primers used for tar-
geting the 16S rRNA gene were 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1541R (5’-
AAGGAGGTGATCCANCCRCA-3’) [29,30]. The PCR reaction mix consisted of: dNTPs,
MgCl2, Taq DNA polymerase, DNA template, forward and reverse primers, and nuclease-free
water. The thermal cycler conditions were as follows: initial denaturation for 2 min at 95˚C, 30
cycles of denaturation for 30 s at 94˚C, annealing for 30 s at 55–60˚C, extension for 30 s at
72˚C, and final extension for 7 min at 72˚C. Positive controls (E.coli ATCC
1
25922) and
blanks (DNA-free templates) were included. PCR products were subjected to agarose gel elec-
trophoresis (AGE) to detect target DNA band. PCR products were then sent to Macrogen
(South Korea) for DNA purification and sequencing. PreGap4 and Gap4 (Staden Package 2.0)
were used to obtain the consensus sequences [31]. Sequence homologies were determined
using NCBI BLASTn search and further analyses were done using BioEdit [32,33].
Kirby-Bauer Disk Diffusion Assay [34] was performed to determine the sensitivity of the
bacterial isolates to antibiotics (Table 1). These antibiotics were chosen based on (1) inclusion
in the priority list of WHO for antibiotic resistance research; (2) known use in agriculture and
aquaculture; (3) reported susceptibility profiles of bacteria isolated from marine animals
worldwide; (4) use during rehabilitation of stranded marine mammals; and (5) known spec-
trum activity [5,35–38]. To ensure that only acquired resistances will be observed, antibiotics
Table 1. Antibiotics used in the Kirby-Bauer Disk Diffusion Assay.
Antibiotic class Antibiotics
Carbapenems Imipenem
Meropenem
Ertapenem
Doripenem
Penicillins Ampicillin
Cephems Cephalothin
Ceftriaxone
Cefoxitin
Fluoroquinolones Moxifloxacin
Ciprofloxacin
Ofloxacin
Aminoglycosides Amikacin
Gentamicin
Tetracyclines Tetracyclines
Oxytetracyclines
Phenicols Chloramphenicol
Folate pathway inhibitors Trimethoprim-sulfamethoxazole
Macrolides Erythromycin
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to which the bacterial isolates have intrinsic resistances were excluded in the assay. The reac-
tions of the isolates to the antibiotics were described as Susceptible (S), Intermediate (I), or
Resistant (R) based on Clinical and Laboratory Standards Institute (CLSI) M31-A2 (2002),
M100-S24 (2014), and European Committee on Antimicrobial Susceptibility Testing
(EUCAST) v 8.0 (2018). E.coli ATCC125922 was used as the control [39–41]. Multiple Anti-
biotic Resistance (MAR) Index values were computed using the formula: (# of resistant antibi-
otics / total # of antibiotics tested) [40]. MAR indices greater than 0.2 were interpreted to
come from sources where antibiotics are often used [35,42,43]. Also, MAR isolates were inter-
preted as those that are resistant to three or more antibiotic classes [44].
Results
In this study, tissue samples and bacterial isolates were obtained from 21 stranded cetaceans
representing eight species (Feresa attenuata,Kogia breviceps,Globicephala macrorhynchus,
Grampus griseus,Lagenodelphis hosei,Peponocephala electra,Stenella attenuata, and Stenella
longirostris) (Fig 1). Of the 21 select cases sampled, 15 were originally live stranders and six (6)
fresh dead. These stranded cetaceans came mainly from Luzon (n = 14) and Mindanao (n = 7).
The stranded marine mammals sampled consisted of four (4) Fraser’s, four (4) Risso’s, three
(3) spinner, and three (3) pantropical spotted dolphins, and one (1) short-finned pilot, three (3
pygmy sperm, and two (2) melon-headed whales. Samples came from 16 females, four (4)
males and two (2) undetermined. By age class, the samples were composed of 15 adults, five
(5) subadults, and one (1) neonate (Table 2).
A total of 73 tissue samples representing 6 organs (brain, cardiac muscle, kidney, skeletal
muscle, liver, lungs) were obtained from 21 stranded cetaceans: 3 spotted dolphins (S.attenu-
ata), 3 spinner dolphins (S.longirostris), 4 Fraser’s dolphins (L.hosei), 4 Risso’s dolphins (G.
griseus), 3 pygmy sperm whales (K.breviceps), 2 melon-headed whales (P.electra), 1 pygmy
killer whale (F.attenuata) and 1 short-finned pilot whale (G.macrorhynchus). Of these ani-
mals, 19 (90.48%) showed lesions in the organs tissues collected (Table 3 and Fig 2). Most of
these cetaceans were adults; there was only one neonate. Unidentified cysts and putative Sarco-
cystis sp. were observed in some tissues with prevalence rates of 47.62% and 9.52% respectively.
Some of the unidentified cysts are hypothesized to be other coccidian cysts based on observed
structures (e.g., size, shape, thick or thin membrane, etc.) very similar to any stage of reference
species (e.g., Toxoplasma), however in the absence of confirmatory methods such as immuno-
histochemical staining, these cysts are labeled as “unidentified”, as observed in H & E stained
tissues. Also, P.delphini cysts in the muscle-blubber region and nematodes in the stomach
were seen during gross necropsy and the reported identification was confirmed by the authors.
A total of 24 Gram-negative bacteria that belong to the family Enterobacteriaceae were iso-
lated from four cetaceans (S12, S16, S17, S18). Based on 16S rRNA gene, these isolates were
confirmed to have 98–100% sequence similarities to species belonging to the following genera:
Escherichia (n = 6), Enterobacter (n = 8), Klebsiella (n = 5), Proteus (n = 4), and Shigella (n = 1)
(Table 4). These isolates were resistant to at least one antibiotic class tested, and 79.17% were
classified as multiple antibiotic resistant (i.e., resistant to at least three antibiotic classes). The
MAR index values of the isolates ranged from 0.06 to 0.39. (Fig 3).
Discussion
Lesions in tissues of stranded cetaceans
Histopathological assessment of tissues is a very useful tool to identify factors causing the
death of stranded cetaceans or determine the cause of their stranding events [15,45–47]. How-
ever, our histopathological findings were mainly limited to indicative debility and stress during
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the stranding events because of the inadequate gross descriptions available from the stranding
and necropsy reports, prohibiting us to link these findings to disease processes particularly
those contributory to cetacean mortalities. As the stranding network (and the country in gen-
eral) is still building the expertise in performing necropsy for investigating the death of
stranded cetaceans, we tried to gather scientific information by performing histopathological
observations of available tissues as ancillary to the stranding report. We recognize that there is
inadequate information which will help us ascertain the cause of death of the stranded animal,
but at the same time deem our findings useful in providing information about the health of the
cetaceans.
In general, lesions in tissues of cetaceans were associated with bycatch, trauma, parasitic
and bacterial infections, and presence of persistent organic pollutants in stranded cetaceans
[13–15,17,20,48–50]. A previous study in the Philippines involved the histopathological
Fig 1. Sites of cetacean stranding events from February 2017-April 2018. S1 –S21: Cetacean Strander Codes; I-XIII,
CAR, NCR and ARMM: Administrative Regions in the Philippines (Reprinted from Philippines—Subnational
Administrative Boundaries under a CC BY license, with permission from The Humanitarian Data Exchange, original
copyright 2020).
https://doi.org/10.1371/journal.pone.0243691.g001
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assessment of renal tissues which corroborated the results of molecular and culture methods
for a suggested case of leptospirosis in a melon-headed whale (Peponocephala electra) [51].
When a cetacean strands, it is highly likely to have congestion in the liver and other organs
due to the pressure from the weight of its body lying on the thorax as well as immobility pre-
venting venous circulation [52]. It may be noted that organ congestion is the most observed
type of lesion in this study, i.e., observed in at least one organ tissue of 13 cetaceans (62%). Sev-
eral factors can also put cetaceans in stressful situations which can induce stress myopathy and
possibly cause congestion and hemorrhage [53]. The stranding event itself can induce trauma
and stress myopathy on the animal, causing congestion, hemorrhage, and skeletal and cardiac
muscle degeneration such as in the case of Zenker’s necrosis [15,53,54] in the skeletal muscle
of an adult Risso’s dolphin. However, we cannot corroborate these assumptions with other
evidence.
Congestion in brain and kidneys of cetaceans has also been associated with acoustic trauma
[55]. One of the ways to confirm acoustic trauma is through histological observations of the
inner ears [56]. Acoustic trauma was suggested as the cause of some previously reported ceta-
cean stranding events in the Philippines, possibly due to blast fishing activities near the strand-
ing sites [23,57]. There is a growing concern on marine environment being compromised by
human activities (e.g. underwater explosions, seismic exploration, shipping, operation of naval
sonar) which affects the physiology, communication, behavior and energetics of several popu-
lation of marine species [58–60]. Anthropogenic noise is now recognized as a major global
Table 2. Stranded cetaceans sampled for the study (2017–2018).
Strander
Code
PMMSN Code Species Region Date of
Stranding
Stranding
Type
Age Class Condition Sex
S01 Lh03R5270217 Fraser’s dolphin Lagenodelphis hosei V 27-Feb-17 Single Adult Alive Male
S02 Sl21R5040317 Spinner dolphin Stenella longirostris V 04-Mar-17 Single Adult Alive Female
S03 Lh03R11010317 Fraser’s dolphin Lagenodelphis hosei XI 09-Mar-17 Single Adult Dead Female
S04 Gg04R4A290317 Risso’s dolphin Grampus griseus IV-A 29-Mar-17 Single Subadult Alive
(Died)
Male
S05 Fa02R5020517 Pygmy killer whale Feresa attenuata V 02-May-17 Mass Adult Alive
(Died)
Unknown
S06 Gg15R5090517 Risso’s dolphin Grampus griseus V 09-May-17 Single Adult Alive
(Died)
Unknown
S07 Pe04R1300417 melon-headed whale Peponocephala electra I 30-April-17 Single Adult Dead Female
S08 Kb07R11160517 Pygmy sperm whale Kogia breviceps XI 16-May-17 Single Adult Alive Male
S09 Gg10R1150617 Risso’s dolphin Grampus griseus I 15-Jun-17 Single Neonate Alive Female
S10 Sa18R1210617 pantropical spotted dolphin Stenella attenuata I 21-Jun-17 Single Subadult Dead Female
S11 Gg02R3230617 Risso’s dolphin Grampus griseus III 23-Jun-17 Single Adult Dead Female
S12 Pe06R12030717 melon-headed whale Peponocephala electra XII 03-Jul-17 Single Adult Alive Male
S13 Sa03R4A280717 pantropical spotted dolphin Stenella attenuata IV-A 28-Jul-17 Single Subadult Alive Female
S14 Sl06R11310817 spinner dolphin Stenella longirostris XI 31-Aug-17 Single Subadult Alive Female
S15 Sl23R1300917 spinner dolphin Stenella longirostris I 30-Sep-17 Single Subadult Dead Female
S16 Kb02R5091117 pygmy sperm whale Kogia breviceps V 09-Nov-17 Single Adult Alive Female
S17 Lh04R2011217 Fraser’s dolphin Lagenodelphis hosei II 01-Dec-17 Single Adult Alive Female
S18 Gm11R151217 short-finned pilot whale Globicephala
macrorhynchus
I 05-Dec-17 Single Adult Alive Female
S19 Sa03R9160118 pantropical spotted dolphin Stenella attenuata IX 16-Jan-18 Single Adult Alive Female
S20 Lh01R9170418 Fraser’s dolphin Lagenodelphis hosei IX 17-Apr-18 Single Adult Dead Female
S21 Kb01R9260418 pygmy sperm whale Kogia breviceps IX 27-Apr-18 Single Adult Alive Female
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pollutant and is acknowledged as an environmental stressor [58]. Thus, future efforts should
include histopathological examinations of the inner ear.
Glomerulopathy was observed in 10 out of 17 cetaceans (59%) with kidney tissues available
for observation, and is the most observed kidney tissue lesion (10 out of 14 with lesions or
71%). Comparably, membranous glomerulonephritis was a common finding among stranded
cetaceans in Brazil [17]. This lesion was suggested in other studies to be associated with micro-
bial infections or chronic exposure of cetaceans to metals such as cadmium, copper, and zinc,
Table 3. Histopathological findings/remarks on cetaceans that stranded in the Philippines from February to April 2018.
Strander
No.
Species Sex Age Class Findings
S10
1
Stenella attenuata Female Subadult moderate congestion, hemorrhage, and membranous glomerulopathy in the kidney; unidentified cysts
in the skeletal muscle; moderate to severe congestion in the liver; atelectasis in the lungs; no apparent
lesion in brain and cardiac muscle
S13 Stenella attenuata Female Subadult glomerulopathy and edema in the kidney; no apparent lesion in brain and cardiac muscle
S19 Stenella attenuata Female Adult severe congestion in the cardiac muscle; hemorrhage, severe congestion, glomerulopathy in the kidney
S02 Stenella longirostris Female Adult moderate congestion in the brain; no apparent lesion in cardiac muscle, kidney, and skeletal muscle
S14 Stenella longirostris Female Subadult moderate congestion for cardiac muscle; glomerulopathy with lymphocytic aggregation and unidentified
cysts in the kidney; unidentified cyst and Sarcocystis cyst in the skeletal muscle
S15
2
Stenella longirostris Female Subadult no apparent lesions in the brain, cardiac muscle, skeletal muscle, liver, and lungs
S01
3
Lagenodelphis hosei Male Adult moderate congestion in the brain; unidentified cyst in the skeletal muscle; no apparent lesions in the
cardiac muscle and kidney
S03
4
Lagenodelphis hosei Female Adult moderate congestion in the brain and cardiac muscle; unidentified cysts in the cardiac muscle; severe
congestion, hemorrhage, and edema in the kidney; no apparent lesion in skeletal muscle
S17 Lagenodelphis hosei Female Adult glomerulopathy and edema in the kidney; unidentified cyst in the skeletal muscle;
S20 Lagenodelphis hosei Female Adult severe congestion in the brain, cardiac muscle and kidney; glomerulopathy in the kidney
S04 Grampus griseus Male Subadult severe congestion in the cardiac muscle and kidney; no apparent lesion in brain and skeletal muscle
S06 Grampus griseus Unknown Adult atrophy and Zenker’s necrosis in the skeletal muscle; no apparent lesion in brain and cardiac muscle
S09 Grampus griseus Female Neonate severe congestion in the cardiac muscle; unidentified cyst in the skeletal muscle; severe diffused hepatic
sinusoidal congestion in the liver; severe congestion and focal pulmonary edema in the lungs; no
apparent lesion in kidney
S11
5
Grampus griseus Female Adult no apparent lesion in cardiac muscle
S07 Peponocephala electra Female Adult swollen glomerulus and hemosiderosis in the kidney; unidentified cysts in the skeletal muscle; no
apparent lesion in cardiac muscle
S12 Peponocephala electra Male Adult membranous glomerulopathy in the kidney; hepatic edema in the liver; pulmonary edema in the lungs;
no apparent lesion in brain, cardiac muscle, and skeletal muscle
S08
6
Kogia breviceps Male Adult putative Sarcocystis cyst in the skeletal muscle; no apparent lesion in brain and cardiac muscle
S16 Kogia breviceps Female Adult severe congestion in the brain; unidentified cyst in the cardiac muscle; hemorrhage and severe
congestion in the kidney
S21 Kogia breviceps Female Adult moderate congestion in the cardiac muscle; hemorrhage and glomerulopathy in the kidney
S05 Feresa attenuata Unknown Adult moderate to severe congestion; hemorrhage and hemosiderosis in the brain; unidentified cysts and
hemosiderosis in the kidney; no apparent lesion in skeletal muscle
S18 Globicephala
macrorhynchus
Female Adult moderate congestion in the cardiac muscle; hemorrhage and glomerulopathy in the kidney; unidentified
cyst in the skeletal muscle; no apparent lesion in the brain
1
acoustic trauma likely cause of stranding.
2
only subadult animal without any apparent lesions in organs examined.
3
unidentified parasites on eyes and P.delphini cyst in the skeletal muscle seen during necropsy.
4
shark attack likely cause of stranding.
5
only adult animal without apparent lesions.
6
P.delphini cysts in the muscle-blubber and nematodes in the stomach seen during necropsy.
Observed tissues include brain, cardiac muscle, kidney, skeletal muscle, liver, and lungs tissues; tissues not mentioned in the findings are those that were not available
for histopathological observation.
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but this remains speculative in our case due to the lack of toxicological analyses and conclusive
diagnoses of infections or diseases [50,61].
Parasites in cetaceans may predispose these animals to bacterial infections, cardiovascular
complications, septicemia and other conditions, which are also frequently reported as probable
causes of death during their stranding events [16,62,63]. Here, we are reporting the detection
of cysts in the observed tissues of cetaceans. There is no known histopathological report on
cysts such as for example, T.gondii and Sarcocystis sp., in tissues sampled from cetaceans that
stranded in different sites in the Philippines, although there are earlier reports on T.gondii
Fig 2. Histopathological lesions observed in tissues of 21 cetaceans that strandedin the Philippines (2017–2018). (A) hemorrhage in S03
kidney; (B) severe congestion in S11 liver; (C) edema in S12 liver; (D) hemosiderosis characterized by the presence of brown granular pigments
in S05 kidney; (E) glomerulopathy in S14 kidney; (F) Zenker’s necrosis characterized by hyaline degeneration, loss of striations, and muscle fiber
waviness in S06 skeletal muscle; (G) atelectasis (collapsed alveoli) in S10 lungs; (H) unidentified cyst in skeletal muscle of S14; and (I) putative
Sarcocystis cyst in skeletal muscle of S08.
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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”. A better understanding of the biology, epidemiology,
and pathogenesis of tissue-encysting coccidian organisms that parasitize marine mammals is
needed to properly assess the risks and burden of protozoal disease in aquatic ecosystems [65–
67]. The transmission of these parasites is still poorly understood in marine mammals,
although it is known that they are found in striated muscles of intermediate hosts [68–71]. The
most likely modes of transmission of these parasites to aquatic animals are via ingestion of
water-borne oocysts or sporocysts originating from sewage runoff or through infected prey
[65,66,72–74]. During the past two decades, coccidian infections have been detected in
marine mammals that stranded along the coast of the northeastern Pacific Ocean [65,75].
These infections include encephalitis, myositis, hepatitis and myocarditis [66,67].
In addition, the presence of P.delphini in the muscles and blubber of stranded Fraser’s dol-
phin (L.hosei) and pygmy sperm whale (K.breviceps) was reported in the necropsy reports.
This parasite has been documented in many cetacean species, commonly in the subcutaneous
blubber with typical concentration in the perigenital region [76]. Siquier and Le Bas (2003)
suggested that Fraser’s dolphins (Lagenodelphis hosei) could act as intermediate or accidental
hosts for P.delphini, and that definitive host infection could occur through predation. There is
a need for more evidence to confirm the role of cetaceans in the life cycle of this parasite [77].
The consumption of muscles containing these parasites is one of the major routes of
Table 4. Genotypic identification of bacteria isolated from stranded cetaceans.
Source Cetacean (Code) Swab Site Isolate Code Nearest Phylogenetic Affiliation (% Sequence Similarity) NCBI�Accession Number
S12 (Peponocephala electra, adult) urine S12-A Enterobacter cloacae (99%) MH101512.1
S12-B Klebsiella aerogenes (99%) CP024883.1
S12-C Escherichia hermannii (98%) JN644551.1
S12-D Enterobacter sp. (99%) KC236445.1
S12-E Enterobacter cloacae (100%) KY492312.1
S12-F Enterobacter cloacae (99%) JN644583.1
S16 (Kogia breviceps, adult) blowhole S16-G Enterobacter ludwigii (99%) JQ659806.1
S16-H Escherichia hermannii (99%) JN644551.1
S16-I Enterobacter cloacae (99%) KM538690.1
S16-J Klebsiella pneumoniae (99%) FO203501.1
S16-K Klebsiella pneumoniae (99%) KJ803907.1
S16-L Shigella sp. (99%) KU362661.1
S17 (Lagenodelphis hosei adult) genital Slit S17-M Klebsiella pneumoniae (99%) CP020847.1
S17-N Escherichia coli (99%) AP017620.1
blowhole S17-O Enterobacter cloacae (99%) CP010512.1
S17-P Escherichia coli (99%) JQ661149.1
wound S17-Q Klebsiella quasipneumoniae (99%) CP014696.2
S18 (Globicephala macrorhynchus, adult) anus S17-R Proteus mirabilis (99%) CP015347.1
brainstem S17-S Proteus mirabilis (99%) CP015347.1
cerebellum S17-T Proteus mirabilis (99%) CP004022.1
lungs S18-U Escherichia fergusonii (99%) KJ803900.1
blowhole S18-V Enterobacter tabaci (99%) NR_146667.2
S18-W Proteus mirabilis (99%) CP015347.1
S18-X Escherichia coli (99%) CP027060.1
�National Center for Biotechnology Information.
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transmission to humans. This route of transmission is unlikely to involve cetaceans in the Phil-
ippines, as hunting and killing of marine mammals are prohibited under Section 4 of Republic
Act 9147 (Wildlife Resources Conservation and Protection Act of the Philippines). Still, there
were local reports of fishermen butchering cetaceans for food consumption (pers comm.,
BFAR Region V).
Antibiotic resistant bacteria from stranded cetaceans
Overall, the bacterial isolates have resistances to carbapenems and third-generation cephalo-
sporins. Enterobacteriaceae resistant to carbapenems and third-generation cephalosporins are
considered a research priority for the discovery of new antibiotic agents [38]. As the “last line
of defense” against multiple antibiotic resistant bacteria, the detection of carbapenem-resistant
strains is a troubling point of concern as carbapenems are fourth- generation antibiotics rec-
ommended for critical Gram-negative infections [78]. To the best knowledge of the authors,
only Greig et al. (2007) had so far used imipenem and meropenem for antibiotic susceptibility
tests on bacteria isolated from cetaceans. Greig et al. reported imipenem-resistant E.coli in
bottlenose dolphins, but all of their isolates were still susceptible to meropenem at the time [5].
All isolates were most resistant to erythromycin. The high frequency of resistance against this
antibiotic is said to be due to acquired macrolide–lincosamide–streptogramin B (MLS) resistance
genes, which is common among Enterobacteriaceae [79,80]. More than 50% of the isolates were
also resistant to cephalothin, ampicillin, and moxifloxacin. It must be noted that the isolated Kleb-
siella spp., and Escherichia spp., bacterial species often reported as pathogenic to cetaceans, were
resistant to erythromycin [81,82]. Similarly, high resistance to erythromycin, cephalothin, and
ampicillin of E.coli isolated from bottlenose dolphins in Florida and South Carolina was reported
[5,6]. Extra-intestinal pathogenic E.coli isolated from resident killer whales of San Juan Islands,
Fig 3. MAR index values of Enterobacteriaceae from sampled cetaceans.
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Washington, were found to be resistant to aminoglycosides, sulfonamides, and tetracycline [83].
Resistances against cephalothin and ampicillin were also observed in bacteria isolated from dol-
phins, whales, and seals in the Northeastern United States Coast [35]. An overall high prevalence
(88%) of resistance to at least one antibiotic was found among bacteria isolated from wild bottle-
nose dolphins in Florida, with highest resistances against erythromycin followed by ampicillin
[6]. A previous study on antibiotic susceptibility patterns of bacteria isolated from stranded ceta-
ceans in the Philippines reported the highest resistance (47%) to cefazolin [12]. Susceptibilities to
amikacin and gentamicin were also reported among bacteria isolated from marine mammals in
Florida, South Carolina, and Northeastern US Coast [5,35].
Based on these findings, the choice of antibiotics for treating bacterial infections (most
commonly pneumonia: pers comm., PMMSN veterinarians) caused by Enterobacteriaceae in
locally stranded cetaceans under rehabilitation should consider the susceptibility and/or resis-
tance profiles of bacteria. This may be possibly done through the stranding response being car-
ried out by PMMSN, wherein such profiles can be provided by the collaborating
microbiologists (e.g., the authors of this study) to the veterinarians handling the medical man-
agement of cetaceans. In the case of the bacteria isolated from cetaceans sampled in the present
study, carbapenems are the most effective antibiotic. However, this information must be inter-
preted with caution, as the bacteria were not significantly associated with any clinical presenta-
tion of infection or disease in the cetacean.
The cetacean species sampled in this study generally inhabit deep waters, but their physiol-
ogy entails a regular need to surface to sequester oxygen from the air for breathing, thus expos-
ing themselves to sewage outflows and other forms of pollution that eventually reach them
from the nearby coast [84–86]. The presence of bacteria (and associated antibiotic resistances)
in these cetaceans indicate biological pollution and presence of antibiotic resistance in their
habitats [87–89]. In this study, 33.33% of the isolates from cetaceans had MAR indices greater
than 0.2, suggesting that the isolates may have developed resistance from sources that the ceta-
ceans were exposed to, such as bodies of water highly polluted with antibiotics, including
domestic, industrial and hospital sewage outflows, water-treatment facilities, and the like [85,
86]. As the use of antibiotics stems from anthropogenic activities, this implies the need to regu-
late and monitor the use and improper disposal of antibiotics to water bodies.
Conclusion
Twenty-one cetaceans that stranded in different parts of the Philippines were sampled for bac-
terial isolation and antibiotic resistance screening as well as histopathological assessment of
available tissues. In the absence of conclusive data on the specific causes of the mortality or
morbidity of the cetaceans in relation to the stranding event, the histopathological findings
just provide clues on possible involvement of factors (e.g., acoustic trauma, stress, etc.) that
may have affected the health of cetaceans rendering them to strand or die, or possible effects of
the stranding event itself on the animal. Bacteriological findings showed more than 50% of the
isolated bacteria are multiple antibiotic resistant and that all of them are resistant to erythro-
mycin and susceptible to imipenem, doripenem, ciprofloxacin, chloramphenicol, and genta-
micin. While these information may be helpful in the medical management of stranded
cetaceans during rehabilitation, they also indicate the extent of antimicrobial resistance in the
marine environment. As sentinels, cetaceans demonstrate the threats faced by their popula-
tions in the wild, and monitoring their health through stranded representatives is a practical
approach that can help improve conservation efforts. As local stranding network expands and
veterinary and research expertise improve, more robust data from bacteriological and histo-
pathological assessments of cetaceans are expected to be available in the coming years.
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Acknowledgments
We thank the Philippine Marine Mammal Stranding Network (PMMSN) and the Bureau of
Fisheries and Aquatic Resources (BFAR) for the nationwide cetacean stranding response. Like-
wise, we thank Honey Leen M. Laggui for help in the preparation of Fig 1 and Christopher
Torno, DVM for valuable comments.
Author Contributions
Conceptualization: Marie Christine M. Obusan, Cristina C. Salibay, Maria Auxilia T. Sirin-
gan, Windell L. Rivera, Joseph S. Masangkay, Lemnuel V. Aragones.
Data curation: Marie Christine M. Obusan, Jamaica Ann A. Caras, Erika Joyce S. Calderon,
Ren Mark D. Villanueva, Lemnuel V. Aragones.
Formal analysis: Marie Christine M. Obusan, Ren Mark D. Villanueva, Cristina C. Salibay,
Maria Auxilia T. Siringan, Windell L. Rivera, Joseph S. Masangkay, Lemnuel V. Aragones.
Funding acquisition: Marie Christine M. Obusan, Lemnuel V. Aragones.
Investigation: Marie Christine M. Obusan, Jamaica Ann A. Caras, Lara Sabrina L. Lumang,
Erika Joyce S. Calderon, Ren Mark D. Villanueva, Lemnuel V. Aragones.
Methodology: Marie Christine M. Obusan, Jamaica Ann A. Caras, Lara Sabrina L. Lumang,
Erika Joyce S. Calderon, Ren Mark D. Villanueva, Cristina C. Salibay, Maria Auxilia T. Sir-
ingan, Windell L. Rivera, Joseph S. Masangkay, Lemnuel V. Aragones.
Project administration: Marie Christine M. Obusan, Jamaica Ann A. Caras, Lara Sabrina L.
Lumang, Erika Joyce S. Calderon, Ren Mark D. Villanueva, Lemnuel V. Aragones.
Resources: Marie Christine M. Obusan, Cristina C. Salibay, Maria Auxilia T. Siringan, Wind-
ell L. Rivera, Joseph S. Masangkay, Lemnuel V. Aragones.
Supervision: Marie Christine M. Obusan.
Validation: Marie Christine M. Obusan, Cristina C. Salibay, Maria Auxilia T. Siringan, Wind-
ell L. Rivera, Joseph S. Masangkay, Lemnuel V. Aragones.
Writing – original draft: Marie Christine M. Obusan, Jamaica Ann A. Caras, Lara Sabrina L.
Lumang, Erika Joyce S. Calderon, Ren Mark D. Villanueva, Cristina C. Salibay, Maria Aux-
ilia T. Siringan, Windell L. Rivera, Joseph S. Masangkay, Lemnuel V. Aragones.
Writing – review & editing: Marie Christine M. Obusan, Jamaica Ann A. Caras, Lara Sabrina
L. Lumang, Erika Joyce S. Calderon, Ren Mark D. Villanueva, Cristina C. Salibay, Maria
Auxilia T. Siringan, Windell L. Rivera, Joseph S. Masangkay, Lemnuel V. Aragones.
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