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Comparative Pathology (RCP)/AF1P accession numbers (Inskeep, 1990), Smithsonian catalog numbers (Kuehl
et al., 1991), or Mote Marine Laboratory case numbers (Rawson et al., 1991) for reference. Barros and Odell
(1990) provided both field numbers and Regional Stranding Network numbers for each case they discussed. The
use of identifying codes for each animal should aid in the analysis of stranding data and allow for the cross-
referencing of multidisciplinary studies and the archiving of materials at different facilities.
The most ambitious study of a mass mortality of dolphins was reported by Geraci (1989). The
conclusions reached were also the most controversial. Data or specimens from 347 T. truncates that stranded
on the U.S. east coast from June 1987 to March 1988 were available for study. Gross findings from 240 partial
and 46 complete necropsies were presented in the report in a table by the number of animals with lesions,
although no information was available on exactly which animals had multiple lesions. Lesions from 95 dolphins
were examined histologically, 48 dolphins were examined for bacteria, and 42 for chlamydia. Additional studies
using electron microscopy, virology, and chemical analyses were performed on subsets of dolphins by various
research groups and institutions around the country. The animals had suffered a variety of diseases and lesions,
including unusual epidermal blistering and sloughing of the skin, massive systemic bacterial infections, and
necrosis of liver, lung, pancreas, and heart. Viruses, however, were not found in any unusual types or numbers,
although serological tilers to antibodies of a morbillivirus, canine distemper virus, were found in 6 of 13 blood
samples from dolphins captured alive off Virginia Beach in October 1987 (no active viral infection was present).
No single pattern of pathogens or parasites was evident from the studies. Chemical analyses performed on 75
dolphins (organochlorines) and 68 dolphins (heavy metals) showed that detectable amounts of DDTs, chlordanes,
and PCBs were present in every blubber and liver sample. The concentrations found in one animal were among
the highest values ever recorded in any animal tissue.
Based on suspected confounding factors, 17 dolphins were tested for red tide toxins (Geraci 1989).
Brevetoxin was found in eight of those dolphins, hi one menhaden from the stomach of one dolphin, and in one
fresh-caught fish. Other fish species tested from the study area did not have this red tide toxin. The research
team concluded that the brevetoxin poisoning was responsible for the death of the dolphins (since an unusual
bloom of Ptychodiscus brevis occurred off North Carolina in October of 1987). The dolphins appeared
emaciated; utilization of their blubber reserves had reduced their buoyancy and insulation and released stored
pollutants. It was speculated that the combined stresses had led to decreased immunocompetence, and the
animals had succumbed to opportunistic bacterial infections (Geraci, 1989). However, this scenario was
challenged by a number of researchers, resulting in a congressional hearing (Foglietta, 1989). Critics noted that
the animals had begun dying before the red tide occurred and that many red tides occur in the Gulf of Mexico
without such effects on the bottlenose dolphins there. The effects of brevetoxin on marine mammals are
unknown, but PCBs can impair functioning of the immune system and liver and cause skin lesions in terrestrial
mammals and humans. Although PCBs were found in a large subsample of animals, only a few animals and fish
were tested for the red tide toxins and the results were not significant. In defending the study's conclusions, Dr.
Geraci noted that little is known of the effects of toxic contaminants on dolphins, how much contamination can
occur before effects are observable, and whether the animals can metabolize the compounds. He also noted that
it would be impossible to perform appropriate lexicological tests on marine mammals. The 1989 report had
recommended further studies on biotoxins (such as toxin from red tide) in marine mammals and on whether
chemical contaminants at the levels found in this study could have affected susceptibility to biotoxins or
pathogens. Early necropsies and microbiological analyses had revealed that the animals had died of generalized
infections and septicemia, and had been weakened by immunosuppression or some malfunction of a major organ
before infection by microorganisms (Brody, 1989). Smith (1990) noted that over 50 percent of the bacteriologic
isolates were vibrios and hypothesized that sewage dumping (washing up on beaches at the tune of the
strandings) could have increased the numbers of vibrios to levels that could not be tolerated by the dolphins,
changing the normally saprophytic bacteria to a pathogen.
Additional studies have been conducted on chemical contaminants in marine mammals. In addition to
the dangers of exposure to floating or fouling oil and inhalation of volatile organics (Geraci and St. Aubin, 1982;
Geraci, 1990) and other air pollutants (Rawson et al., 1991), pollutants such as nutrients, microbial pathogens,
and toxic organic and inorganic chemicals are discharged into estuaries, nearshore coastal waters, and the open
ocean from municipal and chemical point source effluents, agricultural and urban nonpoint source runoff, and
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60
ocean dumping (Gaskin, 1982; Haebler and Moeller, 1993). As noted by Haebler and Moeller (1993), many
complex and varied factors affect the acute and long-term biological effects of contaminants, their fate and
transport, their accumulation in the food web, and synergistic interactions with environmental conditions that can
result in an increased incidence of disease in marine organisms. Among the suite of proposed biomarkers
(biochemical, physiological, and histological indicators to assess exposure to, or effects of, xenobiotic chemica s
on organisms) are a number of assays to analyze chemically-induced toxic effects on immunocompetent cells
(McCarthy and Shugart, 1990; Weeks et al., 1992). Measurements of either individual components or the entire
immune system may be used to monitor changes that could adversely affect the health of aquatic animals. The
Laboratory for Marine Mammal Immunology, University of California, Davis, California, is developing functional
assays for blood samples to measure immune status in several species of marine mammals (J.L. Stott, D. Ferrick,
Department of Microbiology/Immunology, University of California, personal communication).
It is important to note that all studies will be more valuable from a comparative standpoint if similar
methods and techniques are used. Kuehl et al. (1991) used standard procedures for chemical analysis of tissues
developed by EPA's Environmental Research Laboratory in Duluth, Minnesota. The NMFS is also working on
procedures for these chemical analyses (Calambokidis et al., 1984; see below). Details of the procedures must
be jnvcn in reports to assist in the interpretation of the results. Aguilar (1985) noted that there may be problems
in sampling different tissues. In particular, he noted that the blubber is not a homogeneous tissue and
orEanochlorines may be differentially distributed in the lipids. Borrell and Aguilar (1990) examined the problem
of^analyzing chemicals in a decomposing stranded dolphin over a period of 55 days. They noted that some
disease states may lead to abnormal rates of metabolization and excretion of pollutants; fat reserves may have
been mobilized as the animal's health declined; and pollutants in the carcass, may be affected by direct exposure
to sun high temperatures, wind, bacterial activity, and other factors before sampling occurs, thus altering the
composition and concentration of chemicals originally present in the tissues. They found that concentrations of
oreanochlorine pollutants (PCBs and DDTs) in muscle varied widely during the study, probably as a result of
weather conditions and variation in water content of the tissue. The lipid content of, the blubber progressively
decreased with time, perhaps due to leaking and volatilization of lipids during direct exposure to the sun Tissue
residue levels generally decreased" over time, but the decreases were not identical for all of the organochlorines
studied. Thus, badly preserved or unpreserved stranded cetaceans should be considered unreliable for this type
^The presence of lipophilic xenobiotics in blubber samples will also vary because of changes in the
animal's diet and nutritional status, its sex and age, and its reproductive status Females may transfer
contaminants to the fetus during gestation and to the offspring during lactation. This fact leads to the result flia
levels of contaminants in mature males are higher than those observed in mature females (a relationship that
will be more pronounced for organochlorines than for heavy metals), although there may be other confoundmg
factors (Tanabe et al., 1982; Reijnders, 1988, Cockcroft et al., 1989). Law et al. (1991) examined concentrations
of seven trace metals in the livers of seals, porpoises, 5. coeruleoalba, T. truncatus, L acutus, L. albirostns, and
D. delphis near the British Isles, finding elevated concentrations of mercury and lead in animals from the
Liverpool Bay area. They urged the development of more structured studies to assess the real risks to coastal
marine mammal populations. Other issues in the study of ^^^^°^^ T^TrUB
impacts of tissue contamination in cetaceans have been discussed by Risebrough (1978), Tanabe et al. (1983,
1988), Reijnders (1986,1988), and Granby and Kinze (1991).
Other questions remain. Landy (1980) reviewed reports of neoplasms in marine mammals, listing eight
cases in T. truncates. He observed that most cases in cetaceans were found in the whales collected during
whaling expeditions or from sporadic necropsies of beached animals. Since carcinogens had been linked to the
induction of neoplasms in fish, he proposed that the increasing pollution of the sea might lea1 toa sirmla
situation in marine mammals. Howard et al. (1983b) noted that so few tumors were found '" cetacean
because they were not susceptible to neoplasia) that it would be difficult to correlate observed e
exposure to carcinogens in these organisms and appropriate experimental studies arc not feas.ble Geraa et aL
(1987) rcanaly/ed 52 tumors reported in cetaceans, finding that 23 reports had been sufficiently deta.led to
onfirm the £oposcd diagnoses'while 15 tumors had been inadequately described but did have acceptable
diagnoses. They disputed 12 diagnoses, but confirmed 4 of those cases by combimng origmal descriptions and
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61
illustrations with contemporary information on etiology and classification. Adding 14 more cases from their files
brought the total to 41 confirmable tumors. Geraci et al. (1987) noted that the distribution of tumors in
cetaceans was most likely the result of biased sampling and the difficulties of examining all organ systems in large
animals or mass-stranded herds. In addition to the concern that neoplasms could be related to environmental
pollutants, they discussed other etiological scenarios, such as hormonal influences and viruses, and urged caution
in seeking causative agents while carefully documenting each case for future examination.
Haebler and Moeller (1993) reiterated the concerns of Geraci et al. (1987) regarding sampling biases
and disagreements over diagnosis and interpretation of tumors among pathologists. They called for a centralized
registry to archive microscopic slides and biological data from marine mammals with tumors in order to provide
an opportunity for more pathologists to examine the material and pool scientific information, standardize tumor
nomenclature and diagnosis, and compare data to improve our understanding of the temporal and geographic
distribution of tumor incidence. Although the AFIP maintains an appropriate facility for such materials, many
cases reported in the literature (cases of neoplasms as well as other diseases and lesions) have not been archived
there. (The AFIP is a voluntary program.) The use of new techniques to search for DNA-carcinogen adducts
may provide additional insights into the presence of pollutant or nonpollutant mechanisms for genetic damage
in these animals leading to the formation of neoplasms (Ray et al., 1991).
Interpreting the disease findings for stranded animals remains difficult. In reviewing the literature on
diseases of stranded animals, Simpson and Cornell (1983) noted that heavy parasite loads were often seen in such
animals; but since other debilitating diseases were seen as well, the major cause of the stranding was not easy
to determine. The pathogenicity of the parasite depends on location in the body and number of parasites,
general health and nutritional state of the host, and other endogenous or exogenous chemical or immune effects
that the parasite may present to the host. They suggested that investigators need to arrange parasites in order
of "known lethal influence" and then come to a diagnostic consensus. However, the extent of pathogenicity of
one or more parasites may not be well-defined. Is the host's condition a result of the parasitism or is the
parasitism the result of the host's condition? The contribution of parasites to morbidity and mortality is often
treated as a matter of relative probabilities because the prior condition of the animal is not known. Howard et
al. (1983a) stated that it was also difficult to attribute the cause of death to bacterial infections because growth
of natural or pathogenic endogenous bacteria can occur quickly, especially in warm temperatures. Most stranded
cetaceans are already dead when they wash ashore, live stranded cetaceans are usually already diseased and die
from overheating and cardiovascular collapse. Stranding stress may also increase opportunistic infections.
Moreover, bacterial diseases or septicemias may occur secondarily hi other diseases such as malnutrition,
parasitosis, and neoplasia. Swabs need to be taken during necropsy from all organs and tissues, not only from
obvious lesions, and from heart blood. Pyothorax or deep tissue lesions or wounds should also be examined for
anaerobes.
Cowan et al. (1986) examined beached dolphins collected over 4 years from a 100-mile stretch of
coastline near Los Angeles, California. Fifty-one of the 81 animals recovered by the Marine Mammal Disease
Surveillance Program were suitable for pathological examination. Cowan et al. (1986) divided their findings into
three categories:
1) Residual old diseases or minor active diseases probably unrelated to stranding;
2) Severe or fulminant processes probably causing the stranding; and
3) Processes related to tumbling about in the surf and lying up out of the water on the beach.
Although a variety of lesions were related to the latter problem, they noted that cardiovascular system
disease, found widely in this sample, was probably unrelated to stranding. They suggested that the cause of these
subepicardial scar lesions was periods of vasospasm, with no evidence of viral or toxin involvement. The mixing
of pre-existing and acute disease was most readily seen in the liver. A number of hypotheses were proposed to
explain the various findings, including nutritional or diffuse toxic etiology, particularly in regard to the liver and
mammary gland changes that appeared to be similar to those found in cattle exposed to highly chlorinated
organic compounds. They reported that no liver disease had been found in 68 individuals of 2 pelagic species
of dolphins that would not have been exposed to significant environmental contamination (Cowan and Walker,
1979). Parasitic damage was described and appropriately discussed, with events reconstructed, as a cause of
stranding mortalities. Cowan et al. (1986) concluded that catastrophic diseases causing immediate death would
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62
result in loss at sea, so the diseases found in this study represent those that allow dolphins to survive long enough
to reach^1g^n (1986y report provided a careful analysis of the possible set of lesions occurring in
critical areas that maybe the proximate cause(s) of stranding. Toxins were postulated to P^y a larger role than
S be determined by morphological methods alone, and should be investigated m ^ure studies ^0= tuches
have reported links to nutritional disorders in stranded animals except for the occurrence of malnutrition at the
S Snding, which may have been caused by pathogens, parasites, or exposure to contaminants Mead and
Patter (1990) however, noted no apparent differences in species composition between stomach contents o tm
sbaufed IT truncates and 6 dolphins captured incidentally in nets (admittedly a very limited comparison).
S^«*S£ai be undertaken £ this area. With the postulated loss of 50 percent of the estonated
boulcnose dolphin (T. truncatus) migratory stock from the U.S. Atlantic coast during the 1987-1988 mass
moctaJky (Scott and Burn, 1987; Scott et al, 1988), additional studies of diseases and the role of envnx^ental
(factors will be necessary to understand their influence on mortalities in this and other species of dolphm.
ONGOING RESEARCH PROGRAMS
Following the 1987-1988 bottlenose dolphin mass mortality on the Atlantic coast, additional efforts have
been made to support research on dolphin physiology, nutrition, biochemistry, immunology, ' "*£***£
These programs are designed largely to establish baseline levels of tasue contammants and to determnie the role
of StaS bioaccumulaTion and exposure to biological toxins in relation to dolphin leases and stranding
£ Sul information can be used to better assess risks to the health of various dolphin populations
S to continued studies undertaken at captive dolphin facilities, disease research performed on stranchng.
Mnson 1991), and long-term monitoring studies on feral populations in the field wdl continue. A few of the
projecTie ^cussf d in this section. As mentioned previously, the IAAAM Tissue Registry provides a
moi^
archived in their laboratories. While many of the requests are for »o«ntf
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63
manual for use by stranding network members. This manual will include an inventory and description of all
marine mammals in U.S. and Canada coastal waters, detailed information on handling stranded marine
mammals, and various sample collection protocols.
NMFS has also revised the Regional Stranding Network program to include the originally separate
National Marine Mammal Tissue Bank (NMMTB), located in Gaithersburg, MD. Although initially proposed
by Risebrough (1978), this program was set up in response to the 1987-1988 east coast dolphin die-off to obtain
baseline levels of environmental contaminants and biotoxins in marine mammals from the Atlantic, Pacific, and
Gulf of Mexico coasts of the United States. The combined program consists of four components—Stranding,
Monitoring, Tissue Bank, and Quality Assurance-with activities recommended by the NMMTB Team of
Scientists. The Stranding component is designed to improve the reporting of basic data from marine mammal
standings, to upgrade the capacity of Stranding Networks to respond more effectively to mortality events, and
to provide data that can be used for management purposes. The Monitoring portion of the program will conduct
a standard suite of analyses on 10 to 20 marine mammals from incidental fisheries catches or mass strandings
in each region, depending on the availability of funds. The normal suite of analyses will include organic and
inorganic compounds and toxins from blubber and liver tissue, necropsy, and histopathology. The Tissue Bank
will collect and store selected marine mammal tissues on a regular basis to be used in the monitoring studies,
as well as archiving them for future comparisons (NMFS 1992a).
The Quality Assurance component of the combined program was developed by the National Institute
of Standards and Technology (MIST) in collaboration with the NMFS's Environmental Conservation Division
in Seattle, Washington. This program will test and evaluate analytical methods for organic contaminants in lipid-
rich tissue matrices, conduct and evaluate interlaboratory comparison exercises with NIST and other NOAA
laboratories involved hi marine mammal tissue analyses, and develop Standard Reference Materials (SRMs) of
blubber and liver for use in the analysis of marine mammal tissues. A team of scientists drawn from marine
mammal research, analytical chemistry, chemical contaminants, toxicology, and specimen banking backgrounds
was consulted during the development of the program (Lillestolen et al., in press). Duplicate « 150 g samples
are banked, with one duplicate homogenized and the other remaining in bulk form. Fifty percent of each
specimen will be available to the scientific community upon written request to the Director of NMFS/OPR (and
with informal review by three members of the NMMTB's Team of Scientists), and 50 percent is intended for
long-term storage.
For Fiscal Year 1991, full analyses were initiated on 11 freshly dead stranded T. truncytus from the
southeast region. The Alaska Marine Mammal Tissue Archival Project was established in 1987 by the MMS,
as part of the National Biomonitoring Specimen Bank (NBSB) program at the NIST, to establish a representative
collection of Alaskan marine mammal tissues taken during native subsistence hunts for future contaminant
analysis and documentation of long-term trends in environmental quality. This program is now being managed
by the NMMTB & SN Program (NMFS 1992a). Currently, samples from 72 animals representing 8 species have
been collected, and some samples have been analyzed for inorganic and organic compounds (Becker et al., 1992;
NMMTBB & SN Program 1992 Update). Another program for analyzing levels of pollutants and
biotoxins in marine mammals is under way following a workshop held by the MMS Gulf of Mexico Region in
the summer of 1989 (Tucker and Associates, Inc., 1990).
A study of Sarasota Bay, FL, bottlenose dolphins (ongoing since 1970) is the longest running study
involving repeated sampling of dolphins hi the wild. This project has been conducted by a number of scientists
and veterinarians, with the assistance of over 200 volunteers from Earthwatch, New College, the University of
Florida, the University of South Florida, and the University of California at Santa Cruz. This research has been
supported by funding from Mote Marine Laboratory, the MMC, NMFS, the Inter-American Tropical Tuna
Commission, the University of California at Santa Cruz, the Denver Wildlife Research Center, Earthwatch, the
Office of Naval Research, and Woods Hole Oceanographic Institution, as well as donations of funds and
equipment to Dolphin Biology Research Associates (Wells, 1991). The behavior and ecology of the Sarasota
group of dolphins have been studied, particularly the social system and patterns of social development (reviewed
in Scott et al., 1990). As of 1988, 85 of the approximately 100 community members were readily recognizable,
65 had been handled since the sampling program began hi 1984, 74 were of known gender, 56 were of known
age, and 53 were of known gender and age. The sampling procedure, which has been described by Wells (1991),
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64
then the dolphin is freeze-branded, and pictures are taken of fins and scars.
«^^^
^
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immunosuDoressive pollutants in diseases and standings of dolphins.
In Se^ake^f the 1987-1988 bottlenose dolphin mass mortalities, additional support for studies to
the assessment of the health and health trends of marine mammal
between environmental parameters and marine mammal
healt fel nan adequate understanding of the causes of marine mammal brandings and
of the presence, levels, and effects of potentially harmful contaminants
SarSd methods for reporting stranded, dying, dead, or otherwise mcapacitated
. oormyt5 for the collection, preparation, and archiving of marine mammal tissues; and
. A need for broad access to data through a central data base to gather information on marine
mammals and analyses of their tissues. introduced bv
parameters.
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65
CONCLUSIONS
Investigations of diseases and causes of mortalities in captive and stranded dolphins have established that
a variety of pathogenic microorganisms, parasitic infestations, and nutritional disorders can adversely affect the
health of these mammals. The quality of studies being performed has improved over the years, with greater
emphasis being placed on testing hypotheses, examining larger numbers of animals, and designing studies to
include control animals. The need for adequate, long-term sources of support and continued training
opportunities for scientists and veterinarians involved in dolphin disease research has been recognized.
The necessity of integrating our knowledge of hazards in the marine environment with a thorough
understanding of mammalian diseases and the basic biology of healthy "normal" animals has also been noted.
New studies testing for the bioaccumulation of toxic pollutants, as well as naturally occurring toxic substances,
will provide additional information on the role of environmental contamination in susceptibility to pathogens.
Investigations are also under way to characterize the immune system of dolphins and other cetaceans that should
aid in the treatment of captive and live-stranded animals. Many of the reports on captive and stranded dolphins
noted that animals appeared to have suppressed immune systems, but further understanding of the cetacean
immune system will be necessary to interpret the disease state (Bossart, 1984; Lahvis et al., 1992).
Diagnoses of diseases in captive animals, however, may provide little insight into the prevalence and
distribution of different, often unrelated, diseases found in wild animals. Social groupings, individual and herd
behavior, and migration patterns, as well as the effects of other factors (pathogens, physiology, toxic chemicals,
environmental conditions) on young or adult animals, must be taken into account when examining diseases in
stranded or incidentally captured dolphins. Studies of physiology, biochemistry, and pathogenesis in captive
animals can provide information essential to understanding mechanisms of disease (Dr. J.R. Geraci, Ontario
Veterinary College, personal communication). Thus, research on captive and stranded dolphins should
complement, but not replace, one another, and both must be continued to provide the strongest program for
disease studies.
RECOMMENDATIONS
NMFS is continuing to resolve issues arising from the operation of the stranding networks. There
appear to be problems, however, with inconsistencies in the types and numbers of tissue samples collected from
each stranded animal, proper labeling and handling of tissues for chemical analyses, and the collection of
appropriate materials to obtain the age, gender, and reproductive status of the specimen (teeth and reproductive
organs). Additional information on the taxonomic status of the species (e.g., inshore/offshore/embayment
populations of T. truncatus) and their natural histories (e.g., feeding, diet, nutritional requirements, migrations)
is needed. Better coordination of collecting activities will also be required to ensure that all interested
investigators can obtain exactly what they need, preserved by the appropriate method, as soon as possible
following notification that a stranding has occurred. Several recommendations for improving the Regional
Marine Mammal Stranding Networks were developed at the Second Marine Mammal Stranding Workshop
(Reynolds and Odell, 1991). Interagency and individual cooperation should be encouraged to make the best use
of these relatively scarce research materials and to develop the broadest base of information for the identification
and interpretation of dolphin diseases and then- contributions to stranding mortalities.
Based on the published reports reviewed here, it is evident that the more tests and supporting
information gathered for each animal—whether captive, stranded, or collected—the more useful the information
will be. While the NMMTB program performs chemical analyses, necropsy, and histopathology on tissues from
each animal to be examined, apparently microbiological examinations are not included; such examinations may
be helpful, however, to properly interpret the histopathological findings in freshly dead animals. Furthermore,
skeletal remains, in particular the head, reproductive organs, and other materials, have not been collected for
each specimen. At the very least, the proper curation and archiving of tissues and organs collected before the
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condition of the carcass deteriorates or, in the case of skeletal structures before the carcass is buried with
maintenance of all information on each case, should permit qualified investigators to continue multidisciphnary
studies. following recommendations ^ proposed to strengthen and enhance the study of diseases in
dolphm ^
discussed in Wilkinson, 1991). All interested parties, identified previously for each region and/or
technical specialty, should be notified immediately when captive, stranded, or collected dolphins are
available for study, and all participants in a case should be kept informed about the studies being
conducted to maximize cooperation and minimize redundancy. Concurrent studies on apparently
healthy dolphins, as well as those showing signs of disease, must be performed to provide valuable
baseline comparative data (e.g., Rommel et al., 1991).
The latest techniques and equipment should be used whenever possible, following established quahty
assurance procedures for each, and standardized methods need to be established whenever possible
to increase the validity of comparisons with other studies. (NMFS is working with Regional
Stranding Networks to develop standardized protocols for conducting necropsies on stranded
animals and to collect more extensive data and tissues for analyses.) This will require interagency
cooperation in the development of such methods (e.g., chemical analyses) and the training of
appropriate personnel. New methods that could increase the amount of information such as
microbiological cultures, biochemical tests, tissue cultures and biomarker techniques, should be also
ae currently in operation (e.g., MMEP and NMMTB) should be fully supported with
appropriate computer equipment and personnel. If new databases should be required for storing
the data collected during these studies (morphological, chemical, pathological, other), and for
tracking transfers of materials and locations of archived material (as in § 307 of the 'Marine
Mammal Health and Stranding Response Act), they must be carefully developed and should be
identified for, and be accessible by, researchers. Some of these functions coul(f probably be added
to the databases maintained by MMEP andNMMTB, provided adequate equipment, personnel, and
Ions-term support are provided.
Types of disease data collected and research to be performed on each case should be standardized.
NMFS plans to establish a national information database to include information on collected tissues,
disposition of the collected tissues, and a summary of research conducted on these tissues to support
the Monitoring and NMMTB components of the Regional Stranding Networks program. The issue
of whether Level B and C data (Appendix A) are proprietary and, thus, may be released at the
discretion of the researcher (Wilkinson, 1991) needs further clarification. If data are not published
within a certain period of time, then the data should be entered into the appropriate database (e.g
in particular, other data, not in the researcher's specialty, collected during a study but not intended
for publication) All data collected on one animal should be identified by field and archiving
numbers (assigned by the stranding network regional director) for easy cross-referencing.
Disease researchers must be aware of current taxonomic revisions and systematics research since
this information may aid in establishing parasite or pathogen relationships, or new discoveries may
be made concerning distributions of different populations of dolphins or migration patterns that
could provide additional information on exposure to environmental stresses contributing to
mortalities. The latest review of marine mammal diseases by Dierauf (1990) contamed several
references to Stenella plagiodon, although this species had been synonymized with S. frontahs by
Perrin and colleagues in 1987. Research should be continued on the inshore/offshore/embayment
populations of T. truncatus (Mead and Potter, 1990), as well as other systematic problems. Because
Sstematics research may result in new species names and changes in current names as our
understanding of species and their evolutionary relationships is refined, appropriate voucher
materials from each carcass must be collected and archived to aid in this research and prevent the
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67
loss of costly data to future investigators (Lee et al., 1982; Heyning, 1991; Association of Systematics
Collections Alert).
• A more rapid method of dissemination of research results should be pursued. In addition to
professional meetings, workshops, and peer-reviewed publications, an accessible database could
provide up-to-date information on all aspects of disease research for these'animals. The location
of archived tissues, organs, and skeletal remains available for study could also be stored on this
system, and studies in progress or results of studies from laboratories around the country could be
cross-referenced to provide a more complete picture of disease factors and processes for each
animal examined. Annual reviews of the results of these studies should also be published to provide
a continuing record of the status of dolphin disease research, along the lines of Greenwood and
Taylor (1977, 1978, 1979), but with more detail.
• Archiving of histological preparations of diseased and normal tissues needs to be improved.
Researchers should submit materials to the AFJP so that they are available to all qualified
investigators for study. Workshops and courses utilizing these materials should be presented. In
particular, a workshop on neoplasms in marine mammals should be held to improve diagnosis of
neoplastic diseases and related disorders of these animals and to standardize tumor nomenclature,
as has been recently implemented for fish and invertebrate neoplasms. Publication of an atlas of
marine mammal neoplasms would also be useful for comparative studies.
Clearly, there have been problems in the past with the completeness of published studies, but the value
of multidiscipunary investigations of dolphin diseases has been recognized. The Marine Mammal Health and
Stranding Response Act may help the field in terms of providing additional support, especially funding for quick
response to mass mortality events, as well as improving facilities and training for marine mammal disease
specialists. However, support must be provided for more comprehensive studies. Furthermore, there must be
more equitable sharing of funds by agencies and researchers. While it is difficult to predict the availability of
dolphins for studies on their health or assessments of stranding mortalities, qualified investigators should be able
to receive prior peer review and approval of proposed research strategies, and then be notified and allowed to
participate in field or oceanarium studies as opportunities arise. Funds could be made available for this type
of research as proposed by the investigator (such as travel to/from site within the investigator's home region,
expendable supplies, certain analytical procedures). Databases storing the information collected from individual
and mass strandings, analytical procedures performed, or archiving will also require additional support. These
databases will continue to be important for comparative studies and for analyzing long-term trends. Because
funding is limited, further discussions of the appropriate use of available monies should be encouraged to identify
and support the most promising research leads while maintaining valuable archives for disease research.
Recently, mass mortalities of 150 striped dolphins (S. coeruleoalba) occurred in the summer of 1991 on
beaches of the Greek island of Zakinthos and around the coast of Italy and Sicily. Investigators have found
animals infected with same morbillivirus that caused striped dolphin mortalities off the Mediterranean coasts of
Spain and France during the_summer of 1990 (Jones, 1991a; Domingo et al., 1992). Currently, a reduced food
supply is suspected of leading to starvation, with suppression of the immune system and release of
immunodepressant PCBs, which may increase the susceptibility of the animals to the virus. Or, possibly a highly
pathogenic virus has been introduced into an immunologically naive population. As in the 1987-1988 mass
mortality of T. truncatus on the U.S. east coast, some animals contained unusually high levels of these
compounds. Mass strandings of T. truncatus occurred in February and March of 1990 along the coast of Texas,
with 112 animals in a two county area affected. Scientists are continuing investigations of environmental stress,
toxic contaminant or biotoxin exposure, and immune system dysfunction as possible factors in these mortalities
(NMFS, 1992b). As this apparent epizootic of morbillivirus spreads eastward in the Mediterranean (Jones,
1991b) and strandings continue along the coasts of the United States, scientists and veterinarians need to have
access to programs and funding that will allow rapid response and thorough analysis of the health of stranded
dolphins and that can be integrated with disease research programs at captive facilities.
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LIST OF ACRONYMS
AHP
AWA
CITES
DDTs
EPA
ESA
FWS
GC/MS
IAAAM
MABs
MMC
MMEP
MMIG
MMPA
MMS
MST
NOAA
NMFS
NMFS/OPR
NMMTB&SN
NMMTB
NMNH
PCBs
PCDDs/PCDFs
QA/QC
SEAN
SRM
USDA/APHIS
Armed Forces Institute of Pathology
Animal Welfare Act
Convention on International Trade in Endangered Species of Wild Fauna and
Flora
dichlorodiphenyltrichloroethane and related compounds
Environmental Protection Agency
Endangered Species Act
United States Fish and Wildlife Service
gas chromatography/mass spectrometry
International Association of Aquatic Animal Medicine
monoclonal antibodies
Marine Mammal Commission
Marine Mammal Events Program (Smithsonian Institution)
Marine Mammal Interest Group
Marine Mammal Protection Act
Minerals Management Service (Department of the Interior)
National Institute of Standards and Technology
National Oceanic and Atmospheric Administration (Department of
Commerce)
National Marine Fisheries Service (NOAA)
National Marine Fisheries Service, Office of Protected Resources
National Marine Mammal Tissue Bank and Stranding Network Program
(Oceans Act of 1992)
National Marine Mammal Tissue Bank
National Museum of Natural History (Smithsonian Institution)
polychlorinated biphenyls
polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans
quality assurance/quality control
Scientific Event and Alert Network (Smithsonian Institution)
standard reference materials (NMMTB)
United States Department of Agriculture/Animal and Plant Health Inspection
Service
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APPENDIX A
DATA TO BE COLLECTED
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Level A Data: Basic minimum data from all stranding events
(to be submitted to the National Office)
1.
2.
3.
4.
5.
6.
7.
8.
9.
Investigator
— name,
— address (institution)
Reporting source
Species
— preliminary identification (by qualified personnel)
— voucher (supporting material)
a) photograph—full lateral view (cetaceans); dorsal view (pinnipeds); dorsal, lateral, ventral views of whole
carcass, with close-up of head (when possible). Include a card with field number in each photo.
b) specimens—canine tooth or entire mandible (pinnipeds); 2 pieces of midrow baleen, or faulla if baleen
missing (mysticetes), tooth counts and samples, or entire skull for difficult species (odontocetes).
Field number
'Number of Animals
— total
— sub-groups (fragmented mass stranding)
Location
— preliminary description (local designation)
— latitude and longitude (to 0.1 minute, if possible) with closest named cartographical feature (USGS 1: 250,000
series) as determined subsequently in the lab.
Date, time
— first discovery
— of data and specimen recovery
Length .(Girth and Weight, when possible)
a) cetaceans and sirenians—tip of rostrum to fluke notch
b) pinnipeds—tip of rostrum to tip of tail, lying on back.
Condition—recorded for bodi discovery and recovery times. Categories as follows-
1) alive
2) freshly dead (i.e. edible)
3) decomposed, but organs basically intact
4) advanced decomposition (i.e. organs not recognizable, carcass intact)
5) mummified or skeletal remains only
10. Sex
a) cetaceans—probe genital slit (anteriorly directed are female, posteriorly directed are male)
b) pinnipeds—position of apertures
c) sirenians
From Appendix A, Hofmann (1991)
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Level B Data: Supplementary onsite information
(Augments data' on life history and the stranding event)
1, Weather and tide conditions
2. Orientation of carcasses
3. Offshore human/predator activity
4. Presence of prey species
5, Behavior
— pre-stranding
— stranding (on beach)
— after return to sea
6. Samples collected for subsequent analysis
A. Age Determination
a) odontocetes—4-5 adjacent teeth from the middle of the left lower tooth row.
b) mysticetes—minimum of one ear/plug, preferably in situ in a sample of externalauditory meatus, or
in a glove finger.
c) pinnipeds—minimum of 1 canine tooth - claw
d) sirenians—tusk, where present
B. Reproductive Tracts
a) females—both ovaries, uterus, fetus (if any) and measurements and samples of mammary glands.
b) males—one testicle with epididymis, or samples with weights and measurements, baculum (when pres-
ent), vas deferens.
C. Stomach Contents
— weigh contents, if possible
— preserve in alcohol (never in formalin)
— freeze whole, if possible
7. Disposition of carcass
From Appendix A, Hofmann (1991)
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Level C Data: Necropsy Examination and Parasite Collection
1. Necropsy
Precise recording of findings and appropriate preservation of tissue are of great importance to an understanding
of disease conditions. The most important characteristics of an abnormality are its SIZE and LOCATION Also
important are features such as COLOR, TEXTURE, and SHAPE, as well as the nature of the transition from
normal to abnormal tissue, that is, whether the boundaries are sharp or vague. All findings are described in
STANDARD ENGLISH using NON-TECHNICAL TERMS. Lesions are described using terms such as raised,
flat, depressed, rough, smooth, velvety, warty, yellowish, round, irregular, etc. Photographs should be made
whenever possible, and should include a ruler or some other non-ambiguous reference object.
External Examination—
Describe all unusual features such as marks, abrasions, parasites; examine mouth and teeth, etc.
Internal Examination—
Samples are to be taken routinely from all organs including brain, muscle, endocrine glands and viscera.
When an organ is normal, a random section should be preserved in formalin. Any abnormality should
be sampled with an adjacent piece of normal tissue. If an organ is: studded with many discrete lesions,
all apparently identical, sample only two or three. Describe organs as normal appearing, if that is the
case. Vessels and ducts are normally opened throughout their length. While this is in theory desirable
for the intestine, sampling of two or three tubular sections may be adequate. AH major organs are
weighed after cleaning of excess fat and extraneous tissue. Large organs are weighed in pieces, and'
the partial weights added. Hearts are normally weighed with a short cuff of aorta.
Preservation of Tissue ,
Formalin (10% neutral buffered) is the standard fixative. Tissue taken for histology should be fixed in for-
malin of a volume 20 times the volume of tissue. Tissues should be sliced thin—about 3 mm. Other dimen-
sions are not critical; 3x3 cm is a convenient size. Larger pieces of tissue do not fix well.
Whole lesions, e.g., stomach ulcer, may be taken and fixed, with good results as the wall of the organ
is thin. When possible cysts and cavities in tissue, pus-filled lesions and fluid found in body cavity should
be cultured for bacteria. Commercial holding media are excellent for the purpose, and their use is recom-
mended. Special requests for research material such as whole organ preparations should only be honored
if accompanied by detailed protocols.
Collection of Toxicology Specimens •
Tissue samples collected for pesticide and heavy metal analyses may be wrapped in aluminum foil or placed
in plastic bags. For prolonged storage, glass containers with teflon-lined lids are recommended. The samples
should be frozen as soon as possible, but may be transported on ice without significant loss of residues.
Samples of blubber, brain, liver, kidney and muscle should be collected routinely. Single assays may
be performed with as little as 10-20 g of tissue, but samples weighing 200 g or more are necessary for a
complete spectrum of analyses.
From Appendix A, Hofmann (1991)
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84
•> Parasite Collection
Parasites may be found anywhere within the body, but problem areas are identified as follows:
Head
— sinuses
— ears
— brain
Skin, Blubber
Muscle, Fascia
G. I. Tract
— including fecal sample
— liver, gallbladder, duct
— pancreas, duct
Respiratory
— major airways (opened)
— lungs
Uro-genital
— kidneys
— genital organs
— ureters, bladder
Blood
— sample or smear
Fixatives
A) Alcohol-Formalin Acetic Acid (AFA)-40 mL of 70% alcohol, 10 mL of 5 % formalin, 2 mL of acetic
acid, 48 mL of distilled water
B) Glycerin-Alcohol—5 mL of glycerin in 95 mL of 70% alcohol
C) Potassium Bichromate—2% aqueous
D) Formalin—5% solution
E) Ethanol—70% solution
Sampling Procedures
— subsample when large numbers are present
— do not distort
— ensure collection of head and tail
— sample portion of infected tissue when a parasite reaction is observed. Fix m A if possible
— measure and photograph, when possible
1) Nematodes
— fix in hot (16°C, 60° F) fixative B or
— place in tap water in cooler for 12 hours, then fix in solution A
2) Trematodes, Cestodes, Acanthacephalans
— place in tap water in cooler for 12 hours, then fix in solution A
3) Lice, Mites, Copepods, Barnacles
— fix in either D or E
4) Stool Sample
— preserve in fixative C
From Appendix A, Hofmann (1991)
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85
MARINE MAMMAL STRANDING REPORT
OMB#0648-0178, expires 01/31/94
SIDtt
FIELD NO. :
NMFS REGISTRATION NO.
(NMFS OSE)
COMMON NAME:.
OBSERVER
Name:
GENUS:
SPECIES:
Agency:.
Phone:
Address:
LOCATION
State:
County:.
City:
Locality Details:
* Latitude:.
* Longitude:.
TYPE OF OCCURRENCE
Mass Stranding: (Yes) / (No) # Animals.
Human Interaction: (Yes) / (No) / (?)
Check one: 1. Boat collision
2. Shot
4. Fishery interaction
5. Other
How determined:
Other Causes (if known):.
DATE OF INITIAL OBSERVATION:
Ifr Mo Day ..
CONDITION: Check one: 1.Alive
2.Fresh dead
3.Moderate decomp.
4.Advanced decomp.
5.Mummified
? Unknown
DATE OF EXAMINATION:
Yr ' Mo
Day
CONDITION: Check one: 1.Alive
2 .Fresh dead
3 . Mode ra te de comp.
4 .Advanced decomp.
5.Mummified
? Unknown
LIVE ANIMAL - Condition and Disposition:
3ieck one l.Released at site
or more: 2.Sick
3.Inj ured
4.Died
5.Euthanized
6.Rehabilitated and released
? Unknown
TAGS APPLIED?: (Yes) / (No)
TAGS PRESENT?: (Yes) / (No)
Dorsal
Left
Right
Pransported to:
(Died) / (Released) Date:.
Tag No. (s) :.
Color(s) :
Type:
Placement
Front/Reaar Front/Rear
ZARCASS - Disposition, check one:
rheck one: l.Left at site
2.Buried
3.Towed
4.Sci. collection (see below)
S.Edu. collection (see below)
_1_6.Other ;
? Unknown
reCROPSIED? (Yes) / (No)
MORPHOLOGICAL DATA:
Sex - Check one: I.Male
2.Female
? Unknown
Straight Length:.
*Weight:
.(cm)/(in)/test)
.(kg)/(lb)/(est?)
PHOTOS TAKEN? (Yes) / (No)
REMARKS:
DISPOSITION OF TISSUE/SKELETAL MATERIAL:
•Record data if available
It is estimated that completion
of this form requires 20 minutes.
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APPENDIX B
EXTENDED BIBLIOGRAPHY
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89
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