Leptospira spp. And Toxoplasma gondii in stranded representatives of wild cetaceans in the Philippines

Abstract

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.

Full text

R E S E A R C H A R T I C L E Open Access
Leptospira spp. and Toxoplasma gondii in
stranded representatives of wild cetaceans
in the Philippines
Marie Christine M. Obusan
1,2*
, Ren Mark D. Villanueva
1,2
, Maria Auxilia T. Siringan
3
, Windell L. Rivera
1,2
and
Lemnuel V. Aragones
2,3
Abstract
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.
Keywords: Leptospira spp., Toxoplasma gondii, Cetaceans, Stranding events, Philippines
Background
The waters of the Philippine archipelago harbor a
diverse array of marine mammals. To date, 30 marine
mammal species, including 28 cetaceans, the dugong
(Dugong dugon) and the Asian clawless otter (Aonyx
cinereus), have been confirmed in the Philippines [1].
Based on limited surveys, opportunistic sightings, and
stranding events, most of these species range from very
rare to common in the Philippines [2]; and regarded as
data deficient, endangered, threatened, and vulnerable to
extinction, globally. In general, marine mammals live
long, grow slowly, and have low fecundity. These fea-
tures make them not only prone to over-exploitation
and exposed to anthropogenic impacts but also good
sentinel species [3,4]–i.e. indicators of oceans and hu-
man health. The utility of these species as sentinels for
ocean and human health stems from their physiological
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
* Correspondence: mmobusan@up.edu.ph
1
Institute of Biology, College of Science, University of the Philippines,
Diliman, Quezon City 1101, Philippines
2
Natural Sciences Research Institute, College of Science, University of the
Philippines, Diliman, Quezon City 1101, Philippines
Full list of author information is available at the end of the article
Obusan et al. BMC Veterinary Research (2019) 15:372
https://doi.org/10.1186/s12917-019-2112-5
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similarity with humans and their ability to “sample”or
concentrate toxins and pathogens from their habitats
[5]. Thus, based on the types of diseases and pathogens
found in their wild populations, they can indicate human
health risks posed by common water resource use [6–9].
Knowledge of their diseases and pathogens is valuable to
understand the impacts of subclinical or overt diseases
in their populations, routes of infection in marine
ecosystems, and risks to other marine and terrestrial
vertebrates [10]. This information is needed to prevent
the transmission of zoonotic diseases especially at the
human-wildlife interface.
One of these zoonotic diseases is leptospirosis, endemic
in most tropical and subtropical regions. Southeast Asia is
reported as one of its most significant foci regions, and
Philippines (with 4.8 annual incidence per million) is 26th
among the 28 countries with highest incidence of the dis-
ease in humans [11]. The disease is caused by pathogenic
spirochetes of the genus Leptospira and is propagated in
nature through chronic renal infection of carrier animals
[12]. Rodents, pigs, dogs, and cattle serve as Leptospira
reservoirs but different wild and domestic mammals act as
accidental hosts for various serotypes of this pathogen
[13–15]. Antibodies against Leptospira serovars were also
detected in reptiles such as snakes, lizards, and turtles
[16]. Although it is well documented and characterized in
terrestrial species including humans, less information is
available regarding its distribution and impact in marine
mammals [17]. Previous studies reported the prevalence
of leptospirosis or seropositivity to Leptospira spp. in the
sirenian Peruvian Amazon manatees (Trichechus inunguis)
[18] as well as in pinnipeds including harbor seals (Phoca
vitulina)[19,20], Northern elephant seals (Mirounga
angustirostris)[21], California sea lions (Zalophus califor-
nianus)[22–26,17], and Chilean South American sea
lions (Otaria byronia)[27]. Most recently, two serovars –
Pomona and Calicola –of Leptospira interrogans were de-
tected in serum samples of endangered Caspian seals
(Pusa caspica) in the Caspian Sea off Northern Iran [28].
Information on the prevalence of Leptospira in cetaceans
is scarce, with the first isolation of the proposed L. brihue-
gai sp. nov from Southern Right Whale (Eubalaena aus-
tralis) that stranded in Argentina reported by Loffler et al.
(2015) [29]. Bik et al., (2016) also reported detecting
several bacteria with Leptospira sequence types in appar-
ently healthy bottlenose dolphins (Tursiops truncatus)in
California, although none of these sequence types were
close to that of pathogenic L. interrogans [30].
Another zoonotic disease, toxoplasmosis, is caused by
Toxoplasma gondii, a coccidian parasite of mammals
with cats as definitive host [31]. Previous knowledge
considers T. gondii as a land-based parasite, until the im-
portance of its transmission by water [32] was implicated
by waterborne outbreaks [33] and reports of infections
or prevalence in marine mammals including cetaceans
[34–43], 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].
The stranding events of cetaceans in the Philippines
provide opportunities for gathering biological informa-
tion and specimens, especially from the pelagic forms.
Based on Aragones et al., (2017), the trend in the
frequency of local marine mammal stranding events in
the Philippines has been increasing through the years,
with a total of 713 strandings from 2005-August 2016
and an annual average of 65 events. These strandings
are most likely to be responded in the so-called regional
hotspots, administrative regions with highest stranding
frequencies. As an archipelago, the Philippines is divided
into 17 regions for administrative purposes, and Regions
I, II, III, V, and VII, are the marine mammal stranding
hotspots [52]. Cetacean stranding events have been asso-
ciated with infection by pathogenic agents occurring
during or after periods of immune suppression [53,54].
However, proving this, as well as identifying the specific
cause of a stranding event is a difficult task, as there is
usually a synergy of factors that may cause an animal
to strand. While the presence of pathogens (and the
diseases associated with them) does not necessarily
explain the causation of a stranding event, it indicates
the health status of wild cetacean populations as well
as the conditions of their habitats. As part of an ef-
fort to monitor the health of cetaceans found in the
Philippines, this study detected Leptospira spp. and T.
gondii in different biological samples obtained from
individuals that stranded in the country from October
2016–August 2018.
Results
Stranded cetaceans
Forty (40) cetaceans that stranded in Philippine waters
from October 2016 to August 2018, were sampled for
biological materials (Table 1). Thirty-seven (37) of these
were involved in single stranding events. Three (3) ceta-
ceans were from mass stranding events; two of which
were sampled from one event while one came from a
separate event. Stranded individuals represented 14 cet-
acean species (Fig. 1). The majority of these individuals
were alive when they stranded (n= 26); 21 of them died
while being responded or rehabilitated while three were
released back into the wild.
Stranding events were recorded in Luzon (n= 25) and
Mindanao (n= 15) Islands (Fig. 1). More than half (n=
21) of these strandings occurred in administrative
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regions included in the top five stranding hotspots: Re-
gion I (n= 9), Region II (n= 3), Region III (n= 1), and
Region V (n= 8). The rest occurred in Region IV-A (n=
4), Region X (n= 3), Region IX (n= 6), Region XI (n= 2),
Region XII (n= 2), and Region XIII (n= 2). Most of the
strandings (n= 16) occurred during the lull period before
southwest (SW) monsoon, while only one was recorded
during the lull before northeast (NE) monsoon. The rest
occurred during SW monsoon (n= 13) and NE monsoon
(n= 10).
The following biological samples were obtained (with
n= number of cetacean individuals): brain tissues (n=
10), cardiac muscle tissues (n= 14), and skeletal muscle
tissues (15) were used for molecular detection of T. gon-
dii while kidney tissues (n= 12) were used for molecular
detection, histopathological examination, and isolation
of Leptospira spp. Bacterial isolation was also done using
urine (n= 2) and blood samples (n= 22). Moreover, all
blood samples were subjected to molecular detection of
both target pathogens. Serum samples (n= 7) were used
to detect T. gondii IgG antibodies and Leptospira IgM
antibodies. The number of detection methods to which
each cetacean was subjected depended on the type of
biological sample/s collected considering the physical
preservation and condition of the animal.
T. gondii detection
For the detection of T. gondii, 15 individuals (S1, S2, S3,
S4, S5, S10, S11, S12, S13, S16, S18, S21, S22, S24, and
S25)hadtissue/spositiveforthetargetRE gene and six
(S15, S24, S36, S37, S39, and S40) were seropositive for
IgG antibodies against the protozoan parasite. One in-
dividual (S24) was both sero- and RE gene- positive.
Another individual (S25) was RE-gene positive but
sero-negative. Among 28 cetaceans with biological
samples subjected to either gene-specific PCR assay or
agglutination-based serological assay, T. gondii was de-
tected in 20 (71%) individuals (Table 2).
Leptospira spp. detection
Leptospira was detected in the blood samples of nine in-
dividuals (S2, S3, S4, S10, S15, S16, S19, S20, and S22)
through 16S rDNA amplification. This detection repre-
sents both pathogenic and non-pathogenic species of
the genus as targeted by the primers used. Seven indi-
viduals (S15, S24, S25, S36, S37, S39, and S40) were
sero-positive for Leptospira IgM antibodies. Two (2)
were successfully sequenced from 15 putative lepto-
spires that were isolated: isolate 4KT1.2 (from the
kidney of S4) and isolate 6KT1.2 (from the kidney of S6)
has 98 and 99% sequence similarity respectively to L. inter-
rogans serovar Copenhageni strain FDAARGOS_203
(NCBI Accession No. CP020414). S6 exhibited
leptospirosis-associated tubulointerstitial nephritis (Fig. 2),
characterized by mild thickening of basement membrane
capillaries and necrosis of convoluted tubular epithelium
[22]. As this lesion was observed concurrent to bacterial
isolation, it is likely that this cetacean individual had recent
Leptospira infection. In addition, hemosiderosis was ob-
served (Fig. 3). Both the isolates were found to tolerate dif-
ferent seawater concentrations (1, 3, 5, 7 and 10%) up to 2
days of incubation when grown in EMJH Media and
Korthof Media, indicating their ability to survive in the
marine environment. Out of 28 cetaceans with biological
samples subjected to any of the detection methods (culture,
gene-specific PCR assay, or ELISA-based serological assay),
18 (64%) individuals were positive for Leptospira spp.
(Table 2).
Discussion
The detection of potentially pathogenic Leptospira spp.
in cetaceans underscores the need to understand how
this bacterial group moves through hosts and environ-
ments that are not usually identified in its cycle of
transmission. Prager et al. (2013) reported the asymp-
tomatic carriage of Leptospira in both wild and captive
sea lions, giving clues on the long-term circulation of
leptospirosis in their habitats [26]. Leptospirosis was
also significantly associated with close proximity to
dog parks as well as high dog-park density in Califor-
nia sea lions [62]. However, such case represents a
coastal environment that directly receives land-based
effluents. How species of Leptospira become transmitted
to pelagic cetacean species (e.g., in this study, Fraser’s
dolphin (L. hosei), melon-headed whale (P. electra), and
others) that stay in the open sea remains to be understood
in relation to the ability of these bacterial group to remain
viable in saltwater conditions. Elsewhere, reports on lepto-
spirosis and seroprevalence to Leptospira were mostly
on pinniped species and involved cases prompted by
epizootics [24]. Among cetaceans, Leptospira spp. were
only reported in Southern Right Whale (E. australis)in
Argentina [29] and in bottlenose dolphins (T. trunca-
tus) in California [30].
While leptospirosis in marine mammals is not yet
substantially characterized, interpretations of detection
methods may use as reference, the descriptions in
humans and other mammals. Leptospirosis in humans
has two phases: (1) acute phase, which is usually the
first 7 days of illness (may end 3–7days), when lepto-
spires can be cultured and detected from the blood;
and (2) immune phase, which can last for 4–30 days,
when antibodies can be detected in the blood and lep-
tospires can be cultured from the urine [63]. The limi-
tations of serology include (1) lack of antibodies at the
acute phase; (2) “anamnestic response”or the rise in
antibody titer that is directed against a previous
infecting serovar; (3) high degree of cross-reactions
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Table 1 Stranded cetaceans that were sampled from October 2016–August 2018
Strander
No.
Common name Sex Age Class Physical
Preservation
Code
Type of
Stranding
Date of
Stranding
Season of
Stranding
Region of
Stranding
S1 Grampus griseus (Risso’s dolphin) Female Adult 2 Single 19 October
2016
Lull before
NE
Region IV-A
S2 Lagenodelphis hosei (Fraser’s
dolphin)
Male Adult 2 Single 27 February
2017
NE Region V
S3 Stenella longirostris (spinner dolphin) Female Adult 2 Single 04 March
2017
NE Region V
S4 Lagenodelphis hosei (Fraser’s
dolphin)
Female Adult 2 Single 09 March
2017
NE Region XI
S5 Grampus griseus (Risso’s dolphin) Unknown Subadult 2 Single 29 March
2017
NE Region IV-A
S6 Peponecaphala electra (melon-
headed whale)
Female Unknown 2 Single 30 April 2017 Lull before
SW
Region I
S7 Feresa attenuata (pygmy killer
whale)
Unknown Adult 2 Mass 02 May 2017 Lull before
SW
Region V
S8 Stenella attenuata (Pantropical
spotted dolphin)
Female Adult 2 Single 07 May 2017,
0800H
Lull before
SW
Region XIII
S9 Stenella attenuata (Pantropical
spotted dolphin)
Male Adult 2 Single 07 May 2017,
1400H
Lull before
SW
Region XIII
S10 Grampus griseus (Risso’s dolphin) Unknown Adult 2 Single 09 May 2017 Lull before
SW
Region V
S11 Kogia breviceps (pygmy sperm
whale)
Male Adult 2 Single 16 May 2017 Lull before
SW
Region XI
S12 Grampus griseus (Risso’s dolphin) Female Neonate 1 Single 15 June 2017 SW Region I
S13 Stenella attenuata (Pantropical
spotted dolphin)
Female Subadult 2 Single 21 June 2017 SW Region I
S14 Grampus griseus (Risso’s dolphin) Unknown Adult 2 Single 23 June 2017 SW Region III
S15 Lagenodelphis hosei (Fraser’s
dolphin)
Unknown Unknown 1 Single 02 July 2017 SW Region I
S16 Peponocephala electra (melon-
headed whale)
Male Adult 2 Single 03 July 2017 SW Region XII
S17 Stenella attenuata (Pantropical
spotted dolphin)
Female Subadult 1 Single 28 July 2017 SW Region IV-A
S18 Stenella longirostris (spinner dolphin) Female Subadult 2 Single 31 August
2017
SW Region XI
S19 Stenella longirostris (spinner dolphin) Female Subadult 2 Single 30
September
2017
SW Region I
S20 Kogia breviceps (pygmy sperm
whale)
Female Adult 2 Single 09 November
2017
NE Region V
S21 Lagenodelphis hosei (Fraser’s
dolphin)
Female Adult 2 Single 01 December
2017
NE Region II
S22 Globicephala macrorhynchus (short-
finned pilot whale)
Female Adult 2 Single 05 December
2017
NE Region I
S23 Tursiops aduncus (Indo-Pacific
bottlenose dolphin)
Female Adult 1 Single 15 January
2018
NE Region IX
S24 Stenella attenuata (Pantropical
spotted dolphin)
Male Adult 2 Single 16 January
2018
NE Region IX
S25 Stenella coeruleoalba (striped
dolphin)
Female Adult 1 Single 15 February
2018
NE Region V
S26 Lagenodelphis hosei (Fraser’s
dolphin)
Female Adult 2 Single 17 April 2018 Lull before
SW
Region IX
S27 Kogia breviceps (pygmy sperm Male Subadult 2 Single 26 April 2018 Lull before Region IX
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between serogroups especially during the acute phase;
and (4) differences in the sensitivity of tests, for ex-
ample, earlier detection by ELISA from day 6–8(which
may cover the acute phase) compared with MAT [64].
In the case of the nine stranders (S2, S3, S4, S10, S15,
S16, S19, S20, and S22) that were positive in blood
PCR detection, the presence of acute infection cannot
be confirmed since 16S rDNA targeted both patho-
genic and non-pathogenic Leptospira spp. Included in
these stranders are S15 and S4, which had IgM in the
blood and bacterium isolated from the kidney respect-
ively. It is possible that a non-pathogenic Leptospira
species was detected by PCR in the blood of these indi-
viduals. If this is the case, then the detected IgM in
S15 was against a pathogenic serovar, or if indeed such
serovar was amplified in the blood, then the IgM might
have been detected early in the acute phase given the
reported early detection by ELISA. As the kidney
isolates from S4 and S6 were most phylogenetically
related to L. interrogans, these cetaceans may have
chronic renal carriage of leptospires (immune phase of
infection) or active infection if presented with clinical
symptoms such as in the case of dogs [65]. The seven
stranders (S15, S24, S25, S36, S37, S39 and S40) that
were sero-positive for IgM might be in the immune
phase of leptospirosis. The presence of the anti-
leptospiral IgM may be attributed to the persistence of
the antibody after infection, frequent reinfection with
leptospires in endemic areas, or cross-reaction with
other infectious agents [66]. Overall, there is evidence
for the exposure of sampled cetaceans to pathogenic
Leptospira spp., but it is rather difficult to confirm the
phase of infection given the limitations in the detection
tests and biological samples.
It must be noted that there is 100% sero-positivity in
all sera qualified for the detection of IgM antibodies
against Leptospira. However, this result is limited by the
fact that SERION Leptospira IgM-ELISA was only evalu-
ated to detect the complexes formed by human IgM and
Leptospira antigens representing known serovars bound
to goat antihuman IgM. The test was used as an ac-
cepted surrogate to the gold standard but laborious and
time-consuming Microscopic Agglutination Test (MAT)
which requires the maintenance of live serovars [63].
The test’s protocol claims the likelihood of cross-reactivity
of goat antihuman IgM with IgM from other species [63].
While specific information on cross-reactivity of cetacean
and human antibodies to Leptospira antigens are yet to be
Table 1 Stranded cetaceans that were sampled from October 2016–August 2018 (Continued)
Strander
No.
Common name Sex Age Class Physical
Preservation
Code
Type of
Stranding
Date of
Stranding
Season of
Stranding
Region of
Stranding
whale) SW
S28 Kogia breviceps (pygmy sperm
whale)
Female Adult 2 Single 27 April 2018 Lull before
SW
Region IX
S29 Balaenoptera omurai (Omura’s
whale)
Female Neonate 2 Single 30 April 2018 Lull before
SW
Region X
S30 Balaenoptera sp. (unidentified
baleen)
Unknown Adult 2 Single 03 May 2018 Lull before
SW
Region V
S31 Kogia breviceps (pygmy sperm
whale)
Male Adult 2 Single 06 May 2018 Lull before
SW
Region IV-A
S32 Kogia breviceps (pygmy sperm
whale)
Female Adult 2 Single 10 May 2018 Lull before
SW
Region X
S33 Peponocephala electra (melon-
headed whale)
Female Adult 2 Single 17 May 2018 Lull before
SW
Region X
S34 Stenella attenuata (Pantropical
spotted dolphin)
Unknown Subadult 2 Single 25 May 2018 Lull before
SW
Region I
S35 Tursiops aduncus (Indo-Pacific
bottlenose dolphin)
Male Adult 2 Single 28 May 2018 Lull before
SW
Region IX
S36 Steno bredanensis (rough-toothed
dolphin)
Female Adult 1 Single 02 July 2018 SW Region I
S37 Balaenoptera edeni (Bryde’s whale) Unknown Neonate 1 Single 03 July 2018 SW Region V
S38 Stenella attenuata (Pantropical
spotted dolphin)
Male Adult 1 Single 08 August
2018
SW Region I
S39 Feresa attenuata (pygmy killer
whale)
Male Adult 1 Mass 17 August
2018
SW Region II
S40 Feresa attenuata (pygmy killer
whale)
Female Adult 1 Mass 17 August
2018
SW Region II
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Fig. 1 Cetacean stranding sites. Forty individuals confirmed to belong to 13 cetacean species that stranded in Philippine waters from October
2016 to August 2018, were sampled for biological materials
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Table 2 Summary of results for detection of target pathogens
Strander
Code
Cetacean Species (and common name) T. gondii detection by PCR T. gondii
detection
by LAT
Leptospira
detection by
PCR
Leptospira
culture
Leptospira
detection
by ELISA
Blood Kidney
Blood Cardiac Skeletal Brain
S1 Grampus griseus (Risso’s dolphin) –*+** –*–*
S2 Lagenodelphis hosei (Fraser’s dolphin) –*++* +–+
C
*
S3 Stenella longirostris (spinner dolphin) –+++* +–+
A
*
S4 Lagenodelphis hosei (Fraser’s dolphin) + + + + * + –+
A
*
S5 Grampus griseus (Risso’s dolphin) –+–** –– **
S6 Peponecaphala electra (melon-headed whale) * * * * * * * +
B
*
S7 Feresa attenuata (pygmy killer whale) * * * * * * * * *
S8 Stenella attenuata (Pantropical spotted dolphin) * * * * * * * * *
S9 Stenella attenuata (Pantropical spotted dolphin) * * * * * * * * *
S10 Grampus griseus (Risso’s dolphin) + + + + * + –– *
S11 Kogia breviceps (pygmy sperm whale) * + + + * * * * *
S12 Grampus griseus (Risso’s dolphin) + * * * * –*–*
S13 Stenella attenuata (Pantropical spotted dolphin) * + + * * * * +
B
*
S14 Grampus griseus (Risso’s dolphin) * –*** **+
A
*
S15 Lagenodelphis hosei (Fraser’s dolphin) –***+ +*–+
S16 Peponocephala electra (melon-headed whale) + + * + * + –+
D
*
S17 Stenella attenuata (Pantropical spotted dolphin) * * * * * * * * *
S18 Stenella longirostris (spinner dolphin) + –++* –– – *
S19 Stenella longirostris (spinner dolphin) –**** +*–*
S20 Kogia breviceps (pygmy sperm whale) –– *–*+–– *
S21 Lagenodelphis hosei (Fraser’s dolphin) –++** –– +
A
*
S22 Globicephala macrorhynchus (short-finned pilot
whale)
++ –+* + * * *
S23 Tursiops aduncus (Indo-Pacific bottlenose dolphin) * * * * * * * * *
S24 Stenella attenuata (Pantropical spotted dolphin) + + + * + –*–+
S25 Stenella coeruleoalba (Striped dolphin) + * * * ––*–+
S26 Lagenodelphis hosei (Fraser’s dolphin) * * * * * * * * *
S27 Kogia breviceps (pygmy sperm whale) –– – ** –– – *
S28 Kogia breviceps (pygmy sperm whale) * * * * * * * * *
S29 Balaenoptera omurai (Omura’s whale) * * * * * * * * *
S30 Balaenoptera sp. (unidentified baleen) * * * * * * * * *
S31 Kogia breviceps (pygmy sperm whale) –*––*–– – *
S32 Kogia breviceps (pygmy sperm whale) * * * * * * * * *
S33 Peponocephala electra (melon-headed whale) –**** –*–*
S34 Stenella attenuata (Pantropical spotted dolphin) –*–** –– – *
S35 Tursiops aduncus (Indo-Pacific bottlenose dolphin) –**** –*–*
S36 Steno bredanensis (rough-toothed dolphin) * * * * + * * * +
S37 Balaenoptera edeni (Bryde’s whale) –***+ –*–+
S38 Stenella attenuata (Pantropical Spotted dolphin) * * * * * * * * *
S39 Feresa attenuata (pygmy killer whale) * * * * + * * * +
S40 Feresa attenuata (pygmy killer whale) * * * * + * * * +
Obusan et al. BMC Veterinary Research (2019) 15:372 Page 7 of 14
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available, it is said that humans follow the classical IgM
response to Leptospira antigens similar to animals [63].
The suitability of cetaceans as sentinels for marine zoo-
noses such as leptospirosis may be supported by evidences
of cross-reactivity of (1) antibodies to human antigens and
tissues of the bottlenose dolphin (T. truncatus)[67]sug-
gesting that the applied serological assay has a reasonable
sensitivity at least for many cetacean species; (2) human
and bovine antibodies in paraffin-wax embedded tissues
of striped dolphin (S. coeruleoalba)[63]; and (3) commer-
cially available terrestrial-specific antibodies (from pig, rat,
mice, and humans) to dolphins, allowing the
characterization of the immune cell subsets of under hu-
man care and free-ranging dolphins [63].
In the Philippines, the detection of pathogenic Leptos-
pira spp. in coastal soil after the storm surge brought
about by typhoon Haiyan that devastated the Eastern
Visayas part of the Philippines was reported by Saito
et al. (2014) [63]. Their report confirmed the survival of
pathogenic Leptospira sp. in seawater for 4 d, showing
the ability of soil-inhabiting leptospires to persist even
after a storm surge, and thus, the likelihood of a lepto-
spirosis outbreak during seawater inundation episodes
brought about by natural disasters. Khairani-Bejo (2004)
reported the short survival of an isolate identified as L.
interrogans serovar Hardjo in a medium with 3.78 and
3.85% salt content and pH of 6.5 to 6.8 [63]. The novel
Leptospira spp. strain Manara isolated from Southern
Right Whale tolerated at least 5% seawater in medium
for 48 h [29]. Likewise, the present study supports the
survival of Leptospira spp. survival in seawater as the
two isolates from stranded cetaceans were found to
tolerate up to 10% seawater in media for 2 d.
Seawater-tolerant leptospires may gain entry in
Table 2 Summary of results for detection of target pathogens (Continued)
Strander
Code
Cetacean Species (and common name) T. gondii detection by PCR T. gondii
detection
by LAT
Leptospira
detection by
PCR
Leptospira
culture
Leptospira
detection
by ELISA
Blood Kidney
Blood Cardiac Skeletal Brain
Total positive results out of screened cetaceans 8/22 10/14 10/15 8/10 6/7 9/22 0/12 15/23 7/7
+ positive for T. gondii or Leptospira spp.
- negative for T. gondii or Leptospira spp.
+
A
one putative leptospire isolate
+
B
two putative leptospire isolates
+
C
three putative leptospire isolates
+
D
four putative leptospire isolates
*biological sample for testing not available/enough
Fig. 2 Interstitial nephritis in the kidney of a melon-headed whale (S6). The kidney tissue exhibited leptospirosis-associated tubulointerstitial
nephritis, characterized by mild-thickening of basement membrane capillaries and necrosis of convoluted tubular epithelium
Obusan et al. BMC Veterinary Research (2019) 15:372 Page 8 of 14
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cetaceans through direct contact with infected urine of
infected or reservoir animals, or exposure to soil,
water, and food that have been contaminated with in-
fected urine. Among different hosts, transmissions
through bites, tissue ingestion, sexual contact, breast
milk, and placenta were also reported [63].
The known symptom of leptospirosis in marine mam-
mals is interstitial nephritis, which is presented with
clinical signs of impaired renal function including dehy-
dration, polydipsia or excessive thirst, muscular tremors,
abdominal pain, vomiting, and depression [24,22]. The
renal lesions in the melon-headed whale (P. electra) were
consistent with those associated with leptospirosis in
California sea lions (Z. californianus) and Northern ele-
phant seals (M. angustirostris) that stranded along the
coast of California [21–23]. The hemosiderosis observed
in this particular cetacean, characterized by the presence
of hemosiderin pigments from hemoglobin degradation,
may result from infection, dietary deficiencies, excessive
dietary iron (which increases susceptibility to bacterial
infections and organ dysfunction), corticosteroids, and
other toxins [63]. As bacterial infection can cause hemo-
siderosis, there is reason to suspect that this, together
with tubulointerstitial nephritis, resulted from the infec-
tion of Leptospira sp. isolated from the kidney of the
cetacean.
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. As it is in the first study, the cet-
acean species where T. gondii was detected in this study
were also pelagic such as the Risso’s dolphin (G. griseus),
Fraser’s dolphin (L. hosei), spinner dolphin (S. longiros-
tris), and others. This pose an interesting question as to
how these cetaceans become exposed or infected with
the parasite. The prevalence of toxoplasmosis in their
populations is possible, given that T. gondii was detected
in tissues of stranders through PCR and serological as-
says. However, the present interpretations are limited by
the fact that only T. gondii specific IgG was detected,
and that the presence of this type of antibody alone can-
not unequivocally indicate a chronic infection. Recent
reports suggest T. gondii specific IgM and/or IgG fail/s
to differentiate between acute (3–6 months) and chronic
(beyond 6 months) phases of toxoplasmosis as they are
detected in both phases (80). In the case of humans, it is
suggested that diagnosis for toxoplasmosis must be
interpreted based on a combination of serological and
molecular detection methods. For example, an acute
infection can be indicated by concurrent IgM and low
IgG avidity or a chronic infection can be indicated by
concurrent IgM and high IgG avidity or IgG and high
IgG avidity [63]. In addition, the use of molecular detec-
tion such as gene-specific PCR is helpful for confirming
disseminated infection due to the systemic nature of
toxoplasmosis as well as propagation of infection
through body fluids [84; 81]. There is one cetacean (S24)
that was positive for both IgG in the serum and RE gene
in the blood and cardiac and skeletal muscles, indicating
a disseminated infection. Another individual (S25) was
RE-gene positive but sero-negative; in this case, there is
the likelihood that IgM antibodies were present but were
not detected given the limitations of testing kits [63].
The other 15 cetaceans that were positive in PCR might
either have disseminated infection (i.e., positive detection
in blood of cetacean stranders (S4, S10, S12, S16, S18,
S22, S24, and S25), and in both blood and muscles of
cetacean stranders (S1, S2, S3, S4, S5, S10, S11, S12, S13,
S16, S18, S21, S22, S24, and S25) or latent infection (i.e.,
positive detection of tissue cysts only in muscles of cet-
acean stranders S1, S2, S3, S5, S11, S13, and S21). The
other five sero-positive cetaceans (S15, S36, S37, S39,
and S40) that are negative in PCR detection can be safely
said to have been exposed to the parasite.
Infection by T. gondii can occur transplacentally, or
through the ingestion of food or water contaminated by
oocysts as well as consumption of tissue with the brady-
zoite stage of the parasite [44]. It is interesting to note
that dolphins drink very small amount of water [44] and
cetaceans in general are known to consume cephalo-
pods, shrimps, and fishes [63] poikilothermic preys that
are not hosts to T. gondii. However, Massie et al. (2010)
proved that northern anchovies (Engraulis mordax) and
Fig. 3 Hemosiderosis: brown granular pigments (black arrows).
Hemosiderosis was observed in the kidney tissue of a melon-headed
whale (S6), characterized by the presence of hemosiderin pigments
from hemoglobin degradation
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Pacific sardines (Sardinops sagax) serve as biotic vectors
for T. gondii transmission in marine environment [63].
With the elimination of carnivory feeding as the possible
source of T. gondii in cetaceans, it is likely that oocyst
contamination of marine water and prey item is a risk
factor for infection, thus supporting pollution of their
habitat by land to sea movement of the parasite. Such
contamination is said to be coming from effluents as
well as ship runoff waters containing oocysts [8,37],
which can survive in the environment for years (Black
and Boothroyd, 2000). Di Guardo and Mazzariol (2013)
asserted that direct oocyst contamination of seawater
from land-based effluents may explain the infection of
coastal species such as bottlenose dolphins, however, in
the case of T. gondii detection in pelagic species, the
possibility of an “open sea T. gondii life cycle”that is dif-
ferent from the known land and benthic protozoan cy-
cles must be considered [63]. It is also possible that
transmission of the parasite or infection happened dur-
ing migration. For example, toxoplasmosis is known to
affect striped dolphins (S. coeruleoalba) in the Mediter-
ranean region [37]. A stranded striped dolphin was also
one of the samples in this study, and T. gondii was amp-
lified from its blood. Striped dolphins are widely-
distributed worldwide; they are found in warm temper-
ate and tropical waters of Atlantic, Indian and Pacific
Ocean [63]. Cetaceans are known to migrate, but infor-
mation is lacking regarding the migration patterns and
abundance ranges of many cetacean species in the
Philippines.
Screening stranded cetaceans for the presence of target
pathogens may help explain the possible cause/s of their
stranding events and guide decisions in cases of medical
intervention or rehabilitation. For example, the melon
headed whale or P. electra (S6) may have stranded due
to leptospirosis evidenced by tubulointerstitial nephritis
with concurrent isolation of Leptospira sp. However,
predation may have also contributed to the debility of
the animal as shark bite was seen in its body. The dol-
phin was rehabilitated but died on 22 May 2017 (more
information on this strander can be accessed through
http://newsinfo.inquirer.net/895271/whale-nursed-back-
to-health-in-la-union)[63]. Another strander, the rough-
toothed dolphin or S. bredanensis (S36) was found to be
seropositive for T. gondii and Leptospira spp., confirm-
ing exposure to the pathogens. For its rehabilitation, the
dolphin was first brought to a fish tank in BFAR-
RMaTDeC (Regional Mariculture Technodermo Center)
in Lucap Wharf, Alaminos, and then to the sea pen in
Cariaz Island of Hundred Islands National Park, Pangasi-
nan, Philippines. During the early days of rehabilitation,
the animal showed symptoms of health problems which
include diarrhea and expulsion of placental-like tissues
indicative of either recent calf delivery or abortion prior
to the stranding event. The dolphin was also observed to
have abnormally short respiratory intervals, followed by
straining and flexing, which could be an effort to expel
placenta. Thus, antibiotics, pain relievers, oxytocin,
dinoprost, and calcium were given to ease the symp-
toms and facilitate expulsion of any remaining placen-
tal tissues (L.J. Suarez, pers. comm., July 2018). While
toxoplasmosis and leptospirosis are reported to cause
abortion in animals [63], the limited serology cannot
conclusively support such in the absence of corrobor-
ating findings due to limitations in the collected bio-
logical samples (e.g., available for PCR assay and
histopathological analyses). The dolphin had IgG anti-
bodies against T. gondi, which cannot differentiate be-
tween acute or chronic toxoplasmosis, and had IgM
antibodies against Leptospira, which more likely indi-
cate immune rather than acute phase of leptospirosis.
As time progressed, continuous improvement in the
dolphin’s health and physical condition was observed
until it was successfully released back into the wild on
21 August 2018 at Lingayen Gulf (news story on this
strander can be accessed through http://www.pna.gov.
ph/articles/1045798)[63]. Considering the foregoing
cases, active infections cannot be confirmed in the
absence of supporting pathological observations and
detection tests.
Conclusions
Leptospira spp. and T. gondii were detected in ceta-
ceans that stranded in the Philippines from October
2016–August 2018. This confirmed the plausibility of
leptospirosis and toxoplasmosis in their populations,
and the possible role of these infections in their local
stranding events. Further studies should explore the
specific mechanisms by which pelagic cetacean species
become infected by Leptospira spp. and T. gondii,as
well as the routes of transmission of these microorgan-
isms in the marine environment. 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 through their stranded representa-
tives. Moreover, experiences in sampling and rehabili-
tating stranded cetaceans should guide future practices
to prevent zoonotic transmissions at the human-
animal interface.
Methods
Stranded cetaceans
Cetacean stranding events that occurred in the
Philippines from October 2016–August 2018 were moni-
tored and responded through collaboration with Philip-
pine Marine Mammal Stranding Network (PMMSN) as
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well as Department of Agriculture’sBureauofFisheries
and Aquatic Resources (DA-BFAR). PMMSN has 12 re-
gional and 32 provincial chapters that have marine mam-
mal stranding response teams mandated by BFAR
regional offices. The members and volunteers of the teams
report any stranding event and the communication is
coursed through channels until the information is relayed
to the research team. Whenever logistically possible,
stranding sites were reached by the researchers through
land, air, or water travel. In cases wherein the stranding
site was very remote and could not be reached immedi-
ately, biological material collection proceeded in coordin-
ation with PMMSN members who trained on medical
aspects of marine mammal stranding response. All provin-
cial chapters of DA-BFAR in different administrative re-
gions have at least one veterinarian who completed such
an intensive training course.
Stranded cetacean individuals were characterized in
terms of: (1) species; (2) sex (based on genital and/or
mammary slits); (3) age class (inferred from length rela-
tive to the species); (4) disposition (dead or alive); (5)
stranding type (single or mass); (6) stranding site; and
(7) stranding season (based on the scheme provided by
Wang, 2006) [55]. Biological material collection was
done based on the Code system established by the
Smithsonian Institution’s Marine Mammal Events Pro-
gram [56]: Code 1- live animal; Code 2 –fresh (carcass
in good condition); Code 3- fair (decomposed, but or-
gans basically intact); Code 4- poor (advanced decom-
position); and Code 5 –mummified or skeletal remains.
Biological materials
Blood was extracted either from the fluke vasculature
(Code 1 specimen) or vena cava (Code 2 specimen). For
serum recovery, whole blood was placed in serum separ-
ator tubes or kept warm until clotted for 30 min and
centrifuged at 280 x g for 7 min. Sera were stored at 4 °C
- 8 °C and processed within 48 h or stored in a −80 °C
freezer for further analysis. Tissue samples (< 1 cm
3
each) from kidney (Codes 2–4 specimens), brain (Code
2 specimen only), heart, and skeletal muscles (Codes 2–
3 specimens) were obtained by performing necropsy.
Urine samples (< 3 mL) were collected from Codes 1–3
specimens. Following necropsy procedure, urine was as-
pirated from the exposed bladder with a syringe or
squeezed through the penis of male individuals [57]. All
biological samples were placed in sterile plastic bags,
stored at 4 °C while on field work, and transferred im-
mediately (preferably < 12 h) to a −80 °C freezer for ana-
lyses within 6 months.
Serological assays
Antibodies against Leptospira spp. were detected using
enzyme-linked immunosorbent assay (SERION ELISA
classic Leptospira IgM (Institut Virion\Serion GmbH,
Warburg, Germany) following manufacturer’sinstruc-
tions. IgM-ELISA used antigens from L. biflexa serovar
Patoc strain Patoc I that contains genus specific epi-
topes for all Leptospira serovars. The test was devel-
oped to detect the complexes formed by human IgM
and Leptospira antigens bound with goat antihuman
IgM. The use of this test for non-human hosts relies
on cross-reactivity of the goat antihuman IgM with
IgM from other mammals. On the other hand, detec-
tion of IgG antibodies against T. gondii was done using
Toxocell Latex Agglutination Test (LAT: BIOKIT
Manufacturing Company, Barcelona, Spain), again, fol-
lowing manufacturer’s instructions. The test used a
suspension of polystyrene latex particles of uniform
size coated with soluble T. gondii antigen.
Leptospira culture
Leptospira spp. were isolated from blood, urine, and kid-
ney samples using Ellinghausen-McCullough-Johnson-
Harris medium (EMJH) following the procedure of Loffler
et al. (2015) [29]. Cultures were incubated at 28–30 °C for
a maximum of 3 months with dark-field microscopy exam-
ination every 15 d to check for turbidity and dinger ring
formation as well as characteristic motility of Leptospira.
Subcultures were prepared in case of positive Leptospira
spp. growth with simultaneous testing of bacterial survival
in halophilic condition through the addition of different
seawater concentrations (1, 3, 5, 7 and 10%, v/v) [29].
Histopathological examination
Kidney tissues were placed in 10% neutral-buffered for-
malin (with a tissue to fixative ratio of 1:10), embedded
in paraffin, and sectioned at 5 μm using a microtome.
The tissue sections were then mounted on a slide, and
subjected to hematoxylin and eosin staining [58]. Tissue
lesions associated with leptospirosis were observed
through microscopy.
Molecular analyses
Extraction of DNA from urine, kidney, blood, brain, and
muscle samples proceeded using a commercially avail-
able kit (Promega, A1120: Wizard Genomic DNA Purifi-
cation Kit). Extracted DNA samples were quantified
using a spectrophotometer and then polymerase chain
reaction (PCR) analyses were performed.
Pathogenic and non-pathogenic Leptospira spp. were
targeted through nested PCR that amplified 525-bp (first
round) and 289-bp (second round) fragments of the 16S
rRNA gene [50]. For first amplification, the primers used
were: 5′-GGCGGCGCGTCTTAAACATG-3′and 5′-
GTCCGCCTACGCACCCTTTACG-3′while for second
amplification, the primers were 5′CAAGTCA AGCGG
AGTAGCAA-3′and 5′-CTTAACCTGCTGCCTCCCG
Obusan et al. BMC Veterinary Research (2019) 15:372 Page 11 of 14
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TA-3′[59]. For both amplifications, the thermocycler
conditions used were: 94 °C for 5 min, 30 cycles of 60 °C
for 2 min, 72 °C for 1.5 min, and 94 °C for 1 min,
followed by 60 °C for 2 min and 72 °C for 15 min [59].
The 164 bp region within the 529 bp of the T. gondii RE
gene was targeted by nested amplifications using primer
pairs (1) 5′-TGACTCGGGCCCAGCTGCGT-3′and 5′-
CTCCTCCCTTCGTCCAAGCCTCC-3′;and(2)5′-AGG
GACAGAAGTCGAAGGGG-3′and 5′-GCAGCCAAGC
CGGAAACATC-3′[60]. The thermocycler conditions
used were: (1) for first amplification, 94 °C for 5 min, 30
cycles of 94 °C for 20 s, 55 °C for 20 s, 72 °C for 20 s and
72 °C at 5 min final extension; and (2) for second amplifica-
tion, 94 °C for 5 min, 35 cycles of 94 °C for 20 s, 55 °C for
20 s, 72 °C for 20 s, and 72 °C at 5 min final extension [61].
Reactions were performed in 25 μl volume with the
following concentrations of components: 1X PCR Master
Mix (Vivantis: contains Taq DNA Polymerase, dNTPs,
MgCl2), 1.0 μM assigned primers, 1.5–3.0 μLDNAtem-
plate, and nuclease-free water adjusted accordingly. Nega-
tive controls excluded DNA template. Positive controls
included either DNA from T. gondii (Su, The University of
Tennessee, Knoxville) or reference clinical strain of L.
interrogans (Rivera, University of the Philippines, Diliman).
Electrophoresis of PCR products in TAE (Tris-acetate-
EDTA) buffer was performed on agarose gels (2% for 16S
rRNA and 1.5% for flaB gene) at 8 V/cm with DNA ladder
(Vivantis, 100 bp Plus DNA ladder). Following electro-
phoresis, the gels were stained using GelRed and
viewed through UV light exposure. PCR-positive sam-
ples were processed for purification, DNA quantifica-
tion, and sequencing.
Abbreviations
BFAR-RMaTDeC: Bureau of Fisheries and Aquatic Resources - Regional
Mariculture Technodermo Center); bp: base pair; DA-BFAR: Department of
Agriculture’s Bureau of Fisheries and Aquatic Resources;
dNTPs: deoxyribonucleotide triphosphate; ELISA: enzyme-linked
immunosorbent assay; EMJH: Ellinghausen, McCullough, Johnson and Harris;
flaB: flagellin B; IgG: immunoglobulin G; IgM: immunoglobulin M; LAT: Latex
Agglutination Test; MAT: Microscopic Agglutination Test; MgCl
2
: magnesium
chloride; NCBI: National Center for Biotechnology Information; NE: northeast;
PCR: Polymerase Chain Reaction; PMMSN: Philippine Marine Mammal
Stranding Network; rDNA: ribosomal DNA; rRNA: ribosomal RNA;
SW: southwest; TAE: Tris-acetate-EDTA; Taq:Thermus aquaticus; UV: Ultraviolet
Acknowledgements
The authors thank Dr. Joseph S. Masangkay (College of Veterinary Medicine,
University of the Philippines Los Ba os) for the shared expertise in
histopathology, Dr. Leo Jonathan Suarez (Ocean Adventure, Subic) and Dr.
Sandy Ling Choo (College of Veterinary Medicine, University of the
Philippines Los Ba os) for the veterinary expertise; Dr. Chunlei Su
(Department of Microbiology, University of Tennessee Knoxville) for T. gondii
DNA samples; Darahlyn B. Romualdo (Institute of Environmental Science and
Meteorology (IESM), UP Diliman), Jamaica Ann A. Caras (IESM, UP Diliman),
Erika Joyce S. Calderon (Institute of Biology, UP Diliman), and Honey Leen M.
Lagui (IESM, UP Diliman) for the technical assistance; and PMMSN members
as well as DA-BFAR officers and veterinarians for the nationwide stranding
response.
Authors’contributions
MCMO, MATS, WLR, and LVA conceived and designed the methodology. LVA
led the cetacean stranding response and rehabilitation. MCMO, RMDV, MATS,
and WLR performed the microbiological protocols. MCMO, RMDV and LVA
performed sampling protocols. MCMO, RMDV, MATS, WLR, and LVA prepared
the manuscript. All authors have read and approved the final version of the
manuscript.
Funding
This study was funded by the Natural Sciences Research Institute (Project No.
BIO-17-1-02) and Office of the Vice Chancellor for Research and Development
(Project No. 171714 PhDIA), University of the Philippines Diliman. The funding
agencies facilitated the release of funds and supervised the procurement of
research materials and equipment, but were not involved in the study design,
sample collection, data gathering and analysis, and manuscript writing.
Availability of data and materials
All data generated or analyzed are included in the article. Other relevant
data may be requested through the corresponding author.
Ethics approval and consent to participate
The collection of biological specimens and information during cetacean
stranding events was done in collaboration with a non-governmental
organization, the Philippine Marine Mammal Stranding Network (PMMSN),
and a government agency, the Department of Agriculture-Bureau of Fisheries
and Aquatic Resources (DA-BFAR), which has the jurisdiction over cetacean
species in the Philippines by virtue of Republic Act (RA) 8550 (amended as
RA 10654). An active Memorandum of Agreement (MOA) that exists between
these two organizations covers the response and sample collection protocols
during marine mammal stranding events nationwide. The proposal for study
was evaluated by the Research Committee of the Institute of Biology prior to
submission to funding agencies. The study is exempted for clearance from the
Institutional Animal Care and Use Committee (IACUC) of the University of the
Philippines Diliman since cetaceans were not handled or maintained inside the
premises
of the university.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Institute of Biology, College of Science, University of the Philippines,
Diliman, Quezon City 1101, Philippines.
2
Natural Sciences Research Institute,
College of Science, University of the Philippines, Diliman, Quezon City 1101,
Philippines.
3
Institute of Environmental Science and Meteorology, College of
Science, University of the Philippines, Diliman, Quezon City 1101, Philippines.
Received: 27 September 2018 Accepted: 24 September 2019
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