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

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  • Institute of Biology, University of the Philippines

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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.
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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 2016August
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 sampleor
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 [69].
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
[1315]. 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)[2226,17], 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
[3443], fissipeds [44,45], pinnipeds [4649,21,36],
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
2016August 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
Obusan et al. BMC Veterinary Research (2019) 15:372 Page 2 of 14
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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, Frasers
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 37days), when lepto-
spires can be cultured and detected from the blood;
and (2) immune phase, which can last for 430 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 responseor 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 2016August 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 (Rissos dolphin) Female Adult 2 Single 19 October
2016
Lull before
NE
Region IV-A
S2 Lagenodelphis hosei (Frasers
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 (Frasers
dolphin)
Female Adult 2 Single 09 March
2017
NE Region XI
S5 Grampus griseus (Rissos 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 (Rissos 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 (Rissos 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 (Rissos dolphin) Unknown Adult 2 Single 23 June 2017 SW Region III
S15 Lagenodelphis hosei (Frasers
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 (Frasers
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 (Frasers
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 68(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 tests 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 2016August 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 (Omuras
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 (Brydes 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 (Rissos dolphin) *+** **
S2 Lagenodelphis hosei (Frasers dolphin) *++* ++
C
*
S3 Stenella longirostris (spinner dolphin) +++* ++
A
*
S4 Lagenodelphis hosei (Frasers dolphin) + + + + * + +
A
*
S5 Grampus griseus (Rissos 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 (Rissos dolphin) + + + + * + –– *
S11 Kogia breviceps (pygmy sperm whale) * + + + * * * * *
S12 Grampus griseus (Rissos dolphin) + * * * * **
S13 Stenella attenuata (Pantropical spotted dolphin) * + + * * * * +
B
*
S14 Grampus griseus (Rissos dolphin) * *** **+
A
*
S15 Lagenodelphis hosei (Frasers 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 (Frasers 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 (Frasers dolphin) * * * * * * * * *
S27 Kogia breviceps (pygmy sperm whale) –– – ** –– – *
S28 Kogia breviceps (pygmy sperm whale) * * * * * * * * *
S29 Balaenoptera omurai (Omuras 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 (Brydes 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 [2123]. 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 Rissos dolphin (G. griseus),
Frasers 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 (36 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
Obusan et al. BMC Veterinary Research (2019) 15:372 Page 9 of 14
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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 cyclethat 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
dolphins 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
2016August 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 2016August 2018 were moni-
tored and responded through collaboration with Philip-
pine Marine Mammal Stranding Network (PMMSN) as
Obusan et al. BMC Veterinary Research (2019) 15:372 Page 10 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
well as Department of AgriculturesBureauofFisheries
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 Institutions 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 24 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 13
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 manufacturersinstruc-
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 manufacturers 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 2830 °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-3and 5-
GTCCGCCTACGCACCCTTTACG-3while for second
amplification, the primers were 5CAAGTCA AGCGG
AGTAGCAA-3and 5-CTTAACCTGCTGCCTCCCG
Obusan et al. BMC Veterinary Research (2019) 15:372 Page 11 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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-3and 5-
CTCCTCCCTTCGTCCAAGCCTCC-3;and(2)5-AGG
GACAGAAGTCGAAGGGG-3and 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, 1.53.0 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
Agricultures 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.
Authorscontributions
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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... T. gondii or T. gondii-like protozoan parasite exposure have been reported in numerous cetacean species (Table 1) (reviewed in [27,[31][32][33][34]). These cases were recorded in certain regions, including North America, Western Europe, and Oceania ( Figure 4). ...
... In previous studies, T. gondii has been detected in several organs in cetaceans, including the brain, heart, skeletal muscle, mesenteric lymph nodes, liver, spleen, lung, and kidney [32,33,35,49,50]. The tissue samples from stranded cetaceans used in this study were collected from CNS, heart and skeletal muscle. ...
... T. gondii or T. gondii-like protozoan parasite exposure in cetaceans[27,[31][32][33][34]. ...
Article
Full-text available
Toxoplasmosis is a zoonotic disease with veterinary and public health importance worldwide. Toxoplasma gondii infection in cetaceans is an indicator of land-to-sea oocyst pollution. However, there is a critical knowledge gap within the distribution of the T. gondii infection in cetaceans. To facilitate the global surveillance of this important zoonotic pathogen, we developed a field-deployable duplex insulated isothermal PCR (iiPCR) with automated magnetic bead-based DNA extraction for the on-site detection of T. gondii in stranded cetaceans. It targets the B1 gene of T. gondii combined with β2-microglobulin (B2M) gene of cetaceans as an internal control. Compared with the conventional qPCR assay, B1/B2M duplex iiPCR assay showed comparable sensitivity (21~86 bradyzoites in 25 mg of tissue) to detect spike-in standard of T. gondii DNA in cerebrum, cerebellum, skeletal muscle and myocardium tissues. Moreover, the overall agreement between the duplex iiPCR and qPCR was in almost perfect agreement (92%; 95% CI: 0.78–0.90; κ = 0.84) in detecting a synthetic spike-in standards. The B1/B2M iiPCR assay coupled with a field-deployable system provides a prompt (~1.5 h), feasible, highly sensitive and specific on-site diagnostic tool for T. gondii in stranded cetaceans. This platform provides one approach to evaluating aquatic ecosystem health and developing early warnings about negative impacts on humans and marine animals.
... Amazon River dolphins (Inia geoffrensis) in Brazil (Santos et al., 2011;Marigo et al., 2013), Mediterranean fin whale (Balaenoptera physalus) and striped dolphins (Stenella coeruleoalba) in Italian coasts (Guardo et al., 2010;Profeta et al., 2015), and common bottlenose dolphins (Tursiops truncatus) in the Eastern Mediterranean Sea (Bigal et al., 2018), among others. It has also been detected in several cetaceans (Stenella attenuata, Tursiops truncatus, T. aduncus, Kogia breviceps, Grampus griseus, Lagenodelphis hosei, S. longirostris, Globicephala macrorhynchus, S. coeruleoalba, and Mesoplodon sp.) in the Philippines (Obusan et al., 2015;Obusan et al., 2019). Toxoplasmosis in cetaceans has often been considered a secondary disease, usually associated with immunosuppression, encephalitis, and abortion in stranded individuals (Grattarola et al., 2016;Mazzariol et al., 2012;Resendes et al., 2002). ...
... Overall, T. gondii was detected in 21% of 19 select cetaceans that stranded during the year 2019. Previous studies reported the local detection of the parasite in 71% of 28 cetaceans that stranded from 2016-2018 (Obusan et al., 2019) and in 3% of 23 cetaceans that stranded from 2012-2013 (Obusan et al, 2015). The differences in the prevalence could be due to the detection ...
... Pantropical spotted dolphins and pygmy sperm whales were reported in previous studies (Obusan et al., 2015;Obusan et al., 2019) as among cetacean species in the Philippines found to have T. gondii (Table 3). To the best knowledge of the authors, the present study is the first to report the detection of the parasite in a Cuvier's beaked whale (Ziphius cavirostris). ...
Article
Full-text available
Toxoplasma gondii infections affect marine mammal species worldwide. Investigating the presence of the protozoan parasite in marine mammals is crucial to understanding land-sea connection in relation to the movement of pathogenic and potentially pathogenic microorganisms in the marine environment. The main objective of this study was to detect T. gondii, through nested PCR targeting the RE gene of the parasite, in select cetaceans (n=19) that stranded in different parts of the Philippines from January to December 2019. T. gondii was detected in four cetaceans, specifically, in the brain tissue of a pantropical spotted dolphin (Stenella attenuata), brain and stomach tissues of a Cuvier’s beaked whale (Ziphius cavirostris), brain and skeletal tissues of a pygmy sperm whale (Kogia breviceps), and lung tissue of another pantropical spotted dolphin. No statistically significant association was established between the stranding parameters and presence of T. gondii DNA in tissues of cetaceans. To the best knowledge of the authors, this study is the first to report the presence of T. gondii in a Cuvier’s beaked whale (Ziphius cavirostris). The detection of T. gondii in deep dwelling cetacean species supports the claim that toxoplasmosis may have extended beyond coastlines where pathogen run-off is likely. T. gondii prevalence among cetaceans in the Philippines has received attention for the past five years, and there is a need to continue the surveillance of T. gondii among local cetacean populations given its implications in the conservation and management of these marine mammals.
... Islands (Obusan et al., 2019) and Sub-Antarctic area (Michael et al., 2016), which raises concerns of the adverse impact of T. gondii infection on the health of marine mammals and humans. Previous studies have brought new insights to T. gondii epidemiology and its negative impact on wild marine mammals and public health, highlighting the need to generate more data and tools to understand the extent of and variables associated with T. gondii infection in wild marine animals. ...
Article
Toxoplasma gondii infection in wild marine mammals is a growing problem and is associated with adverse impacts on marine animal health and public health. This systematic review, meta‐analysis and meta‐regression estimates the global prevalence of T. gondii infection in wild marine mammals and analyzes the association between T. gondii infection and epidemiological variables. PubMed, Web of Science, Science Direct, China National Knowledge Infrastructure, and Wanfang Data databases were searched until 30 May 2021. Eighty‐four studies (n = 14,931 wild marine mammals from 15 families) were identified from literature. The overall pooled prevalence of T. gondii infection was 22.44% (3,848/14,931; 95% confidence interval (CI): 17.29% – 8.04%). The prevalence in adult animals 21.88% (798/3119; 95% CI: 13.40 –31.59) was higher than in the younger age groups. North America had a higher prevalence 29.92% (2756/9243; 95% CI: 21.77 – 38.77) compared with other continents. At the country level, the highest prevalence was found in Spain 44.26% (19/88; 95%CI: 5.21 – 88.54). Regarding climatic variables, the highest prevalence was found in areas with a mean annual temperature >20°C 36.28% (171/562; 95% CI: 6.36 – 73.61) and areas with an annual precipitation >800 mm 26.92% (1341/5042; 95% CI: 18.20 – 36.59). The subgroup and meta‐regression analyses showed that study‐level covariates, including age, country, continent, and mean temperature, partly explained the between‐study heterogeneity. Further studies are needed to investigate the source of terrestrial to aquatic dissemination of T. gondii oocysts, the fate of this parasite in marine habitat and its effects on wild marine mammals. This article is protected by copyright. All rights reserved
... Toxoplasma gondii is responsible for toxoplasmosis, one of the most common parasitic infections of warm-blooded animals, including humans [9]. The finding of acute toxoplasmosis and the detection of antibodies against T. gondii in marine mammals in the Eastern, Central and Western Pacific [10], the Canadian Arctic [11], the Northeastern and Western Atlantic [10,12], the Philippine archipelago [13] and the Mediterranean Sea [14] suggests a worldwide contamination of marine habitats. ...
Article
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Toxoplasma gondii is a protozoan parasite that uses felids as definitive hosts and warm-blooded animals as intermediate hosts. While the dispersal of T . gondii infectious oocysts from land to coastal waters has been well documented, transmission routes to pelagic species remain puzzling. We used the modified agglutination test (MAT titre ≥ 10) to detect antibodies against T . gondii in sera collected from 1014 pelagic seabirds belonging to 10 species. Sampling was carried out on eight islands of the Western Indian Ocean: Reunion and Juan de Nova (colonized by cats), Cousin, Cousine, Aride, Bird, Europa and Tromelin islands (cat-free). Antibodies against T . gondii were found in all islands and all species but the great frigatebird. The overall seroprevalence was 16.8% [95% CI: 14.5%-19.1%] but significantly varied according to species, islands and age-classes. The low antibody levels (MAT titres = 10 or 25) detected in one shearwater and three red-footed booby chicks most likely resulted from maternal antibody transfer. In adults, exposure to soils contaminated by locally deposited oocysts may explain the detection of antibodies in both wedge-tailed shearwaters on Reunion Island and sooty terns on Juan de Nova. However, 144 adults breeding on cat-free islands also tested positive. In the Seychelles, there was a significant decrease in T . gondii prevalence associated with greater distances to cat populations for species that sometimes rest on the shore, i.e. terns and noddies. This suggests that oocysts carried by marine currents could be deposited on shore tens of kilometres from their initial deposition point and that the number of deposited oocysts decreases with distance from the nearest cat population. The consumption of fishes from the families Mullidae, Carangidae, Clupeidae and Engraulidae, previously described as T . gondii oocyst-carriers (i.e. paratenic hosts), could also explain the exposure of terns, noddies, boobies and tropicbirds to T . gondii . Our detection of antibodies against T . gondii in seabirds that fish in the high sea, have no contact with locally contaminated soils but frequent the shores and/or consume paratenic hosts supports the hypothesis of an open-sea dispersal of T . gondii oocysts by oceanic currents and/or fish.
... From these same stranding events, nine individuals that underwent rehabilitation were screened to test for bacterial susceptibility patterns to antibiotics (Obusan et al., 2018); these individuals represented five species including the roughtoothed dolphin, which exhibited development of some antibiotic resistance. In a similar study of samples collected from stranding events that occurred between October 2016 to August 2018 (n = 40), T. gondii and bacteria of the genus Leptospira were also detected (Obusan et al., 2019). Opportunistic hematological, macroscopic and microscopic studies of a Blainville's beaked whale and dwarf sperm whale that stranded in Davao City in April and July 2014 were also undertaken (Bondoc et al., 2017), which reported severe iron deficiency in the former. ...
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Global marine mammal research is disproportionately lacking compared to terrestrial mammal research and is strongly biased toward populations in Europe, North America, New Zealand, and Australia. With high extinction risks facing marine mammals in the tropics, we sought to identify potential drivers of research effort and extinction risk evaluations for marine mammals in the Philippines as a model for tropical island nations with limited resources and research capacity. Using a bibliographic approach, we compiled all materials on marine mammal research in the Philippines from 1991 to 2020, which we categorized into eight thematic areas of research focus. We reviewed all materials based on their research focus to assess the current scientific knowledge of local marine mammal populations. Using a simple metric to calculate research effort allocation, we found that all marine mammal species in the Philippines receive inadequate research attention. Using generalized linear models, we analyzed the relationship of potential factors that drive research effort. The model with the lowest Akaike Information Criterion value suggests that frequency of marine mammal stranding incidents may influence an increase in research effort on marine mammals by providing access to biological specimens that would normally be difficult to obtain. Strandings are unfortunate events with often unclear causes, but they provide an opportunity to collect data from behaviorally cryptic animals in areas where financial constraints often hamper scientific progress. We also determined that a national Red List evaluation was predicted by increased research effort. Maximizing local research using all materials from strandings and building research capacity may be an alternative to expensive field-based methods to increase knowledge on local marine mammal populations.
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Toxoplasma gondii is a significant threat to endangered Hawaiian wildlife including birds and marine mammals. To estimate the prevalence of T. gondii in stranded cetaceans from 1997 to 2021 in Hawai‘i, we tested tissues from 37 stranded spinner dolphins Stenella longirostris and 51 stranded individuals that represented 18 other cetacean species. DNA from cetacean tissue extracts were screened using a nested polymerase chain reaction (PCR) assay targeting the Toxoplasmatinae internal transcribed spacer 1 of the nuclear ribosomal DNA. A positive result was obtained in 9 tissues examined for each of 2 spinner dolphins out of 525 tissue samples analyzed by PCR. The PCR-positive spinner dolphins had disseminated acute toxoplasmosis with necrosis, inflammation, and intralesional protozoal cysts and tachyzoites in multiple organs. Discrete positive immunostaining for T. gondii was observed in all tissues tested including the adrenal gland, brain, liver, and lung. Both positive spinner dolphins were negative for cetacean morbillivirus. The T. gondii genotyping was performed by restriction fragment length polymorphism (PCR-RFLP) based on 10 genetic markers. The PCR-RFLP analysis revealed the T. gondii belonged to PCR-RFLP-ToxoDB genotype #24, previously detected in wild pig Sus scrofa in O‘ahu, bobcats Lynx rufus from Mississippi, USA, and chickens Gallus gallus from Costa Rica and Brazil. These cases represent the first report of this genotype in aquatic mammals and the second and third reports of fatal disseminated T. gondii infection in stranded spinner dolphins from Hawai‘i. Nearshore species, like spinner dolphins, may be at increased risk of mortality from this parasite in marine coastal waterways via sewage systems, storm water drainage, and freshwater runoff.
Technical Report
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Stranding of marine mammals is complex and understanding this phenomenon requires continuous surveillance, monitoring, data collection and research. The Philippine Marine Mammal Stranding Network (PMMSN) has collected 1178 records of stranding events nationwide from 2005 to 2020. This Technical Report is a follow-up to the second Report (i.e., Aragones and Laggui 2019). As stated in the second Technical Report the consequent series of Reports will cover two-year periods only. Thus, this third Report covers the stranding dataset from 2019 to 2020. However, as in the first (Aragones et al. 2017) and second Reports, updates on the general trends for the larger data set (2005 to 2020) will also be provided. This Report showcases analyses of the stranding records from 2019 to 2020 (n=220) for trends in stranding frequency by year, region, season, monsoon, species, sex, age class, original disposition, release and rehabilitation success. The spatial coverage presented in this report was specific to regions and provinces primarily for administrative purposes. Identification of more specific or smaller spatial areas (i.e., by municipality/city) for potential stranding hotspots was assessed using Fishnet Tools (using 15 x 15 km grids). Furthermore, seasonality of stranding events was categorized according to the prevailing monsoons. The Northeast (NE) monsoon months are November to February (NDJF), Southwest (SW monsoon) monsoon months are June to September (JJAS), and Spring Inter-monsoon (Spring IM) in October (or Lull before NE monsoon) and the Winter Inter-monsoon (Winter IM) from March to May (MAM, or Lull before SW monsoon). The stranding data was also presented in the more classic seasonal context of DJF, MAM, JJA, SON. As data analytics advances, future reports will be improved further.
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The relatively high frequency of marine mammal stranding events in the Philippines provide many research opportunities. A select set of stranders (n = 21) from 2017 to 2018 were sampled for bacteriology and histopathology. Pertinent tissues and bacteria were collected from individuals representing eight cetacean species (i.e. Feresa attenuata, Kogia breviceps, Globicephala macrorhynchus, Grampus griseus, Lagenodelphis hosei, Peponocephala electra, Stenella attenuata and Stenella longirostris) and were subjected to histopathological examination and antibiotic resistance screening, respectively. The antibiotic resistance profiles of 24 bacteria (belonging to genera Escherichia, Enterobacter, Klebsiella, Proteus, and Shigella) that were isolated from four cetaceans were determined using 18 antibiotics. All 24 isolates were resistant to at least one antibiotic class, and 79.17% were classified as multiple antibiotic resistant (MAR). The MAR index values of isolates ranged from 0.06 to 0.39 with all the isolates resistant to erythromycin (100%; n = 24) and susceptible to imipenem, doripenem, ciprofloxacin, chloramphenicol, and gentamicin (100%; n = 24). The resistance profiles of these bacteria show the extent of antimicrobial resistance in the marine environment, and may inform medical management decisions during rehabilitation of stranded cetaceans. Due to inadequate gross descriptions and limited data gathered by the responders during the stranding events, the significance of histopathological lesions in association with disease diagnosis in each cetacean stranding or mortality remained inconclusive; however, these histopathological findings may be indicative or contributory to the resulting debility and stress during their strandings. The findings of the study demonstrate the challenges faced by cetacean species in the wild, such as but not limited to, biological pollution through land-sea movement of effluents, fisheries interactions, and anthropogenic activities.
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Toxoplasma gondii infections are common in humans and animals worldwide. T. gondii causes mortality in several species of marine mammals, including threatened Southern sea otters (Enhydra lutris) and endangered Hawaiian monk seals (Monachus schauinslandi). Marine mammals are now considered sentinels for environmental exposure to protozoan agents contaminating marine waters, including T. gondii oocysts. Marine mammals also serve as food for humans and can result in foodborne T. gondii infections in humans. The present review summarizes worldwide information on the prevalence of clinical and subclinical infections, epidemiology, and genetic diversity of T. gondii infecting marine mammals in the past decade. The role of genetic types of T. gondii and clinical disease is discussed.
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Background: The Pacific Islands have environmental conditions highly favourable for transmission of leptospirosis, a neglected zoonosis with highest incidence in the tropics, and Oceania in particular. Recent reports confirm the emergence and outbreaks of leptospirosis in the Pacific Islands, but the epidemiology and drivers of transmission of human and animal leptospirosis are poorly documented, especially in the more isolated and less developed islands. Methodology/principal findings: We conducted a systematic review of human and animal leptospirosis within 25 Pacific Islands (PIs) in Polynesia, Melanesia, Micronesia, as well as Easter Island and Hawaii. We performed a literature search using four international databases for articles published between January 1947 and June 2017. We further included grey literature available on the internet. We identified 148 studies describing leptospirosis epidemiology, but the number of studies varied significantly between PIs. No data were available from four PIs. Human leptospirosis has been reported from 13 PIs, with 63% of all studies conducted in Hawaii, French Polynesia and New Caledonia. Animal leptospirosis has been investigated in 19 PIs and from 14 host species, mainly pigs (18% of studies), cattle (16%) and dogs (11%). Only 13 studies provided information on both human and animal leptospirosis from the same location. Serology results were highly diverse in the region, both in humans and animals. Conclusions/significance: Our study suggests that, as in other tropical regions, leptospirosis is widespread in the PIs while showing some epidemiological heterogeneity. Data are scarce or absent from many PIs. Rodents, cattle, pigs and dogs are all likely to be important carriers, but the relative importance of each animal species in human infection needs to be clarified. Epidemiological surveys with appropriate sampling design, pathogen typing and data analysis are needed to improve our understanding of transmission patterns and to develop effective intervention strategies.
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Canine distemper virus (CDV), Leptospira interrogans, and Toxoplasma gondii are potentially lethal pathogens associated with decline in marine mammal populations. The Caspian Sea is home for the endangered Caspian seal (Pusa caspica). In the late 1990s and early 2000s, CDV caused a series of mortality events involving at least several thousand Caspian seals. To assess current infection status in Caspian seals, we surveyed for antibodies to three pathogens with potential to cause mortality in marine mammals. During 2015–2017, we tested serum samples from 36, apparently healthy, Caspian seals, accidentally caught in fishing nets in the Caspian Sea off Northern Iran, for antibodies to CDV, L. interrogans, and T. gondii, by virus neutralization, microscopic agglutination, and modified agglutination, respectively. Twelve (33%), 6 (17%), and 30 (83%) samples were positive for CDV, L. interrogans and T. gondii antibodies, respectively. The highest titers of CDV, L. interrogans, and T. gondii antibodies were 16, 400, and 50, respectively. Frequencies of antibody to these pathogens were higher in seals >1 year old compared to seals <1 year old. Two serovars of L. interrogans (Pomona and Canicola) were detected. Our results suggest a need for additional studies to clarify the impact of these pathogens on Caspian seal population decline and the improvement of management programs, including systematic screening to detect and protect the remaining population from disease outbreaks.
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Background: Leptospirosis in dogs is a disease of global importance. Early detection and appropriate therapeutic intervention are necessary to resolve infection and prevent zoonotic transmission. However, its diagnosis is hindered by nonspecific clinical signs and lack of rapid diagnostic tests of early infection. Recently, 2 rapid point-of-care tests (WITNESS Lepto [WITNESS Lepto, Zoetis LLC, Kalamazoo, MI, USA] and SNAP Lepto [SNAP Lepto, IDEXX Laboratories, Westbrook, ME, USA]) for detection of Leptospira-specific antibodies in canine sera were developed. Hypothesis: Immunoglobulin M-based WITNESS Lepto containing multiple detection antigens can detect Leptospira-specific antibodies to common leptospiral serovars earlier in the course of infection as compared to microscopic agglutination test (MAT) and SNAP Lepto. Animals: Four groups of 8 6- to 8-month-old male Beagle dogs were used. Methods: Thirty-two healthy seronegative dogs were inoculated experimentally with serovars Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona (8 dogs/serovar). Acute-phase sera were collected at regular intervals and monitored for Leptospira-specific antibodies by WITNESS Lepto, MAT, and SNAP Lepto. Results: Seroconversion was detected in all dogs by day 10 by WITNESS Lepto and in 30 of 32 dogs by day 14 by MAT. The SNAP Lepto test detected seroconversion in 3 dogs during the 2 weeks postchallenge. Conclusions: Immunoglobulin M-based WITNESS Lepto detected immune responses specific to multiple leptospiral serovars early in the course of infection and identified seroconversion in all animals earlier than did the gold standard MAT. The SNAP Lepto test displayed considerably lower and inconsistent performance during the study period. At the point-of-care, WITNESS Lepto should be the test of choice for rapid and reliable screening of acutely ill dogs suspected to have leptospirosis.
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Leptospirosis, caused by Leptospira interrogans, is a highly infectious emerging water borne zoonosis of global significance. It is an enigmatic life threatening disease, which results into high morbidity and mortality, particularly in poor resource nations. Disease is endemic in many countries of the world including India. Currently, over one million people are affected with leptospirosis worldwide annually. Leptospirosis presents an occupational hazard of the persons who have direct or indirect contact with the urine of infected animals. The common mode of transmission of the disease is exposure to and ingestion of urine contaminated water. Clinical signs in humans may vary from asymptomatic to severe stage with a range of non-specific symptoms. Rodents are the chief reservoir of Leptospira, and organisms are excreted in urine, thus contaminating the environment including water. Disease is endemic in tropical regions of the world with maximum cases in young male adults during the rainy season. Pollution of city water supply may result in outbreak of disease. Severe epidemics of leptospirosis are related to water recreational activities. The diagnosis of disease is confirmed by detection of serum antibodies against Leptospira and also by isolation of the pathogen from clinical specimens such as blood, cerebrospinal fluid and urine. Early treatment with antibacterial antibiotics may shorten the duration of fever and reduce the severity of the disease. As leptospirosis is attributed to physical contact with contaminated water supplies, environmental detection is important in the development of adequate control measures. There is a need to develop an effective surveillance system to monitor the trends of disease. Sincere attempts should be made to estimate the annual burden of cases and deaths due to leptospirosis. Additional studies on the epidemiology, diagnosis, chemotherapy and vaccines are required to control this life threatening zoonosis.
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Background Studies suggest that free-ranging bottlenose dolphins exhibit a suppressed immune system because of exposure to contaminants or microorganisms. However, due to a lack of commercially available antibodies specific to marine mammal immune cell surface markers, the research has been indecisive. The purpose of this study was to identify cross-reactive terrestrial-specific antibodies in order to assess the changes in the immune cell populations of dolphins under human care and free-ranging dolphins. The blood and PBMC fraction of blood samples from human care and free-ranging dolphins were characterized by H&E staining of cytospin slides and flow cytometry using a panel of terrestrial-specific antibodies. ResultsIn this study, we show that out of 65 terrestrial-specific antibodies tested, 11 were cross-reactive and identified dolphin immune cell populations within their peripheral blood. Using these antibodies, we found significant differences in the absolute number of cells expressing specific markers within their lymphocyte and monocyte fractions. Interestingly, the peripheral blood mononuclear cell profile of free-ranging dolphins retained an additional population of cells that divided them into two groups showing a low (<27%) or high (>56%) percentage of smaller cells resembling granulocytes. Conclusions We found that the cross-reactive antibodies not only identified specific changes in the immune cells of free-ranging dolphins, but also opened the possibility to investigate the causal relationship between immunosuppression and mortality seen in free-ranging dolphins.
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Background: Leptospirosis is a worldwide zoonotic bacterial disease caused by infection with leptospires. Leptospirosis in humans and livestock is an endemic and epidemic disease in Thailand. Livestock may act as reservoirs for leptospires and source for human infection. Methodology/principal findings: Data on leptospirosis infection in humans and livestock (Buffaloes, Cattle, and Pigs) species during 2010 to 2015 were analyzed. Serum samples were examined using Microscopic Agglutination Test (MAT) to identify antibodies against Leptospira serovars using a cut-off titer ≥ 1:100. The seroprevalence was 23.7% in humans, 24.8% in buffaloes, 28.1% in cattle, and 11.3% in pigs. Region specific prevalence among humans and livestock was found in a wide range. The most predominant serovars were Shermani, followed by Bratislava, Panama, and Sejroe in human, Shermani, Ranarum, and Tarassovi in buffaloes, and Shermani and Ranarum in cattle and pigs. Equally highest MAT titers against multiple serovars per one sample were found mainly in buffaloes and cattle showing equally titers against Ranarum and Shermani. The correlations of distribution of serovars across Thailand's regions were found to be similar in pattern for cattle but not for buffaloes. In humans, the serovar distribution in the south differed from other regions. By logistic regression, the results indicated that livestock is more susceptible to infection by serovar Shermani when compared to humans. Conclusions/significance: This study gives a detailed picture of the predominance of Leptospira serovars in relation to region, humans and typical livestock. The broad spatial distribution of seroprevalence was analyzed across and within species as well as regions in Thailand. Our finding may guide public health policy makers to implement appropriate control measures and help to reduce the impact of leptospirosis in Thailand.
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Marine mammals play crucial ecological roles in the oceans, but little is known about their microbiotas. Here we study the bacterial communities in 337 samples from 5 body sites in 48 healthy dolphins and 18 healthy sea lions, as well as those of adjacent seawater and other hosts. The bacterial taxonomic compositions are distinct from those of other mammals, dietary fish and seawater, are highly diverse and vary according to body site and host species. Dolphins harbour 30 bacterial phyla, with 25 of them in the mouth, several abundant but poorly characterized Tenericutes species in gastric fluid and a surprisingly paucity of Bacteroidetes in distal gut. About 70% of near-full length bacterial 16S ribosomal RNA sequences from dolphins are unique. Host habitat, diet and phylogeny all contribute to variation in marine mammal distal gut microbiota composition. Our findings help elucidate the factors structuring marine mammal microbiotas and may enhance monitoring of marine mammal health.
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Toxoplasma gondii has been described in several marine mammals around the world including numerous species of cetaceans, yet infection and transmission mechanisms in the marine environment are not clearly defined. The Israel Marine Mammal Research and Assistance Center has been collating a database of all marine mammal stranding events along the country's national coastlines since 1993. In this study, we describe the molecular detection and characterisation of T. gondii in three common bottlenose dolphins (Tursiops truncatus) including one case of coinfection with herpesvirus. The animals were found stranded on the Mediterranean coast of Israel in May and November 2013. In one of the three cases, the dolphin was found alive and admitted to intensive care. To our knowledge, this is the first report of T. gondii infection of marine mammals in the Eastern Mediterranean Sea. As this parasite acts as an indicator for marine pollution and marine mammal health, we believe these findings add important information regarding the state of the environment in this region.
Book
Satellite observations and computing technology have advanced our understanding of the monsoon climate enormously in the last two decades. The author provides an update of the knowledge gained over this period, presenting the modern morphology and the physical principles of monsoon climate variation on all time scales ranging from intraseasonal to tectonic time scales. He brings new ideas that can be expected to markedly improve the prediction of monsoon climate, and includes contributions by experts who expand our understanding of the monsoon environment by their study of paleoclimate records, who present evidence of human influences on monsoon climate, and who describe the links of the monsoon to the economy and to human health. This is a comprehensive interdisciplinary text book summarizing new knowledge of Asian monsoon climate variability, dynamics, modeling, and prediction from intraseasonal to geological time scales, and human influence and its links to environmetal/economic issues.
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Canine and Feline Infectious Diseases is a practical, up-to-date resource covering the most important and cutting-edge advances in the field. Presented by a seasoned educator in a concise, highly visual format, this innovative guide keeps you current with the latest advances in this ever-changing field. 80 case studies illustrate the clinical relevance of the major infectious disease chapters.