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Assessing Brucella cetl Infections in Oregon and Washington Dolphins that Stranded with Histopathological Lesions Resembling Neurobrucellosis 2006-2014
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2015
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George, Tabitha A
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Environmental Studies
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Assessing Brucella ceti Infections in Oregon and Washington Dolphins
that Stranded with Histopathological Lesions Resembling
Neurobrucellosis, 2006-2014
By
Tabitha George
A Thesis
Submitted in partial fulfillment
of the requirements for the degree
Master of Environmental Studies
The Evergreen State College
June 2015
©2015 by Tabitha George. All rights reserved.
This Thesis for the Master of Environmental Studies Degree
by
Tabitha George
has been approved for
The Evergreen State College
by
________________________
Kevin Francis, Ph. D.
Member of the Faculty
________________________
Date
Abstract
Assessing Brucella ceti Infections in Oregon and Washington Dolphins that Stranded
with Histopathological Lesions Resembling Neurobrucellosis, 2006-2014
Tabitha George
This thesis documents the presence of Brucella ceti in the Pacific white-sided dolphin
(Lagenorhynchus obliquidens), striped dolphins (Stenella coeruleoalba), and shortbeaked common dolphins (Delphinus delphis) that stranded with histopathological
lesions resembling neurobrucellosis in Oregon and Washington between 2006 and 2014.
These Brucella strandings occurred in very specific years (2006, 2012, and 2014) and
seasons (winter and fall), which may have been driven by an increase in the number of
overall strandings, or an environmental influence, altering their susceptibility to the
disease. Further studies on the linkages between climate and disease will provide a better
understanding on factors that might drive the emergence of seasonal or interannual
variations seen within Brucella stranded individuals. Out of fifteen individuals that had
histopathological lesions resembling neurobrucellosis upon histology, fourteen were sent
for further Brucella tests and ten (71%) came back positive. The positive individuals in
this study were confirmed by culture and serology. However, there were a high number
of false negative PCR and IHC results, making me believe that 71% is an underestimate
of the actual percentage of Brucella positive individuals. Demographically speaking, the
striped dolphin (n=6) was the most common species to be infected with Brucella ceti,
followed by the short-beaked common dolphin (n=3). This study is also the first, to my
knowledge, to document Brucella in a Pacific white-sided dolphin. The observed
predilections at this time include male striped dolphins, subadult individuals of all
species, and short-beaked common dolphins that strand in Washington. These observed
predilections are based off of a small sample size and may be subject to change if further
tests are performed on prior individuals that tested negative for Brucella and from future
strandings.
Ultimately, individuals with histopathological lesions suspicious of neurobrucellosis most
often came back positive for Brucella ceti on further tests.
Table of Contents
Chapter One: Introduction
Background…….………………………………………………….………………1
Neurobrucellosis………………….………………………………..………….1
Significance of Research, Research Questions, and Hypotheses…………………3
Chapter Two: Literature Review
Brucella spp……...………………………………………………………………..7
Marine Brucella…………………………………………………………………...8
Brucella ceti…………………………………………………………………...9
Terrestrial Crossover……………………………………………………..10
Brucella ceti Tests……………………………………………………………11
Culture……………………………………………………………………11
Serology………………………………………………………………….12
Polymerase Chain Reaction (PCR)/Molecular Methods………….……..13
Immunohistochemistry (IHC)……………………………………………13
Chapter Three: Methods
Study Area……………………………………………………………….………15
Data Collection…………………………………………………………….…….15
Level A Data………………………………………………………….….…..17
Histology and Lab Results………………………………….………………..17
Brucella Tests……………………………………………………………18
Data Analyses…………………………………………………………………....21
Stranding History and Demographics………………………………………..21
Analyses of Neurobrucellosis Suspicious Individuals……………………….22
iv
Chapter Four: Results
Overall Stranding History……………………………………………...…...……24
Stranding Numbers…………………………………………………………..24
Stranding Demographics……………………………………………………..25
Stranding Location……………………………………………………….25
Age Class………………………………………………………………...26
Sex………………………………………………………………………..26
Stranding Seasons…...……………………………………………………….27
Nervous System Disorders……………………………………………………….29
General Findings……………………………………………………………..29
Brucella ceti Analyses……………………………………………………….30
Stranding Location……………………………………………………….32
Age Class………………………………………………………………...34
Sex……………………………………………………………………….34
Chapter Five: Discussion
Overall Stranding History…...…………………………………………………...36
Stranding Numbers…………………………………………………………..36
Strandings by Species……………………………………………………36
Strandings by Season…………………………………………………….37
Nervous System Disorders……………………………………………………….38
General Findings……………………………………………………………..38
Brucella ceti Analyses……………………………………………………….40
General Findings………………………………………………...……….40
Brucella ceti Tests………………………………………………...……...40
Species………………………………………………………………..….41
v
Location………………………………………………………………….42
Age Class………………………………………………………………...43
Sex………………………………………………………………………..44
Chapter Six: Conclusion
Overall Findings…………………………………….……………………………45
Future Studies and Suggestions………………………………………………….46
Unknown Cases……………………………………………………………...46
Expected Sea Temperature Changes…………………………………………47
Bibliography…………………………………………………………………………….49
Appendix A: Level A Data Sheet………………………………………………………55
Appendix B: Histopathology Results………………………………………………….57
vi
List of Figures
Figure 1: Host Reservoirs for Brucella Species…………………………………………...8
Figure 2: Area Covered by the Northwest Region Marine Mammal Stranding
Network……………………………………...………………………………..15
Figure 3: Total Number of Strandings (1975-2014)……………………………………..25
Figure 4: Reported Dolphin Strandings by Species (1975-2005) and (2006-2014)…......25
Figure 5: Strandings by Season and Species (2006-2014)……………………….………29
Figure 6: Stranding Location of Positive Brucella Cases…………………………....…..33
vii
List of Tables
Table 1: Overall Stranding Demographics (2006-2014)………………………………...27
Table 2: Overview of Stranded Individuals Suspicious of Neurobrucellosis….…...........31
Table 3: Identification Key………………………………………………………………32
Table 4: Stranding Location Comparisons of the Striped Dolphin (2006-2014)…...........33
Table 5: Stranding Location Comparisons of the Short-Beaked Common
Dolphin (2006-2014)…………………………………………………………….33
Table 6: Age Class Comparisons of the Striped Dolphin (2006-2014)...…….……….....34
Table 7: Age Class Comparisons of the Short-Beaked Common Dolphin (2006-2014)...34
Table 8: Sex Comparisons of the Striped Dolphin (2006-2014)………………………...34
Table 9: Sex Comparison of the Short-Beaked Common Dolphin (2006-2014)………..35
viii
Acknowledgements
First off, I would like to thank my thesis reader, Dr. Kevin Francis. Thank you for
your guidance, encouragement, and most of all, your patience throughout this entire
process. You have taught me so much these past two years and I am beyond grateful to
have had you as my thesis mentor.
Second, I would like to thank Dyanna Lambourn with Washington Department of
Fish and Wildlife: Marine Mammal Investigations. Thank you for all the input and advice
on this thesis and for the experiences I‟ve gained throughout my internship. Also, thank
you to Jessie Huggins (Cascadia Research Collective), Jim Rice (Oregon State University
Marine Mammal Institute) and Dr. Debbie Duffield (Portland State University) for
providing me with data, clarifying any questions that I had, and giving valuable feedback
on all my drafts.
Lastly, I would like to thank my family. To my husband, I greatly appreciate the
love and support you give to me every single day. I can honestly say that I would not
have kept my sanity without your help. Also, thank you to my dad, mom, and brother.
Your encouragement and love have gotten me through every challenge I have faced, and
I am elated to complete yet another one with each of you by my side.
ix
______________________________________________________
Chapter One
-Introduction-
_____________________________________________________
Background
Highly contagious infections from the bacterial genus, Brucella spp., are found in
both terrestrial and marine vertebrates and are among the most prevalent worldwide
zoonotic diseases (Foster et al., 2009 and Sohn et al., 2003). Brucella was first isolated
in marine mammals in 1994, but is now endemic in marine mammal populations
worldwide (Sidor et al., 2013). There are two species of Brucella that are specific to
marine mammals, which include Brucella pinnipedialis (i.e. seals) and Brucella ceti (i.e.
cetaceans) (Cloeckaert et al., 2001). Brucella ceti, which this thesis focuses on, often
presents with clinical manifestations that include, but are not limited to, abortion, orchitis,
abscesses, muscoskeletal disorders, and neurological disorders (Cloeckaert et al., 2001;
Maquart et al., 2009; Sidor et al., 2013; and Thakur et al., 2012). For this particular study,
an emphasis was placed on dolphins that stranded with neurological disorders over other
manifestations.
Neurobrucellosis
Neurobrucellosis occurs when there are Brucella caused complications within the
central and/or peripheral nervous system, often presenting as meningitis or
meningoencephalitis (Tuncel et al., 2008). However, inflammatory peripheral
neuritis/radiculitis, inflammatory demyelinative processes, papilledema, and
1
meningomyelitis are also manifestations that have been documented in neurobrucellosis
individuals (Tuncel et al., 2008). These specific pathological changes have been observed
in humans and a small number of dolphin species, but are seldom, if at all, recorded in
terrestrial hosts such as cattle, pigs, goats, and sheep (Gonzàlez-Barrientos et al., 2010).
Although neurobrucellosis is an infrequent complication in humans (roughly 510% of Brucella cases), it is recurrently observed in select dolphin species with a
seemingly large predilection towards the striped dolphin (Alba et al., 2013; Ceran et al.,
2011; Foster et al., 2002; Gonzàlez et al., 2002; Hernàndez-Mora et al., 2008; and Tuncel
et al., 2008). Brucella ceti has been frequently isolated from the central nervous system
of individuals that macroscopically presented with hyperemia of the meninges and brain
(Hernández-Mora et al., 2013). Histologically, these animals had nonsuppurative
meningoencephalomyelitis, meningoencephalitis, meningomyelitis, or meningitis (Alba
et al., 2013 and Hernández-Mora et al., 2013).
Literature detailing neurobrucellosis within striped dolphins is quite common.
However, documentation within other dolphin species is scarce. Other than the striped
dolphin, I personally only came across two articles that discussed neurological
pathologies in conjunction with a Brucella infection. In 2013, Davison et al. reported
meningoencephalitis, along with muscoskeletal pathologies, in a short-beaked common
dolphin that was associated with Brucella ceti. In 2009, Hernàndez-Mora et al. mentioned
three bottlenose dolphins that tested positive for Brucella and had “neurological
symptoms”. However, the authors did not specify whether these “neurological
symptoms” within the bottlenose dolphins were due to neurobrucellosis explicitly.
2
It is important to note that nervous system disorders can also be caused by other
bacterial infections (e.g. staphylococcal infections), viruses (e.g. herpesvirus and
morbillivirus), parasites, and protozoa (e.g. Toxoplasma gondii and Sarcocystis neurona).
For example, viral infections, such as herpesviruses and morbilliviruses, are responsible
for a vast amount of neurological diseases within the striped dolphin (Gonzàlez et al.,
2002). Individuals can also be co-infected with multiple infectious agents that can
adversely affect the nervous system, which might make it difficult to determine the
causative agent of the neural inflammation. For example, encephalitis caused by fungal
origins and Toxoplasma spp. have been documented as secondary complications of
morbillivirus within the striped dolphin (Gonzàlez et al., 2002). Inflammatory lesions
caused by Brucella ceti have been noted to be strikingly different from encephalitis
caused by other pathogens, however (Gonzàlez-Barrientos et al., 2010). According to
McLean et al. (1992), meningeal infection seems to be the common pathogenic thread for
Brucella, despite the difficulty to detect Brucella organisms directly in infected tissues
(Seidel et al., 2003).
________________________________________________________________________
Significance of Research, Research Questions, and Hypotheses
It is crucial to study communicable diseases in dolphins, such as Brucella ceti,
since they have one of the most highly social groups among mammals and are an
effective sentinel for emerging and reemerging infectious diseases (Bossart, 2011 and
Gaspari et al., 2007). Although there has been great insight on disease exposure and
prevalence in potential vulnerable marine mammals for Brucella, information on
transmission, pathogenicity, and susceptibility of individuals are still scarce (Sidor et al.,
3
2013). Since dolphins have large areas of movement that are not dependent on
geographical boundaries, they can introduce Brucella to a wide range of new hosts and
areas (Thakur et al., 2012). Also, due to the zoonotic potential of Brucella ceti, there are
health risks to humans, domestic pets, and wild animals that may come in contact with a
stranded individual. Therefore, more research needs to be conducted to prevent further
terrestrial crossovers of this marine Brucella species.
Worldwide monitoring and research of marine Brucella is also necessary to better
understand this disease (Hernàndez-Mora et al., 2013). There have been multiple studies
conducted on Brucella ceti in an array of regions including, but not limited to, the UK,
Costa Rica, the Mediterranean, and the U.S. East Coast (Alba et al., 2013; Davison et al.,
2013; Gonzàlez et al., 2002; Gonzàlez-Barrientos et al., 2010; Hernàndez-Mora et al.,
2008; Isidoro-Ayza et al., 2014; and Wu et al., 2014). Besides the very first study that
detailed Brucella in an aborted bottlenose dolphin fetus whose mother was held in
captivity in San Diego, California, I personally did not find any other studies that
discussed Brucella in dolphins that stranded along the U.S. West Coast (Ewalt et al.,
1994). For that reason, it is vital to contribute to the existing literature by looking at the
occurrence of Brucella in dolphins that stranded along Oregon and Washington, which
will also be referred to as the Pacific Northwest (PNW).
Although a variety of dolphin species have stranded in Oregon and Washington,
this thesis solely focused on the Pacific white-sided dolphins (PWSD) (Lagenorhynchus
obliquidens), striped dolphins (Stenella coeruleoalba), short-beaked common dolphins
(Delphinus delphis), long-beaked common dolphins (Delphinus capensis), and common
bottlenose dolphins (Tursiops truncatus) that may have stranded with histopathological
4
manifestations resembling neurobrucellosis. These histopathological manifestations
would more specifically include nonsuppurative meningoencephalomyelitis,
meningoencephalitis, meningomyelitis, or meningitis (Alba et al., 2013 and HernándezMora et al., 2013). The latter four species (i.e. striped, short-beaked common, longbeaked common, and bottlenose) are commonly found in warmer waters (e.g. California
waters) and are considered to be unusual sightings in Oregon and Washington (Allen et
al., 2011).
According to the studies of González et al. (2002), Hernàndez-Mora et al. (2008),
and Xavier et al. (2009), dolphins that consistently presented with these meningeal
disorders also ended up presenting with neurological ailments and tested positive for
Brucella, more specifically on serology and immunohistochemistry (IHC). HernàndezMora et al. (2009) also noted a correlation between individuals with neurological
symptoms and having high titers of antibodies against Brucella antigens. However, along
with positive serology and IHC, positive results have also been achieved via culture and
polymerase chain reaction (PCR) (Sidor et al., 2013).
These findings led to the development of the research questions that this thesis seeks to
address:
1) What has the stranding history looked like among these five species in
Washington and Oregon from 1975-2014? From 2006-2014?
2) Out of the individuals in Oregon and Washington that stranded with
histopathological lesions resembling neurobrucellosis, how many
subsequently tested positive for Brucella ceti?
5
3) Out of the Brucella ceti positive individuals, were there any demographic
predilections observed (i.e. predilection towards species, age class,
stranding location, or sex)?
4) Which tests were the most commonly used and/or most successful in
detecting Brucella ceti within this study?
Since information on this topic is scarce, it is important to note that this thesis is
exploratory and attempts to provide insight on this disease specific to individuals that
stranded with neurobrucellosis-like histopathological lesions in the Pacific Northwest.
The goal of this thesis is to provide direction for future studies as more data is collected
on impending strandings, and the conclusions drawn are based on my attempts, as a
graduate student, to provide insight on Brucella ceti in Oregon and Washington. Further
Brucella tests may also be conducted on multiple cases outlined in this study, so results
may be subject to change.
Due to the exploratory nature of this thesis, not every research question has a
hypothesis. This is especially true since I used previously collected data and was able to
see some of the demographics before beginning my analyses. Based on prior studies (e.g.
Gonzàlez et al., 2002 and Hernàndez-Mora et al., 2008), I would suspect predilections
towards the striped dolphin and subadult individuals, but may see other predilections,
such as stranding location or sex, when the data is further analyzed.
6
_____________________________________________________
Chapter Two
-Literature Review-
_____________________________________________________
Brucella spp.
Brucella spp. is a genus of intracellular, gram-negative bacteria that can infect
both terrestrial and marine vertebrates worldwide (Sohn et al., 2003). It does not multiply
within the environment, but is usually transmitted directly from host to host (Xavier et
al., 2009).
Brucella has species-specific primary reservoirs with clinical features that vary
based on the host species (Sohn et al., 2003 and Xavier et al., 2009). There were
traditionally six nomen species of Brucella that included: 1) Brucella abortus; 2)
Brucella melitensis; 3) Brucella suis; 4) Brucella canis; 5) Brucella ovis; and 6) Brucella
neotomae (Cloeckaert et al., 2001 and Young, n.d.). However, two more nomen species
have been recently added that are specific to marine mammals: 1) Brucella pinnipedialis
and 2) Brucella ceti (Cloeckaert et al., 2001) (Figure 1). DNA-DNA hybridization and
other phenotypic characteristics showed that although these two marine mammal species
were a part of the genus Brucella (more than 77% DNA relatedness), there were still
distinctive characteristics that isolated them from the other terrestrial species (Cloeckaert
et al., 2001; Maquart et al., 2009 and Thakur et al., 2012). All species of Brucella have
proven to have zoonotic potential for humans except B. ovis and B.neotomae (Xavier et
al., 2009).
7
Figure 1: Host Reservoirs for Brucella Species (https://online.epocrates.com/u/2924911/Brucellosis)
________________________________________________________________________
Marine Brucella
Brucella was first isolated in marine mammals in 1994, but now appears to be
endemic in marine mammal populations worldwide (Ewalt et al., 1994 and Sidor et al.,
2013). The name Brucella maris was originally suggested for all marine mammal species
with three biovars (Cloeckaert et al., 2001). Biovar 1 would have included seal and otter
isolates, Biovar 2 would have included cetacean isolates, and Biovar 3 would have
included a particular isolate from a California bottlenose dolphin that had a contrasting
dominant antigen from the previous two (Cloeckart et al., 2001). Ultimately, Biovar 3
ended up representing another serotype rather than a biovar, and Biovar 1 and 2 were
distinct enough to be classified as their own species, which ended up being Brucella
pinnipedialis and Brucella ceti (Cloeckaert et al., 2001). Proposals of having three nomen
species of marine Brucella have also been made, which would include Brucella phocae
(seals), Brucella delphini (dolphins), and Brucella phoecoenae (porpoises) (Groussaud et
al., 2007). However, as of this study, this is not absolute.
8
Brucella ceti
Brucella ceti has been described in an array of species within the Delphinidae
family including, but not limited to, the bottlenose dolphin (Tursiops truncatus), Atlantic
white-sided dolphin (Lagenorhynchus acutus), short-beaked common dolphin (Delphinus
delphis), long-beaked common dolphin (Delphinus capensis), dusky dolphin
(Lagenorhynchus obscurus), striped dolphin (Stenella coeruleoalba), killer whale
(Orcinus orca), and pilot whale (Globic ephala) (Gonzàlez et al., 2002). Positive
isolations have been derived from reproductive organs of both sexes, brain, spinal cord,
joints, lungs, spleen, liver, cerebrospinal fluid (CSF), fetal tissues, mammary glands,
milk, multiple lymph nodes, and more (Thakur et al., 2012). Although a lot of isolates
have come from symptomatic animals, Brucella ceti has also been isolated from normal
tissues and asymptomatic animals, indicating that this bacterium can be an opportunistic
invader, or even an unlikely cause of death (Thakur et al., 2012). Besides the striped
dolphin, it is believed that there are low proportions of other cetacean species that show
Brucella associated clinicopathological signs (Isidoro-Ayza et al., 2014). That would
mean most infected animals remain Brucella carriers and shedders due to their ability to
overcome the clinical disease (Isidoro-Ayza et al., 2014).
Within Brucella ceti there are three documented sequence types (ST) or
subgroups: ST23, ST26, and ST27 (Whatmore et al., 2007 and Wu et al., 2014). ST23 is
predominantly found in porpoises, ST26 is predominantly found in striped and common
dolphins, and ST27 was documented in bottlenose dolphins and humans (Alba et al.,
2013 and Whatmore et al., 2007). This is suggestive that ST27 has a higher zoonotic
potential for human infection than the other sequence types described (Wu et al., 2014).
9
-Terrestrial CrossoverBrucella isolates have the potential to infect human and non-human terrestrial
animals (Xavier et al., 2009). Marine Brucella has been induced in cattle, sheep, and
piglets through inoculations, further demonstrating that terrestrial crossovers are possible
(Rhyan et al., 2001 and Thakur et al., 2012).
According to Goodwin et al. (2012), there are two drivers of zoonotic disease
transmission into human populations: 1) occurrence of the disease in animals which may
change due to population dynamics of hosts or vectors and alteration of habitats, and 2)
variations in composition or behavior of human population, altering their susceptibility to
the disease. The latter is more of a concern for transmission of marine Brucella into
human populations due to the desire of many to live by and/or visit the beach, the
curiosity to see or touch a stranded marine mammal, and the culture of some to consume
marine mammal meat. No system of inspection of consumed meats and organs have been
established, despite how frequently specific countries may eat this meat (HernándezMora et al., 2013). It is also common for people to pick up skulls, teeth, and other parts of
the skeleton of a stranded marine mammal as a trophy or souvenir. This is also dangerous
since these skeletal parts may serve as fomites for transmission (Hernández-Mora et al.,
2013).
So far, there have been four human cases that were described to have marine
Brucella isolates. This included one marine lab researcher and three other people who
acquired the infection with no known exposure to any marine mammals (Sohn et al.,
2003). Two of the individuals that acquired the infection without known marine mammal
10
exposure presented with neurological signs and emigrated from Peru, where they
frequently ate raw shellfish and unpasteurized cheese (Sohn et al., 2003). Due to the
extensive Peruvian coastlines, Brucella ceti could have been transmitted to domestic
animals and wildlife that resided nearby (Sohn et al., 2003). The third person that
acquired the infection without known marine mammal exposure developed spinal
osteomyelitis and was a fisherman from New Zealand who regularly handled uncooked
fish bait and raw fish (McDonald et al., 2006 and Thakur et al., 2012). The laboratory
acquired case was determined to be ST23, while the remainder three cases were identified
as ST27 (Whatmore et al., 2008 and Wu et al., 2014).
Brucella ceti Tests
There are a variety of tests used to diagnose a Brucella ceti infection. The most
common tests I have come across throughout literature review included culture, serology,
polymerase chain reaction (PCR), and immunohistochemistry (IHC). These were also the
tests used in this particular study.
-CultureAccording to Thakur et al. (2012), the majority of the culture isolations are “done
on Farrell‟s medium, followed by Columbia sheep blood agar, Brucella agar with
Brucella selective supplement and 1.4% crystal violet and brain heart infusion agar with
5g of yeast abstract” (p.906). Farrell‟s medium is the most highly used medium
worldwide since it inhibits the growth of most contaminants (Vicente et al., 2014).
Cetacean isolates normally are visible within four days of inoculation and can grow well
without increased CO2 (Thakur et al., 2012). It is recommended that cultures be incubated
11
in 10% CO2 at 37 °C (Foster et al., 2002 and Thakur et al., 2012). According to Wu et al.
(2014), microbiologic culture is considered the “gold standard” for a definitive Brucella
diagnosis. However, culturing can take up to two weeks for a definitive diagnosis, has
low sensitivity, and is more hazardous to laboratory personnel (Wu et al., 2014). Poor
postmortem carcasses and prolonged storage of tissues may also prevent successful
isolations of Brucella (Sidor et al., 2013).
-SerologyAlthough there are a variety of serological tests used to detect Brucella antibodies
and agglutinins, each has its advantages and disadvantages when it comes to specificity
or sensitivity (Thakur et al., 2012). Examples of commonly used serological tests include,
but are not limited to, the enzyme-linked immunosorbent assays (ELISA), Rivanol,
Brucella microagglutination test (BMAT), and Fluorescent Polarization Assays (FPA).
Once again, this is not an exhaustive list.
Although serology can support evidence to Brucella exposure (i.e. presence of
antibodies to the Brucella antigen), a major downfall is the inability to differentiate
between a current or prior infection (Krucik, 2012). Current infections, or active
infections, are based on titer levels, so serial blood draws will need to be conducted to see
if the levels are rising, falling, or staying the same (Dyanna Lambourn, personal
statement and Liu, 2014). Unfortunately, serial blood draws are very difficult to obtain in
wild animals. Serological tests also lack validity due to the need for significant numbers
of serum samples from positive infections and negative controls (Hernández-Mora et al.,
2009). Brucella cells‟ immunodominant antigen is the smooth lipopolysaccharide (S-
12
LPS) (Thakur et al., 2012). Since other gram-negative bacterial species can also have
smooth lipopolysaccharides, antibodies can cross-react, leading to false positives or
misdiagnoses (Thakur et al., 2012). Along with false positives, false negatives can also
occur in serology tests. For example, false negatives can occur on ELISA tests due to the
presence of small amounts of agglutinating antibodies that escaped detection (HernàndezMora et al., 2009).
-Polymerase Chain Reaction (PCR)/Molecular MethodsPolymerase Chain Reaction, or PCR, can detect and identify Brucella at the
genus, species, and biovar level. It is considered to be rapid and simple, requires little
manual labor, and is reliable as long as contamination is avoided (Bricker, 2002). PCR
assays can give immediate results but require more extensive sample preparation in order
to remove PCR inhibiting components (Bricker, 2002). Also, additional data is needed
about what is the best choice specimen and how long DNA can be detected over the
course of an infection (Bricker, 2002). Since cell numbers of Brucella in tissues are very
low, higher sensitive assays are needed to detect Brucella within marine mammals
(Bricker, 2002 and Wu et al., 2014).
-Immunohistochemistry (IHC)Immunohistochemistry (IHC) is considered a useful tool at diagnosing infectious
diseases in tissue samples, more commonly formalin-fixed tissue samples. According to
Eyzaguirre and Haque (2008), immunohistochemistry can identify microorganisms that
are present in low numbers, stain poorly, are difficult to grow, are not able to be cultured,
and/or have atypical morphology. However, similar to serology, cross-reactivity can
13
occur since there is widespread occurrence of common antigens among bacteria
(Eyzaguirre and Haque, 2008). Also, it has been recognized that IHC has lower
sensitivity in identifying Brucella antigens in tissues compared to serology (GonzàlezBarrientos et al., 2010).
14
_____________________________________________________
Chapter Three
-Methods-
_____________________________________________________
Study Area
This study looked specifically at dolphin strandings that occurred throughout
Oregon and Washington. These areas included nearshore waters and shoreline of Oregon
and Washington north of 42° N and south of 49°N, also including the inland waters of
Washington (Norman et al., 2004).
Figure 2: Area Covered by the Northwest Region Marine Mammal Stranding Network (Norman et al., 2004)
_______________________________________________________________________
Data Collection
My data was collected from the Northwest Region Marine Mammal Stranding
Network and from stranding coordinators in Washington and Oregon. The Northwest
15
Region Marine Mammal Stranding Network was formed in the early 1980s and is
comprised of volunteers, state and federal wildlife and fisheries agencies, veterinary
clinics, enforcement agencies, and other professionals (Norman et al., 2004). Stranding
network activities are coordinated by the National Marine Fisheries Services, Marine
Mammal Health and Stranding Response Program based in Seattle, Washington (Norman
et al., 2004). For this study, stranding coordinators included, but was not limited to, Jessie
Huggins (Washington; Cascadia Research Collective), Dyanna Lambourn (Washington
Department of Fish and Wildlife: Marine Mammal Investigations), Jim Rice (Oregon
State University Marine Mammal Institute), and Dr. Debbie Duffield (Oregon; Portland
State University).
The data received included Washington and Oregon Level A records for the
specific dolphin species analyzed. Histology reports and laboratory results were also
obtained from the individuals that stranded with histopathological lesions resembling
neurobrucellosis. Affiliated laboratories that performed histology on the tissue samples
are discussed in further detail below. I did not receive histology reports on individuals
that did not have neurobrucellosis-like lesions upon histology. I chose to look at the
striped, short-beaked common, long-beaked common, and bottlenose dolphins since they
are the most common dolphin species, according to literature review, to be infected with
Brucella ceti that have also been documented to strand within the study area. An initial
review of the data also revealed two PWSDs that stranded with histopathological lesions
resembling neurobrucellosis. I decided to add the PWSD to my analyses due to this
finding, as well as their prominent appearance in the Pacific Northwest (Allen et al.,
2011).
16
Level A Data
Level A data, which is collected on marine mammal stranding responses,
includes variables such as stranding date, stranding location, body measurements, body
and carcass conditions, age class, sex, external injuries, etc. (Appendix A). The amount
of information taken is dependent on the status of the individual (live or dead at response)
as well as the level of decomposition and scavenging. The Level A data I received
included individuals that had full examinations as well as non-examined individuals that
only had photographs taken. Although there were an array of demographics and variables
I could have analyzed, I looked specifically at the species, stranding date (year and
month), stranding season, stranding location (Washington or Oregon), sex, and age class,
since those were the most common categories to be assessed by other studies as well.
Histology and Lab Results
Along with Level A data, I also received histology reports and lab results for the
dolphins that had histopathological lesions resembling neurobrucellosis from 2006 to
2014. To reiterate, this included individuals that had nonsuppurative meningitis,
meningoencephalitis, meningomyelitis, or meningoencephalomyelitis upon histology.
Once again, if a dolphin did not have these specific manifestations, I did not receive their
histology reports, only their Level A data. It is important to note that 2006 was the first
year that histology and lab results were available for this study. It does not mean 2006
was the first year an individual stranded with histopathological manifestations resembling
neurobrucellosis within the study area. In fact, in 2006 the Oregon Marine Mammal
Stranding Network began running and started having histology performed on a regular
17
basis. Prior to 2006, Oregon dolphins were generally not examined histologically at all
(Jim Rice, personal communication).
-Brucella TestsAlthough each stranding examiner may perform necropsies in a slightly different
manner, they generally follow the protocols outlined by Pugliares et al. (2007).
Necropsies include an extensive external and internal exam, which are documented and
photographed. Complete necropsies are performed on carcass conditions that are
relatively fresh with minimal scavenging. Decomposition codes, which can be found on
the Level A sheet attached, are described as follows: 1) Alive; 2) Fresh Dead; 3)
Moderate Decomposition; 4) Advanced Decomposition; 5) Mummified/Skeletal; and 6)
Condition Unknown. The more decomposed or scavenged the carcass is, the less likely
they will be necropsied since tissue viability is compromised. If they are necropsied,
however, it is usually considered a limited necropsy rather than complete.
Tissues collected during necropsy for histology were stored in 10% neutral
buffered formalin and tissues collected for bacterial isolation and other tests were frozen
between -30°C and -40°C for Washington samples (Lambourn et al., 2013) and -20°C for
Oregon samples (Jim Rice, personal communication). Although there were histology
results for an array of tissue samples taken during necropsy, I only focused on the
comments relating to the nervous system and the final diagnosis. If histopathological
lesions resembling neurobrucellosis was found during histology, further tests were
conducted to assess whether the individual was infected with Brucella ceti and/or other
pathogens. Not all dolphins had the same Brucella tests performed. However, the most
common tests, along with which laboratories performed them, are outlined below.
18
Brucella cultures for this study were performed at the National Veterinary
Services Laboratory (NVSL; Ames, IA), Colorado Department of Agriculture (CODAG;
Denver, CO), and the Oregon State University Veterinary Diagnostic Laboratory (OSU;
Corvallis, OR). The majority of the cultures were performed at NVSL, which included
the following protocols previously described by Lambourn et al. (2013):
“Tissues were dissected, mixed with approximately 2 mL of sterile phosphate
buffered saline (pH 7.2), macerated, and inoculated onto tryptose agar with 5%
bovine serum and antibiotics (7.5 U/mL bacitracin, 30 mg/mL cycloheximide, and
1.8 U/mL polymyxin B); tryptose agar with 5% bovine serum, antibiotics, and ethyl
violet; Ewalt‟s media; Farrell‟s media; and Columbia agar with 5% blood. Plates
were incubated for 14 days in 10% CO2 at 37 C and observed for growth at 7 and
14 days.” (p.804)
If growth occurred after seven days, the average sized colonies consistent with Brucella
were counted, recorded, and transferred for identification (Mayfield et al., 1990 as cited
in Lambourn et al., 2013). According to Ewalt & Forbes (1987) and Lambourn et al.
(2013), isolates were confirmed with the following tests:
Growth in the presence of basic fuchsin (1:25,000 and 1:100,000), thionin
(1:25,000 and 1:100,000), and thionin blue (1:500,000);
Growth on medium containing penicillin (5 units/mL) or erythritol (1 mg/mL and
2 mg/mL plus 5% bovine serum);
Urease activity;
Catalase activity;
H2S production;
19
and CO2 dependence
Biotyping was conducted as previously described (Alton et al., 1988 as cited in
Lambourn et al., 2013). An agglutination test using A and M- monospecific antisera
(1:50-1:200) and R antiserum (1:25-1:100) determined the dominant antigen and isolates
were tested by the phages Tbilisi (Tb), Firenze (Fi), Weybridge (Wb), S708, Me/75, D,
BK2, R, R/C, and R/O for lysis susceptibility (Lambourn et al., 2013).
Serology tests were performed at the Washington Department of Agriculture
(WDA; Olympia, WA) and the Washington Animal Disease Diagnostic Lab (WADDL;
Pullman, WA). Serology protocols screening for antibodies were as previously outlined
in Garner et al.‟s (1997) article. Individuals were considered “suspect-positive” if the
buffered plate agglutination test antigen (BAPA) or brucellosis card test using buffered
Brucella antigen (BBA) detected antibodies (Lambourn et al., 2013). They were
considered “positive” if they were positive on BAPA or BBA, and subsequently positive
on the complement fixation (CF) and/or the Rivanol (RIV; +50 to 200) precipitation tests
(Lambourn et al., 2013).
Polymerase chain reaction (PCR) was performed at the Animal Health Center
(AHC; Abbotsford, British Columbia, Canada), Mystic Aquarium & Institute for
Exploration (MAIE; Mystic, Connecticut), and University of Iowa (UI; Iowa City, Iowa).
These laboratories “used previously described PCR techniques for Brucella (AHC;
Bricker et al., 2000) and real-time PCR (qPCR) analysis that used primers, probes, and
protocols that targeted the gene for a 31 kDa outer membrane protein bcsp31 specific to
the genus Brucella” (MAIE, Probert et al., 2004, and Sidor et al., 2013 as cited in
Lambourn et al., 2013, p. 804).
20
Immunohistochemistry (IHC) tests were performed at MAIE, United States
Department of Agriculture (USDA; Fort Collins, Colorado), and NVSL. IHC tests were
performed using previously described techniques for Brucella as mentioned in Lambourn
et al.‟s (2013) article.
“Formalin-fixed, paraffin-embedded tissues were stained with hematoxylin and
eosin and select sections were also stained with Giemsa and with Brown and Brenn.
Immunohistochemistry was performed on a subset of culture-positive cases. Tissue
sections were mounted on charged slides, deparaffinized, hydrated with a buffer
(PBS), treated with 3% H2O2 (5 min) to quench endogenous peroxidase, incubated
for 5 min at 37 C with nonimmune goat serum, rinsed, and incubated for 30 min at
37 C with a polyclonal antibody (1:10,000) prepared against B. abortus.
Amplification was conducted with biotinylated, goat origin, anti-rabbit
immunoglobulin (Ig), and peroxidase-labeled streptavidin; the chromagen was 3amino-9 ethylcarbazole in N, Ndimethylformamide. Sections were counterstained
with Gill II hematoxylin. Nonimmunized rabbit Ig fraction was substituted for
primary antibody as a negative control (Garner et al., 1997)” (Lambourn et al.,
2013, p. 804).
________________________________________________________________________
Data Analyses
Stranding History and Demographics
The Level A data I received went back to 1975. The first thing I wanted to do was
get an overall view of the reported stranding patterns that occurred throughout the years
for each analyzed species. The number of reported strandings every year between 1975
21
and 2014 were graphed, while simultaneously identifying how many of each species were
recorded to have stranded in each specific year. Two pie charts were subsequently created
detailing the number and percentage of each species that stranded between 1975-2005
and 2006-2014, providing a rank of which species were reported to be the most and least
common to strand among the two year ranges. Although stranding numbers and species
rank were graphed out beginning in 1975, the remainder of the demographic analyses
only included data beginning in 2006, since reported strandings were considered to be
more consistent and dolphins started to be routinely tested for Brucella.
After graphing out the number of reported strandings by species, a table was
created outlining different demographic factors such as stranding location, age class, and
sex for the individuals that stranded between 2006 and 2014. Stranding seasons were also
graphed out to identify “high strand seasons” for each species. Spring months included
March, April, and May; summer months included June, July, and August; fall months
included September, October, and November; and winter months included December,
January, and February. These results could suggest a more environmental cause behind
the increase in strandings that may, for example, be influenced by water temperature
and/or food source availability.
Analyses of Neurobrucellosis Suspicious Individuals
A table was created detailing demographic data (stranding location, age class, and
sex), the specific Brucella tests performed along with their results, and the
histopathological diagnoses of each individual that had the neurobrucellosis-like lesions
as previously described. Each individual‟s histopathology and lab results are discussed in
greater detail in Appendix B. Outlining these variables in the table allowed for the
22
visualization of potential demographic predilections as well as the most, and least,
successful Brucella tests according to this study
Each demographic (stranding location, age class, and sex) was discussed in
further detail specific to each species that were found to have Brucella. For each
demographic, a table was created comparing the total number of individuals that stranded
based on the demographic, the number of Brucella positive individuals that stranded
based on the demographic, and the total number of individuals minus the known Brucella
positive individuals that stranded based on the demographic. Comparing these numbers
side-by-side helped clarify whether any observed Brucella predilections were indeed
potential predilections, or if the observed trend was simply based off the normal stranding
patterns of that species. Tables were only created for the striped and short-beaked
common dolphin, however, because they were the only two species to have enough
positive individuals to observe a potential demographic predilection.
23
_____________________________________________________
Chapter Four
-Results-
_____________________________________________________
Overall Stranding History
Stranding Numbers
Between 1975 and 2005, there were fifty-three reported strandings of the analyzed
species: thirty-four (64%) PWSDs, thirteen (24.5%) striped, four (7.5%) short-beaked
common dolphins, and two (4%) bottlenose dolphins. Between 2006 and 2014, there
were forty-nine reported strandings: thirteen (27%) PWSDs, twenty-one (43%) striped,
ten (20%) short-beaked common, three (6%) bottlenose, and two (4%) long-beaked
common dolphins. As illustrated in Figure 3, an increase in strandings was noted from
2006, with the highest years in 2006 (n=9), 2012 (n=10), and 2014 (n=13). Besides 2006,
2012, and 2014, the number of reported strandings per year fluctuated between 0-5
individuals.
24
Bottlenose
Long-Beaked
Common
Short-Beaked
Common
Striped
PWSD
1975
1976
1977
1978
1979
1980
1981
1982
1983
1984
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
Number of Individuals
Total Number of Strandings (1975-2014)
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
Year
Figure 3: Total Number of Strandings (1975-2014)
Dolphin Strandings By Species (1975-2005)
Dolphin Strandings By Species (2006-2014)
4%
4%
7.5%
6%
Bottlenose - 3
Bottlenose - 2
20%
PWSD - 13
PWSD - 34
24.5%
27%
Striped - 21
Striped - 13
64%
Short-Beaked
Common - 4
43%
Short-Beaked
Common - 10
Long-Beaked
Common - 2
Figure 4: Reported Dolphin Strandings by Species (1975-2005) and (2006-2014)
Stranding Demographics
-Stranding LocationSince 2006 there were thirty-five (71%) reported strandings in Oregon and
fourteen (29%) reported strandings in Washington. The PWSD had eleven (85%)
reported strandings in Oregon and two (15%) in Washington. The striped dolphin had
sixteen (76%) reported strandings in Oregon and five (24%) in Washington. The shortbeaked common dolphin had seven (70%) reported strandings in Oregon and three (30%)
25
in Washington. There were only two reported long-beaked common dolphin strandings
and both occurred in Washington. Finally, the bottlenose dolphin had one (33%) reported
stranding in Oregon and two (67%) in Washington.
-Age ClassSince 2006 there were twenty-four (49%) reported subadult strandings, twentytwo (45%) adults, and three (6%) unknowns. The PWSD had six (46%) reported subadult
strandings and seven (54%) adult strandings. The striped dolphin had eleven (52%)
reported subadult strandings, eight (38%) adult strandings, and two (10%) unknown age
class strandings. The short-beaked common dolphin had five (50%) reported subadult
strandings and five (50%) adult strandings. The long-beaked common dolphin had one
(50%) reported subadult stranding and one (50%) unknown age class stranding. Finally,
the bottlenose dolphin had one (33%) reported subadult stranding and two (67%) adult
strandings.
-SexSince 2006 there were twenty-three (47%) reported female strandings, eighteen
(37%) male strandings, and eight (16%) unknowns. The PWSD had eight (62%) reported
female strandings, three (23%) male strandings, and two (15%) unknowns. The striped
dolphin had seven (33%) reported female strandings, ten (48%) male strandings, and four
(19%) unknowns. The short-beaked common dolphin had five (50%) reported female
strandings, four (40%) male strandings, and one (10%) unknown. The long-beaked
common dolphin had one (50%) reported female stranding and one (50%) unknown.
26
Finally, the bottlenose dolphin had two (67%) reported female strandings and one (33%)
unknown stranding.
Location
Species
Age Class
Sex
Total Number of Strandings
(2006-2014)
OR
WA
SA
A
U
F
M
U
L.o.
13
11
2
6
7
0
8
3
2
S.c.
21
16
5
11
8
2
7
10
4
D.d.
10
7
3
5
5
0
5
4
1
D.c.
2
0
2
1
0
1
1
0
1
T.t.
3
1
2
1
2
0
2
1
0
Total
49
35
14
24
22
3
23
18
8
Table 1: Overall Stranding Demographics (2006-2014)
*L.o. = PWSD; S.c. = Striped; D.d. = Short-Beaked Common; D.c. = Long-Beaked Common; T.t. = Bottlenose
* OR = Oregon; WA= Washington
* SA = Subadult; A= Adult; U = Unknown
* F= Female; M = Male; U= Unknown
Stranding Seasons
Total, there were six (12%) reported strandings in the spring, seven (14%) in the
summer, fifteen (31%) in the fall, and twenty-one (43%) in the winter between 2006 and
2014. The majority of the fall strandings was comprised of the short-beaked common
dolphin and the majority of the winter strandings was comprised of the striped dolphin.
Between 2006 and 2014 there was no observed temporal trend in PWSD
strandings besides a slight increase in the winter season. There were three (23%)
strandings in the spring, two (15%) in the summer, three (23%) in the fall, and five (39%)
in the winter.
27
Between 2006 and 2014, the striped dolphin stranded between January-March,
July, and October-December. There seems to be an observed temporal trend for winter
strandings since there were only two (10%) reported strandings in the spring, three (14%)
in the summer, three (14%) in the fall, but thirteen (62%) in the winter.
Between 2006 and 2014, the common dolphin stranded in January (one shortbeaked), March (one long-beaked), and every month between September-December (one
long-beaked in November and the remainder were short-beaked common). Season wise,
the short-beaked common dolphin has an observed temporal trend to strand during the
fall, since there were eight (80%) strandings in the fall and two (20%) strandings in the
winter. The long-beaked common dolphin had one (50%) stranding in the spring and one
(50%) in the fall. Since there were only two individuals, there was no observed temporal
trend for the long-beaked common dolphin.
Between 2006 and 2014, there was one reported bottlenose stranding in January
and two in July. Season wise, there were two (67%) reported strandings in the summer
and one (33%) in the winter. Since there were only three individuals, there was no
observed temporal trend.
28
Number of Individuals
Strandings by Season (2006-2014)
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
Bottlenose
Long-Beaked Common
Short-Beaked Common
PWSD
Striped
Spring
Summer
Fall
Winter
Season
Figure 5: Strandings by Season and Species (2006-2014)
________________________________________________________________________
Nervous System Disorders
General Findings
To my knowledge and request, fifteen individuals came back with
histopathological lesions suspicious of neurobrucellosis out of the individuals that had
histology performed between 2006 and 2014. Out of the fifteen individuals, there were
two (13%) PWSDs, nine striped dolphins (60%), three short-beaked common dolphins
(20%), and one long-beaked common dolphin (7%). Two (13%) of these strandings
occurred in 2006, six (40%) occurred in 2012, and seven (47%) occurred in 2014. I did
not receive any bottlenose histology reports that detailed histopathological manifestations
resembling neurobrucellosis. It is known, however, that at least two of the individuals
were too decomposed to be able to perform a complete necropsy and obtain viable tissue
for histology. As previously mentioned, I did not receive any histology reports on
individuals that did not have neurobrucellosis-like lesions, so the total number of
dolphins receiving histology is unknown for this study.
29
Brucella ceti Analyses
Out of the fifteen individuals that had histopathological manifestations resembling
neurobrucellosis, ten (71%) tested positive for Brucella, four (29%) were negative, and
one did not have any specific Brucella tests performed at the time of this study. Brucella
positive individuals included 6/8 (75%) striped dolphins (the ninth was not tested
specifically for Brucella), 3/3 (100%) short-beaked common dolphins, 1/2 (50%)
PWSDs, and 0/1(0%) long-beaked common dolphin. Although different individuals were
tested by culture, PCR, IHC, and/or serology, positive results were only received via
culture and serology (Table 2). All positive cases that received a sequence type (n=6)
were identified as ST26. These included Individuals 6, 7, 9, 10, 11, and 12.
30
Individual
1
Species
Date
L.o.
Jan 16,
2006
State
WA
Age
A
Sex
F
Culture
PCR
Serology
NA
(AHC)
+
(WDA)
Pending
(MAIE)
NA
NA
NA
IHC
Histopathological Diagnosis
NA
Marked, multifocal, necrotising,
nonsuppurative encephalitis with
scattered microgliosis with clusters
of intra and extracellular oblong
basophilic deposits
Severe, multifocal to coalescing,
nonsuppurative
meningoencephalitis with
prominent perivascular
lymphoplasmacytic cuffing,
satellitosis and acute subcortical
hemorrhage
Marked, focally extensive,
necrotising meningoencephalitis
with variably extensive meningeal
fibrosis, numerous acicular clefts,
and multifocal lymphoplasmacytic
perivascular cuffing
2
D.d.
Nov 29,
2006
WA
SA
F
NA
(AHC)
Mes. LN
(MAIE)
3
D.c.
Mar 28,
2012
WA
SA
F
(CODAG)
(AHC)
4
S.c.
July 14,
2012
WA
A
M
(NVSL)
(AHC)
NA
NA
Marked, diffuse, nonsuppurative
meningitis with circumferential,
peripheral myelin vacuolation and
occasional malacia (spinal cord; 56 cervical vertebrae)
5
S.c.
July 23,
2012
WA
SA
M
NA
(UI)
NA
NA
Severe, chronic, nonsuppurative
meningoencephalomyelitis
6
S.c.
Dec 5,
2012
OR
SA
M
+
(NVSL)
(UI)
NA
NA
Moderate to severe,
nonsuppurative
meningoencephalomyelitis
7
L.o.
Dec 10,
2012
OR
SA
F
+
(NVSL)
(UI)
NA
NA
Lymphoplasmacytic
meningoencephalitis
8
S.c.
Dec 10,
2012
OR
SA
M
+
(OSU)
(UI)
NA
NA
Lymphoplasmacytic
meningoencephalitis
9
S.c.
Feb 19,
2014
OR
A
M
+
(NVSL)
NA
NA
NA
Severe, nonsuppurative meningitis,
choroid plexitis, and perivasculitis
10
S.c.
Feb 20,
2014
OR
SA
F
+
(NVSL)
NA
NA
NA
Severe, lymphocytic meningitis,
encephalitis, myelitis, and
radiculoneuritis
11
S.c.
Feb 21,
2014
OR
SA
M
+
(NVSL)
NA
NA
NA
Severe, lymphocytic meningitis
(brain and spinal cord)
12
S.c.
Mar 17,
2014
WA
A
M
+
(NVSL)
NA
NA
(USDA)
13
D.d.
Oct 25,
2014
WA
SA
F
+
(NVSL)
NA
NA
NA
14
D.d.
Nov 9,
2014
WA
SA
M
+
(NVSL)
(AHC)
+
(WADDL)
(NVSL)
15
S.c.
Dec 27,
2014
OR
A
F
NA
NA
NA
NA
Marked, nonsuppurative
meningoencephalomyelitis and
root ganglioneuritis
Severe, nonsuppurative
meningomyelitis and root
ganglioneuritis
Severe, nonsuppurative
meningoencephalomyelitis
Severe, diffuse, lymphocytic
meningitis; Mild, multifocal
lymphocytic encephalitis; Focal
lymph node pyogranuloma
Table 2: Overview of Stranded Individuals Suspicious of Neurobrucellosis
*L.o. = PWSD; S.c. = Striped; D.d. = Short-Beaked Common; D.c. = Long-Beaked Common
* NVSL= National Veterinary Services Laboratory; AHC= Animal Health Center; WADDL= Washington Animal Disease Diagnostic
Lab; WDA = Washington Department of Agriculture; NWZP = Northwest ZooPath; MAIE = Mystic Aquarium & Institute for
31
Exploration; UI = University of Iowa; CODAG = Colorado Department of Agriculture; USDA = United States Department of
Agriculture
* A = Adult; SA = Subadult
* F= Female; M = Male
Individual
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Animal I.D.
CRC-702
CRC-779
CRC-1200
MKH2012-025
PSU-12-07-23Sc
HMSC-12-12-05Sc
HMSC-12-12-10Lo
HMSC-12-12-10Sc
PSU-14-02-19Sc
HMSC-14-02-20Sc
HMSC-14-02-21Sc
MKH2014-002
CRC-1462
MKH2014-29
HMSC-14-12-27Sc
Table 3: Identification Key
-Stranding LocationOut of all the positive Brucella individuals, there were six (60%) strandings in
Oregon and four (40%) in Washington. There were five (83%) striped dolphins that
stranded in Oregon and one (17%) that stranded in Washington. Three out of three
(100%) positive short-beaked common dolphins stranded in Washington and the single
positive PWSD stranded in Oregon. As seen from Table 2, multiple individuals would
strand within the same month in the same state. For example, the three positive 2012
cases all occurred in December and all occurred in Oregon. Also, three individuals
stranded in February of 2014, which also all occurred in Oregon. Contrarily, the
remainder of the 2014 Brucella cases all occurred in Washington.
32
Location
All Strandings
Brucella Positive
All Strandings Excluding Brucella Individuals
Oregon
76%
83%
73%
Washington
24%
17%
27%
Table 4: Stranding Location Comparisons of the Striped Dolphin (2006-2014)
Location
All Strandings
Brucella Positive
All Strandings Excluding Brucella Individuals
Oregon
70%
0%
100%
Washington
30%
100%
0%
Table 5: Stranding Location Comparisons of the Short-Beaked Common Dolphin (2006-2014)
Figure 6: Stranding Location of Positive Brucella Cases
* Yellow= PWSD; Red = Striped; Purple = Short-Beaked Common Dolphin
* Square = 2006; Star = 2012; Circle = 2014
33
Age Class
Overall, 80% of the Brucella positive individuals were reported to be subadults.
This included 3/3 (100%) short-beaked common dolphins, the single PWSD, and 4/6
(67%) striped dolphins.
Age Class
All Strandings
Brucella Positive
All Strandings Excluding Brucella Individuals
Subadult
52%
67%
40%
Adult
38%
33%
47%
Unknown
10%
0%
13%
Table 6: Age Class Comparisons of the Striped Dolphin (2006-2014)
Age Class
All Strandings
Brucella Positive
All Strandings Excluding Brucella Individuals
Subadult
50%
100%
29%
Adult
50%
0%
71%
Unknown
0%
0%
0%
Table 7: Age Class Comparisons of the Short-Beaked Common Dolphin (2006-2014)
Sex
Out of the Brucella positive individuals, there were six (60%) males and four
(40%) females. Out of the striped dolphin, there were five (83%) males and one female
(17%). There were two (67%) female short-beaked common dolphins and one (33%)
male. The sole PWSD was female.
Sex
All Strandings
Brucella Positive
All Strandings Excluding Brucella
Male
48%
83%
33%
Female
33%
17%
40%
Unknown
19%
0%
27%
Table 8: Sex Comparisons of the Striped Dolphin (2006-2014)
34
Sex
All Strandings
Brucella Positive
All Strandings Excluding Brucella
Male
40%
33%
43%
Female
50%
67%
43%
Unknown
10%
0%
14%
Table 9: Sex Comparisons of the Short-Beaked Common Dolphin (2006-2014)
35
_____________________________________________________
Chapter Five
-Discussion-
_____________________________________________________
Overall Stranding History
Stranding Numbers
Reported strandings were inconsistent prior to 2002, but funding from stranding
networks through the “John H. Prescott Marine Mammal Rescue Assistance Grant
Program” has significantly improved stranding responses since then (Jim Rice, personal
communication). Although this could have contributed to the overall increases seen
within the data, the number of strandings in 2006, 2012, and 2014 still seem suspiciously
high, and may have been influenced by an environmental factor and/or an influx of
disease, which is further discussed in the subsequent sections below.
-Strandings by SpeciesBetween 2006 and 2014 the striped dolphin was the most common species to
strand and the short-beaked common dolphin only had three fewer strandings than the
PWSD. Since the striped dolphin and the short-beaked common dolphin do not normally
inhabit Oregon and Washington waters, the amount of strandings as well as their
stranding rank is suspicious. It is important to keep in mind, however, that advancements
in technology (i.e. cell phones) could have contributed to the rise in strandings seen
within the uncommon species. Since high-quality photos of a stranded individual can
now be taken, sent, and received in a matter of seconds, the accuracy of identifying the
36
species of the individual vastly increases or is even absolute, compared to solely relying
on a verbal description from a passerby.
We may also be seeing a high number of striped dolphin strandings in the PNW
due to the possibility of their range expanding northward from warming sea temperatures
(Allen et al., 2011). Since the striped dolphin is associated with convergence zones of
warm and colder waters, they may be on the leading edge of the shift in ranges that is
being seen with warmer water species (Allen et al., 2011). Because of this, we may also
see an increase in strandings for the remainder of the uncommon species, as well as more
strandings in Washington and farther north into Canada due to predicted warmer sea
temperatures and climate change.
-Stranding SeasonsAs discussed in the results, the majority of the strandings occurred in winter
(December-February) and was closely followed by fall (September-November). The
winter strandings were primarily comprised of the striped dolphin, and the fall was
primarily comprised of the short-beaked common dolphin. Environmental factors, such as
changes in sea surface temperature and/or the movement of the dolphins‟ preferred food
sources, could potentially be a reason as to why there has been an increase in reported
strandings of the uncommon species, as well as why we are seeing more of one species
strand in a specific season over others.
Research has, and will continue to be conducted on relationships between
environmental variability and recruitment of an array of fish species, cephalopods,
copepods, etc., focusing on the roles of local upwelling vs. large basin-scale climate
37
cycles (Peterson et al., 2014). These cycles include the El Niño Southern Oscillation
(ENSO), the Pacific Decadal Oscillation (PDO), and the North Pacific Gyre Oscillation
(NPGO) (Peterson et al., 2014). Looking more closely at seasonal and interannual
changes in coastal upwelling and food chain structures of these dolphin species could
provide more insight as to whether environmental factors have influenced the increases in
strandings seen in the data. Unfortunately, due to time constraints, this was not further
assessed within this thesis, but should be in future studies.
________________________________________________________________________
Nervous System Disorders
General Findings
As seen in the results, individuals that stranded with neurobrucellosis-like
histopathological lesions only stranded in 2006, 2012, and 2014. Each subsequent year
had more individuals strand with these lesions and more individuals test positive for
Brucella than the previous. However, 2006, 2012, and 2014 also had the highest number
of reported strandings since 1975 as well. Without seeing the other histology reports, it is
difficult to determine whether this is a meaningful increase in Brucella disorders, or if the
increase is driven by more stranded individuals overall. The increase could have also
been due to more performed necropsies and histology reports. It is suspicious, however,
that the Brucella strandings did not occur sporadically throughout each stranding year,
but rather within the same season, and many times within the same month.
Gonzàlez et al.‟s (2002) study detailed three striped dolphins that stranded in a
period of a month. Their rationale behind this “indicate[d] the contribution of an
unrecognized, perhaps environmental, influence at a given time” (p. 151). As previously
38
discussed, environmental influences can include oil spills or a change in water
temperature and food chain structures, which can alter the susceptibility of individuals to
infection, leading them to strand. It has been known since the beginning of medical
science that a change in weather can lead to the emergence of epidemic diseases
(National Resource Council, Committee on Climate, Ecosystems, Infectious Disease, and
Human Health, 2001). Factors such as temperature, precipitation, and humidity can all
affect the life cycle of pathogens, potentially altering the timing and intensities of disease
outbreaks, and can also increase the introduction of vectors and animal reservoirs
(National Resource Council, Committee on Climate, Ecosystems, Infectious Disease, and
Human Health, 2001; Sachan and Singh, 2010). Since the majority of the striped and
short-beaked common dolphins in this thesis stranded when the water temperatures were
colder, there is a possibility that these individuals swam farther north to the PNW,
thriving during the warmer months, but became more susceptible to infection when the
water temperatures dropped below a certain threshold. Although individuals may be more
susceptible to infection when the water is colder, incubation periods of Brucella ceti is
still unknown and may vary between different individuals and/or species, hence why
multiple individuals strand within a few months, and even days, of each other in very
specific years. However, more data would be needed to support or refute this hypothesis.
Another interesting discovery was that Individuals 2, 3, and 13 all had a previous
shark bite wound and were all common dolphins (two short-beaked and one longbeaked). Besides these three, the only other individual to have a noted shark bite wound
in the Level A database was a PWSD that was too decomposed to perform a complete
necropsy. Shark bite wounds can cause substantial injury and could alter the individual‟s
39
susceptibility to disease or capability to fight off disease. Also, if an individual is
experiencing altered swimming behaviors due to nervous system ailments from an
infectious agent, this could also increase their susceptibility to a shark attack.
Brucella ceti Analyses
-General FindingsIf neurobrucellosis was suspected after histology, then verification of Brucella by
laboratory tests most often came back positive.
-Brucella ceti TestsPositive Brucella cases for this study were reported via culture and serology only.
Culture is considered to be the “gold standard” in testing (Wu et al., 2014), but serology
is more difficult to decipher. Two individuals, 2 and 14, were tested via serology in this
study. Individual 2 was only tested by serology but Individual 14 had a culture and IHC
performed in addition to serology. Both individuals are considered positive rather than
suspect-positive since Individual 2 came back as “Brucella-RAP positive and Rivinol
positive +200” and Individual 14 had positive culture isolates.
Within this study there was a high rate of false negative results, particularly with
PCR. There was not a single PCR test that came back positive, making it the least
effective test out of all four, in my opinion. The AHC performs PCR on pooled tissue
samples, which typically include brain, lung, liver, spleen, and lymph node (Lambourn et
al., 2013). Although pooling techniques can increase analytical efficiency and promote
cost savings, sensitivity is compromised because it is inversely proportional to the
40
number of samples in the pool, and a significant portion of the detectable microbial
community could be masked (Manter et al., 2010 and Sun et al., n.d.). Although not
specific to Brucella, other studies have also noted decreases in sensitivity of detection
when compared to testing the tissues separately (Grmek-Kosnik et al., 2006 and Manter
et al., 2010). IHC also did not provide any positive results despite one individual coming
back positive via serology and the other coming back positive on culture. As previously
mentioned, it has been recognized that IHC has lower sensitivity in identifying Brucella
compared to serology (Gonzàlez-Barrientos et al., 2010).
The amount of false negatives is indicative that the success rate of these tests are
based on how sensitive a particular pathologist‟s or lab‟s tests are, what part/which
tissues are tested (e.g. was the affected part of the nervous system tested or was a sample
taken from an unaffected section?), and how viable the tissue samples are (e.g. was the
tissue frozen and thawed multiple times?). Due to all the factors that can provide a false
negative result, I believe that 71% is an underestimate of how many individuals are truly
Brucella positive. Again, this statistic may be subject to change after further tests are
performed.
-SpeciesIt is believed that there are low proportions of other cetacean species that show
Brucella associated clinicopathological signs, besides the striped dolphin (Isidoro-Ayza
et al., 2014). However, the results of this paper details at least two other species, the
short-beaked common dolphin and PWSD, that stranded with neurobrucellosis-like
41
manifestations and tested positive for Brucella. To my knowledge, this is the first study
to publish confirmation of Brucella within a PWSD.
This study also reconfirmed the well-established fact that Brucella infections are
most prominent in striped dolphin species and supports Hernàndez-Mora et al.‟s (2008)
study that the striped dolphin is a highly susceptible host and even a potential reservoir
for the transmission of Brucella ceti. According to Allen et al. (2011), short-beaked
common dolphins have been periodically observed in schools of striped dolphins. If the
striped dolphin is a reservoir for Brucella ceti and periodically associates with the shortbeaked common dolphin, that could explain why the short-beaked common dolphin is the
second most susceptible dolphin species to Brucella in the PNW. More information on
vertical and horizontal transmission between species would be needed, however, to make
any further claims.
-LocationAlthough the majority (83%) of the positive striped dolphins stranded in Oregon,
the observed trend seems to be influenced by the normal stranding pattern of the species,
and would not be considered a predilection at this time. However, there seems to be an
observed predilection for positive short-beaked common dolphins to strand in
Washington, since 100% of the short-beaked strandings in Washington ended up being
Brucella positive. Although there were only three positive short-beaked dolphins at this
time, the observed predilection should be taken into consideration on impending
strandings.
42
It is important to keep in mind that a carcass might strand hundreds of kilometers
from their normal range and/or from where they actually died, since carcass movement
can be affected by wind and water currents, the height of the carcass above the water line,
upwelling, and downwelling (Norman et al., 2004). However, a carcass that drifts that far
is not normally fresh enough for a complete necropsy or meaningful histopathology
results. Dolphins typically sink when they die, and re-float once gasses build up inside
them from decomposition (Jim Rice, personal communication). Because of this, the
majority of dolphin carcasses never come close to shore and those that do, are normally
found freshly dead and in good enough condition for histopathology (Jim Rice, personal
communication).
Also, although the PWSD and short-beaked common dolphin can generally be
seen in coastal and oceanic waters, the striped dolphin is mainly pelagic (Allen et al.,
2001). Therefore, the presence of them is suspicious and can indicate that they were
neurologically debilitated, venturing into waters that they normally would not venture
into if they were lucid.
-Age ClassThe findings of age class supports this thesis‟ hypothesis as well as Gonzàlez et
al.‟s (2002) study noting the greater probability of subadults to develop neurobrucellosis
compared to adults. Although there were a couple adult striped dolphins that came back
Brucella positive, the remainder were subadult individuals. This does not fit the “normal”
stranding trends observed from the historical data, which showed roughly half of the
strandings being subadult and half being adult. Why we are seeing Brucella more in
43
subadult individuals than adults is still to be determined. There were a decent amount of
unknown age classes, however, which may have changed the results of this study if they
were known.
Lambourn et al. (2013) noted the possibility of subadults to cease producing
Brucella antibodies or even clearing infection, subsequently coming back negative on
serology tests. Although this was pertaining to Brucella pinnipedialis in harbor seals, it
could possibly be applicable to Brucella ceti as well. It is also not known if individuals
can be reinfected and if so, the consequent antibody response to that reinfection
(Lambourn et al., 2013). Although the serology tests came back positive for this study,
this should be taken into consideration in future studies if negative serology tests are
obtained.
-SexThere seems to be an observed predilection towards striped male dolphins. Striped
dolphins have complex systems of individuals organized by age, sex, and breeding status
(Allen et al., 2011). According to Gaspari et al.‟s (2007) study, female striped dolphins
have higher average kinship within groups rather than between groups. Females were
also found to associate more with adult kin than males. Assuming that Brucella ceti can
be passed horizontally, that would make sense why we are seeing more males with
Brucella than females, since the males associate more between different groups and are
not preferring to associate only with adult kin. There did not seem to be an observed
predilection for the short-beaked common dolphin at this time.
44
_____________________________________________________
Chapter Six
-Conclusion-
_____________________________________________________
Overall Findings
This thesis documents the presence of Brucella ceti in dolphin species along the
Oregon and Washington coast. According to the results, there were subsequent increases
in dolphins that stranded with neurobrucellosis-like manifestations in 2006, 2012, and
2014. Since they occurred in very specific years and seasons, the increases may have
been due to an unknown environmental influence, altering their susceptibility to the
disease. Or, the increases may have been driven by an overall rise in strandings, which
could have also occurred due to an unknown environmental influence. This thesis also
outlines the high susceptibility of the striped dolphin to be infected with Brucella ceti
followed by the short-beaked common dolphin. This study is also the first, to my
knowledge, to document Brucella in a PWSD. Other observed predilections at this time
include male striped dolphins, subadult individuals of all species, and short-beaked
common dolphins that strand in Washington. Once again, these observed trends are based
on a small sample size and the results may be subject to change based on re-testing some
negative individuals as well as future strandings.
The high rate of false negative tests (i.e. PCR and IHC tests) reinforces the
suspicion that there is an underestimate in positive Brucella cases among the individuals
that stranded with histopathological manifestations resembling neurobrucellosis. Further
45
tests will be conducted on individuals with available and viable samples, so the current
results may be subject to change. Determining whether or not neurobrucellosis was the
cause of these neurological disorders can also be difficult to conclude, as an array of
other infectious agents can cause similar manifestations. For example, as previously
stated, viral infections are responsible for a vast amount of neurological diseases within
the striped dolphin, and encephalitis caused by fungal origins and Toxoplasma spp. have
been documented as secondary complications of morbillivirus within the striped dolphin
as well (Gonzàlez et al., 2002). This is especially true since it can be difficult to detect
Brucella organisms (as well as other organisms) directly in infected tissues (Seidel et al.,
2003). This thesis does conclude, however, that individuals with histopathological
manifestations suspicious of neurobrucellosis most often come back positive for Brucella
on further tests. Because of this, any individual remotely suspicious for neurobrucellosis
(i.e. strandings of the demographics discussed or suspicion of a debilitated nervous
system) should be deemed high risk for having Brucella and appropriate precautionary
measures should be implemented to avoid zoonotic exposure.
________________________________________________________________________
Future Studies and Suggestions
Unknown Cases
Although this study only went back to 2006, there were two 2003 individuals
found in older records that were suspicious for neurobrucellosis and subsequently tested
positive for Brucella via serology. One case was an adult, male, Northern right whale
dolphin that stranded in Seattle, Washington in March of 2003. If more time was
46
available for this study, I would have liked to have included the Northern right whale
dolphin in my list of analyzed species. The other individual stranded in February of 2003
and was an adult, female, common dolphin. This individual stranded in Long Beach, but
it was unable to be determined if it was Long Beach, Washington or Long Beach, British
Columbia, Canada. There was no record of this individual in my Level A data and I only
received a small excerpt, detailing the histopathology results. Knowing more information
about this individual will be helpful in any future studies.
Expected Sea Temperature Changes
The region of the North Pacific Ocean has been the warmest on record due to
what has been nicknamed by climate scientist, Nick Bond, to be “the blob” (Hickey, 2015
and Milstein, 2014). One factor that has been impacting the California coast is a lowpressure trough between California and Hawaii (Milstein, 2014). The winds that typically
drive upwelling of deep, cold water has been weakened by the low-pressure trough,
resulting in warmer waters that have been persisting longer than usual (Milstein, 2014).
Besides a narrow strip of cold water along the PNW coast that is being fed by upwelling
from the deep ocean, the North Pacific Ocean sea surface temperature has increased as
much as 3°C (Milstein, 2014). This change in water temperature has and will continue to
favor warmer water species, such as the dolphins in this study, and will have detrimental
impacts on marine populations preferring a colder, more productive ocean (Milstein,
2014 and Profita, 2015). A survey of whales and dolphins off the West coast revealed
marine mammals, as well as other marine fauna, farther north from their normal ranges
due to the unusually warm waters (Profita, 2015). In fact, one hundred common dolphins
47
were documented in an area not normally seen due to this warm water surge (CBS SF,
2014).
There is also an estimated 65% chance that El Niño will arrive later in 2015,
which is a separate warming event from the blob (Milstein, 2014). ENSO events can
impact sea surface temperature and current patterns, which can lead to warmer water
temperature and a change in cetacean species distributions (Norman et al., 2004). This
will only reinforce the warming seen from the blob and will further have an impact on
marine ecosystems.
Studying linkages between climate and disease can provide understanding on
factors that might drive the emergence of seasonal or interannual variations in diseases
such as Brucella (National Resource Council, Committee on Climate, Ecosystems,
Infectious Disease, and Human Health, 2001). Because of this, stranding data from this
year and the next couple years will be vital in order to assess stranding patterns based on
this change in water temperature and its potential impacts on the dolphins‟ susceptibility
to infection.
48
_____________________________________________________
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Appendix A: Level A Data Sheet
55
56
Appendix B: Histopathology Results
Individual
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Animal I.D.
CRC-702
CRC-779
CRC-1200
MKH2012-025
PSU-12-07-23Sc
HMSC-12-12-05Sc
HMSC-12-12-10Lo
HMSC-12-12-10Sc
PSU-14-02-19Sc
HMSC-14-02-20Sc
HMSC-14-02-21Sc
MKH2014-002
CRC-1462
MKH2014-29
HMSC-14-12-27Sc
Individual one was an adult, female, PWSD that had marked, multifocal, necrotising,
nonsuppurative encephalitis which was deemed to be severe enough to cause antemortem
morbidity and the death of this individual. The brain also had scattered microgliosis with
intracellular and extracellular clusters of oblong basophilic deposits. This was the only
individual to have encephalitis and not have a meningeal predilection that is commonly
seen in other Brucella cases. According to the pathologist, the intralesional structures
were suggestive of a protozoal infection, which, after a protozoal PCR test, came back
positive for Sarcocystis neurona. PCR was negative for morbillivirus and negative for
Brucella. An aerobic culture was performed on the brain, lung, lymph nodes, and small
intestines of the animal, but the only bacteria to be isolated were a light to moderate mix
of Aeromonas hydrophila, Enterobacter spp, Enterococcus spp., and Rahnella aquatilis.
According to the pathologist, these bacterial isolates most likely occurred postmortem. A
specific Brucella culture was not performed.
Individual two was a subadult, female, short-beaked common dolphin that had severe,
multifocal to coalescing, nonsuppurative meningoencephaltis with prominent
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periviascular lymphoplasmacytic cuffing, satellitosis, and acute subcortical hemorrhage.
According to the pathologist, the meningitis and acute multifocal hemorrhaging attributed
to the death of this animal. PCR attempts came back negative for Brucella and
morbillivirus and there were no bacterial growths seen from the lung, lymph node, brain,
spleen, uterine, or small intestines of the animal. It is important to note that a specific
Brucella culture was not done and PCR was performed on a mesenteric lymph node and
not on nervous system tissue. Virology tests also came back negative for this individual.
This animal did come back Brucella positive via serology (RAP positive and Rivinol
positive +200), however, and IHC tests for Brucella are currently pending as well.
Individual three was a subadult, female, long-beaked common dolphin that had marked,
focally extensive, necrotising meningoencephalitis with variably extensive meningeal
fibrosis, numerous acicular clefts, and multifocal lymphoplasmacytic perivascular
cuffing. The skull had moderate meningeal to periosteal adhesions on gross findings with
an accumulation of clear fluid. According to the pathologist, the meningeal fibrosis and
presumptive adhesion to the skull were severe enough to account for the cerebrospinal
fluid accumulation and the death of this animal. This animal did not come back positive
for Brucella via culture or PCR. IHC and PCR came back negative for protozoa
(Toxoplasma gondii, Sarcocystis neurona, and Neospora caninum), PCR came back
negative for Apicomplexa, and PCR came back negative as well for morbillivrus.
Although no significant bacteria were recovered from sampled tissues, the pathologist
still believes these ailments were due to a bacterial infection.
Individual four was an adult, male, striped dolphin that had marked, diffuse,
nonsuppurative meningitis of the spinal cord at the 5-6 cervical vertebrae level with
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circumferential, peripheral myelin vacuolation and occasional malacia. According to the
pathologist, the cervical meningitis likely contributed to the antemortem morbidity and
death of this animal and an infectious agent is a prime consideration. IHC came back
negative for Toxoplasma gondii, Neospora caninum, and Sarcocystis neurona. Special
histochemical stains did not come back positive for any acid fast bacilli, fungal elements,
or bacteria. No bacteria were recovered from the brain and PCR of the brain came back
negative for Brucella and canine distemper virus. Morbillivirus also came back negative
and further protozoa PCR is pending. Although the brain did not come back positive with
any pathogens, the spinal cord, which was the location of the meningitis, was not tested.
This individual‟s tissues were also submitted for testing after two years of being frozen
and thawed multiple times, which can damage cell structures and denature proteins,
leading to false negatives.
Individual five was a subadult, male, striped dolphin that had severe, chronic,
nonsuppurative meningoencephalomyelitis. PCR came back negative for Brucella but
culture, serology, and IHC were not performed. Although Brucella was not confirmed by
PCR, the pathologist‟s statement was in support of neurobrucellosis since the lesions in
this individual strongly resembled the lesions in other striped dolphins described by the
CDC that did have neurobrucellosis. Other pathogen types were not strong contenders
due to the presence and/or absence of specific microscopic findings subsequently listed:
1) The meningeal predilection was unusually strong for a viral pathogen; 2) there were no
microglial nodules within the lymphocytic infiltrate commonly accompanied by a
protozoal infection; 3) there was not enough mitotic activity to indicate lymphoma; and
4) the presence of mixed character of cells excluded neoplasia.
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Individual six was a subadult, male, striped dolphin that had moderate to severe,
nonsuppurative meningoencephalomyelitis. According to the pathologist, the cervical
spinal cord section showed that this was not merely a cellular residue from a contained
exposure, but ongoing due to the damage seen in the grey and white matter tracts of the
spinal cord. Protozoal infection is less likely since there were no microglial nodules
presenting with the encephalitis and a virus did not seem likely due to the meningeal
predilection over an encephalitis predilection. This individual came back Brucella
negative by PCR, but positive (ST 26) via Brucella culture. Therefore, this individual
would be considered to be Brucella positive despite the false negative PCR result, which
is commonly seen in this study. The pathologist also noted that this individual had a lot of
similarities to individual five, which tested negative for Brucella by PCR. Since this
individual also tested negative by PCR then subsequently tested positive via culture, it
only further supports the belief that individual five will be Brucella positive if culture or
serology is to be conducted in the future.
Individual 7 was a subadult, female, PWSD that had lymphoplasmacytic
meningoencephalitis, which according to the pathologist, was suggestive of
neurobrucellosis and likely accounted for the stranding and contribution to the death of
this animal. An aerobic brain culture came back with 2+ mixed Gram-positive and Gramnegative organisms, which included Aeromonas spp. and Staphylococcus spp. A specific
Brucella culture isolated Brucella in the brain tissue (ST 26), but a PCR came back
negative. Since there were isolates via culture, this animal is considered to be Brucella
positive.
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Individual eight was a subadult, male, striped dolphin that had lymphoplasmacytic
meningoencephalitis. According to the pathologists, the lesions in the brain were
consistent with neurobrucellosis and were severe enough to have led to the stranding
and/or the death of this animal. Brucella was cultured in brain tissue despite a negative
PCR, and was sent to the CDC for sequence typing. This individual also had negative
tests for Leptospira and canine distemper virus.
Individual nine was an adult, male, striped dolphin that had severe, nonsuppurative
meningitis, choroid plexitis, and perivasculitis. According to the pathologist, these lesions
are consistent with those caused by Brucella. Brucella was isolated (ST 26) in the
individual‟s cerebrospinal fluid, lung, and pulmonary lymph node. This was the only
individual in my dataset to have a positive result from an area other than the nervous
system.
Individual ten was a subadult, female, striped dolphin that had severe lymphocytic
meningitis, encephalitis, myelitis, and radiculoneuritis. The pathologist noted that
although some of the changes were consistent with Brucella, a viral infection was also a
consideration. This individual came back positive for Brucella (ST 26) via culture of the
brain but no inclusion bodies typical of Morbillivirus were observed.
Individual eleven was a subadult, male, striped dolphin that had severe lymphocytic
meningitis of the brain and spinal cord, which were noted to be typical of the changes
observed in Brucella infections. This individual came back Brucella positive (ST 26) via
culture in both the brain and spinal cord.
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Individual twelve was an adult, male, striped dolphin that had marked, nonsuppurative
meningoencephalomyelitis and root ganglioneuritis. This individual had a negative
herpesvirus PCR of the spinal cord and was also negative for morbillivirus testing,
despite the pathologist‟s statement that the lesions were suggestive of a viral infection.
According to the pathologist, this individual lacked the microglial nodule formation
typically associated with protozoan infection and the suppurative components typically
associated with most bacterial infections. Brucella came back negative on IHC for this
individual, but did come back with positive isolates in the brain via culture.
Individual thirteen was a female, subadult, short-beaked common dolphin that had severe,
nonsuppurative meningomyelitis and root ganglioneuritis. Lesions were also found in the
meninges and neuropil of the brain, but were much more mild. A small focus of
lymphocytic inflammation was also noted in the meninges of the optic nerve. This
individual came back positive via culture of the brain with a pending sequence type.
Individual fourteen was a subadult, male, short-beaked common dolphin that had severe,
nonsuppurative meningoencephalomyelitis. This individual had negative PCR and IHC
results for Brucella, but came back positive via serology and culture with a pending
sequence type. This indiviudal was also negative for morbillivirus and herpesvirus but
has a pending protozal PCR test.
Individual fifteen was an adult, female, striped dolphin that had severe, diffuse,
lymphocytic meningitis and mild, multifocal lymphocytic encephalitis. Although no
bacterial isolates were recovered from a general aerobic brain culture, a bacterial etiology
is still more likely than viral due to the severe meningeal predilection. Sending brain
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tissue for a specific Brucella culture would be suggested, but no frozen brain was kept
from necropsy.
63
