Toxoplasma gondii, Neospora caninum, Sarcocystis neurona, and Sarcocystis canis-like infections in marine mammals

Toxoplasma gondii, Neospora caninum, Sarcocystis neurona, and S. canis are related protozoans that can cause mortality in many species of domestic and wild animals. Recently, T. gondii and S. neurona were recognized to cause encephalitis in marine mammals. As yet, there is no report of natural exposure of N. caninum in marine mammals. In the present study, antibodies to T. gondii and N. caninum were assayed in sera of several species of marine mammals. For T. gondii, sera were diluted 1:25, 1:50, and 1:500 and assayed in the T. gondii modified agglutination test (MAT). Antibodies (MAT > or =1:25) to T. gondii were found in 89 of 115 (77%) dead, and 18 of 30 (60%) apparently healthy sea otters (Enhydra lutris), 51 of 311 (16%) Pacific harbor seals (Phoca vitulina), 19 of 45 (42%) sea lions (Eumetopias jubatus) [corrected] 5 of 32 (16%) ringed seals (Phoca hispida), 4 of 8 (50%) bearded seals (Erignathus barbatus), 1 of 9 (11.1%) spotted seals (Phoca largha), 138 of 141 (98%) Atlantic bottlenose dolphins (Tursiops truncatus), and 3 of 53 (6%) walruses (Odobenus rosmarus). For N. caninum, sera were diluted 1:40, 1:80, 1:160, and 1:320 and examined with the Neospora agglutination test (NAT) using mouse-derived tachyzoites. NAT antibodies were found in 3 of 53 (6%) walruses, 28 of 145 (19%) sea otters, 11 of 311 (3.5%) harbor seals, 1 of 27 (3.7%) sea lions, 4 of 32 (12.5%) ringed seals, 1 of 8 (12.5%) bearded seals, and 43 of 47 (91%) bottlenose dolphins. To our knowledge, this is the first report of N. caninum antibodies in any marine mammal, and the first report of T. gondii antibodies in walruses and in ringed, bearded, spotted, and ribbon seals. Current information on T. gondii-like and Sarcocystis-like infections in marine mammals is reviewed. New cases of clinical S. canis and T. gondii infections are also reported in sea lions, and T. gondii infection in an Antillean manatee (Trichechus manatus manatus).     

Thoracic auscultation in captive bottlenose dolphins (Tursiops truncatus), California sea lions (Zalophus californianus), and South African fur seals (Arctocephalus pusillus) with an electronic stethoscope

Thoracic auscultation is an important diagnostic method used in cases of suspected pulmonary disease in many species, as respiratory sounds contain significant information on the physiology and pathology of the lungs and upper airways. Respiratory diseases are frequent in marine mammals and are often listed as one of their main causes of death. The aim of this study was to investigate and report baseline parameters for the electronic-mediated thoracic auscultation of one cetacean species and two pinniped species in captivity. Respiratory sounds from 20 captive bottlenose dolphins (Tursiops truncatus), 6 California sea lions (Zalophus californianus), and 5 South African fur seals (Arctocephalus pusillus) were recorded with an electronic stethoscope. The sounds were analyzed for duration of the respiratory cycle, adventitious sounds, and peak frequencies of recorded sounds during expiration and inspiration as well as for sound intensity as reflected by waveform amplitude during the respiratory cycle. In respiratory cycles of the bottlenose dolphins' expiring "on command," the duration of the expiration was significantly shorter than the duration of the inspiration. In the examined pinnipeds of this study, there was no clear pattern concerning the duration of one breathing phase: Adventitious sounds were detected most often in bottlenose dolphins that were expiring on command and could be compared with "forced expiratory wheezes" in humans. This is the first report of forced expiratory wheezes in bottlenose dolphins; they can easily be misinterpreted as pathologic respiratory sounds. The peak frequencies of the respiratory sounds reached over 2,000 Hz in bottlenose dolphins and over 1,000 Hz in California sea lions and South African fur seals, but the variation of the frequency spectra was very high in all animals. To the authors' knowledge, this is the first systematic analysis of respiratory sounds of bottlenose dolphins and two species of pinnipeds.     

Brucella-induced abortions and infection in bottlenose dolphins (Tursiops truncatus).

Two bottlenose dolphins (Tursiops truncatus) aborted fetuses that died as a result of Brucella infection. Brucella placentitis occurred in both cases. Infected placenta and vaginal/uterine fluids may transmit Brucella species to other cetaceans. In a third case, an identical organism was cultured from lung necropsy tissue of an adult female T. truncatus. Microbiology, specific polymerase chain reaction, and pulsed-field gel electrophoresis results supported the designation of an additional genomic group(s), Brucella delphini, for isolates adapted to T. truncatus. Current serologic diagnostic tests reliable for known Brucella species are unreliable in detecting dolphin brucellosis. Our findings, together with previous reports, suggest that dolphin brucellosis is a naturally occurring disease that can adversely impact reproduction in cetaceans. The zoonotic significance of cetacean brucellosis is unknown, although the disease has not been reported in people who have frequent contact with dolphins. Further studies on the zoonotic aspects, distribution, prevalence, virulence, and impact of this disease in cetaceans and other marine mammal species are needed.     

Isolation of culturable microorganisms from free-ranging bottlenose dolphins (Tursiops truncatus) from the southeastern United States

Reports of diseases in marine mammals are increasing worldwide, however our understanding of the microorganisms associated with marine mammals is still limited. In this study, we cultured bacteria and fungi isolated from the upper respiratory tract (blowhole), gastric fluid and anus of 180 wild bottlenose dolphins (Tursiops truncatus) from two estuarine locations along the southeastern Atlantic Coast of the United States. A total of 339 and 491 isolates from Charleston, SC (CHS) and Indian River Lagoon, FL (IRL) dolphins, respectively, were cultured from gastric (70 CHS/82 IRL), fecal (141 CHS/184 IRL), and blowhole (128 CHS/225 IRL) swabs on selective media used for routine clinical microorganisms of human concern. The most frequently cultured Gram-negative bacteria from all sample and study types were Plesiomonas shigelloides, Aeromonas hydrophila, Escherichia coli, and Pseudomonas fluorescens. Among the Gram-positive bacteria, Clostridium perfringens, Bacillus sp., and Staphylococcus Coag. Neg were the predominant organisms. For fungi, the most abundant species were Candida glabrata, budding yeasts, and Candida tropicalis. Of concern, the MRSA strain of Staphylococcus aureus was detected in the blowhole and gastric swabs from CHS dolphins. In general, a greater prevalence of bacteria and fungi (four-fold increase) were cultured from IRL than CHS animals. Together, these culture-dependent studies, coupled to on-going culture-independent approaches, should help establish a baseline of microorganisms associated with bottlenose dolphins and aid in the identification of organisms responsible for infectious diseases(s) in these animals.     

Development and validation of monoclonal and polyclonal antibodies for the detection of immunoglobulin G of bottlenose dolphins (Tursiops truncatus).

Antibodies directed against species-specific immunoglobulin G (IgG) have a broad range of applications in serologic and immunologic research and in the development of clinical assays. Validated anti-IgG antibodies for marine mammal species are in short supply. The objective of this study was to produce and validate antibodies with specificity for IgG of the common bottlenose dolphin (Tursiops truncatus). Bottlenose dolphin IgG was purified using protein G. Two mouse monoclonal antibodies and a rabbit polyclonal antibody were developed from mice and rabbits immunized with bottlenose dolphin IgG. The specificity of the monoclonal antibodies and the polyclonal antibody for bottlenose dolphin IgG was first verified by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). For further validation, both monoclonal antibodies and the polyclonal antibody were incorporated in an indirect ELISA for the detection of the immune response of bottlenose dolphins to a vaccine antigen. Three bottlenose dolphins were immunized with a commercial Erysipelothrix rhusiopathiae vaccine, and serial blood samples were collected from all dolphins for measurement of levels of circulating antibodies. Seroconversion was observed in all 3 dolphins by use of both monoclonal antibodies and the polyclonal antibody. Circulating antibodies were detectable as early as 6 days after immunization in 1 dolphin. Peak antibody levels were detected 14 days after the immunization. The ability to detect seroconversion in all 3 immunized bottlenose dolphins firmly establishes the specificity of the monoclonal antibodies and the polyclonal antibody for IgG of the common bottlenose dolphin.     

Infection with Brucella ceti and high levels of polychlorinated biphenyls in bottlenose dolphins (Tursiops truncatus) stranded in south-west England

Eight bottlenose dolphins (Tursiops truncatus) that stranded in Cornwall, south-west England, between June 2004 and December 2007 were examined using standardised postmortem examination and bacteriological methods. Evidence of Brucella species infection was found in four of these dolphins on culture. In addition, of the eight dolphins, four were positive and two were weakly positive for antibodies to Brucella species on serological analyses of pericardial and other fluids using a competitive ELISA and two indirect ELISAs. High or very high levels of the sum of 25 individual chlorobiphenyl congeners (∑25CBs) were also determined in blubber samples from two of the dolphins (45.5 and 446.6 mg/kg lipid weight).     

Influence of season on the hematological and serum biochemical values of bottlenose dolphins (Tursiops truncatus) housed in a controlled environment in northern Italy

Season appears to influence the physiology of bottlenose dolphins (Tursiops truncatus). From 2000 to 2005, blood parameters of three captive bottlenose dolphins housed in northern Italy were examined to determine if seasonal variation was present. Seasonal variation was observed in the male dolphins, with both males exhibiting aminotransferase levels that were higher in autumn and lower in winter. Mean serum creatinine levels were higher during summer and lower during autumn in the adult male, and mean lactate dehydrogenase higher during summer and lower during spring in the juvenile male. Both males exhibited red blood cell counts and hemoglobin levels that were higher during autumn and lower during summer. This study contributes to the knowledge of baseline hematologic and biochemical values based on seasonality in bottlenose dolphins.     

Haematological findings in captive dolphins and whales

Objective To examine haematological features in five species of healthy, captive marine mammals.
Animals Twenty bottlenose dolphins ( Tursips truncatus ), seven Pacific white-sided dolphins ( Lagenorhynchus obliquidens ), five Risso dolphins ( Grampus griseus ) and five false killer whales ( Pseudorca crassidens ).
Results and conclusion The red blood cell count was 4.21 × 10¹²/L in bottlenose dolphins, 5.32 × 10¹²/L in Pacific white-sided dolphins, 4.35 × 10¹²/L in Risso dolphins and 4.43 × 10¹²/L in false killer whales. The haemoglobin concentration was 1.51 g/L and packed cell volume 44.7% in bottlenose dolphins; the corresponding values were 1.71 g/L and 48.9% in Pacific white-sided dolphins, 1.72 g/L and 49.4% in Risso dolphins, and 1.52 g/L and 47.8% in false killer whales. The white blood cell count was 7.097 × 10⁹/L in bottlenose dolphins, 5.928 × 10⁹/L in Pacific white-sided dolphins, 5.001 × 10⁹/L in Risso dolphins and 7.921 × 10⁹/L in false killer whales. There were no significant differences in these values among bottlenose dolphins and Pacific white-sided dolphins. The proportion of eosinophils in the differential leukocyte count ranged from 10.3% to 11.5% in bottlenose dolphins, Pacific white-sided dolphins and false killer whales, but was only 0.4% in Risso dolphins. The eosinophilic granules were larger in Risso dolphins and false killer whales than in bottlenose and Pacific white-sided dolphins.     

Malignant seminoma with metastasis, Sertoli cell tumor, and pheochromocytoma in a spotted dolphin (Stenella frontalis) and malignant seminoma with metastasis in a bottlenose dolphin (Tursiops truncatus)

Seminoma with metastasis was diagnosed in a spotted dolphin (Stenella frontalis) and an Atlantic bottlenose dolphin (Tursiops truncatus). Sertoli cell tumor and pheochromocytoma were also diagnosed in the spotted dolphin. The spotted and bottlenose dolphins were adult males that stranded and died on the coasts of northwest Florida and southeast North Carolina, respectively. Neoplasia is infrequently reported in cetaceans. This is the first report of seminoma, Sertoli cell tumor, and pheochromocytoma in a dolphin, the first report of three distinct neoplasms in a dolphin, and one of the few reports of malignant neoplasia in dolphins.     

Bronchoscopic and serologic diagnosis of Aspergillus fumigatus pulmonary infection in a bottlenose dolphin (Tursiops truncatus).

A 4-yr-old male bottlenose dolphin (Tursiops truncatus) developed an Aspergillus fumigatus pneumonia. Fungal elements were identified by cytology and microbiology from endoscopic bronchoalveolar lavage and brushings of a raised yellow endobronchial lesion. The results of qualitative immunodiffusion serology, a technique that identifies specific circulating antibodies to Aspergillus fumigatus, were suggestive of an active infection. The dolphin was treated with itraconazole for over 2 yr, which resulted in remission of clinical signs. Pneumonia caused by Aspergillus sp. accounts for the large majority of pulmonary mycoses in marine mammals. Bronchoscopy facilitated an early definitive diagnosis, accurate treatment, and remission.     

Hematologic, biochemical, and endocrine effects of dexamethasone on bottlenose dolphins (Tursiops truncatus).

Two Atlantic bottlenose dolphins (Tursiops truncatus) were given 0.11 mg/kg dexamethasone p.o., and complete blood count and serum chemistry analyses, including insulin, thyroxine (T4) adrenocorticotrophic hormone (ACTH), and cortisol level determinations, were performed at 0 hr, 24 hr, 36 hr, 48 hr, 7 days, and 17 days. Significant changes included neutrophilia, eosinopenia, lymphopenia, elevated insulin, and depressed ACTH and cortisol levels within 24 hr of dexamethasone administration. These effects were rapid, and values returned to normal within 48 hr.     

Toxoplasmosis in Tammar wallabies (Macropus eugenii) in the Budapest Zoo and Botanical Garden (2006-2010)

Smaller macropodid species (commonly referred to as wallabies) are extremely susceptible to toxoplasmosis: in most cases, infection with Toxoplasma gondii leads to death within a short time. Between June 2006 and July 2010, T. gondii was detected by immunohistochemical examination in six Tammar wallabies (Macropus eugenii) that died in the Budapest Zoo and Botanical Garden; in another four specimens histopathology revealed T. gondii-like organisms (which could not be differentiated from Neospora caninum solely by morphology), and in another 11 animals toxoplasmosis as the possible cause of death could not be excluded. The current zoo population of 12 Tammar wallabies was tested for T. gondii IgG antibodies by the modified agglutination test (MAT), with negative results. We suppose that most of the deaths were due to acute toxoplasmosis resulting from a recent infection.     

Saksenaea vasiformis and Apophysomyces elegans zygomycotic infections in bottlenose dolphins (Tursiops truncatus), a killer whale (Orcinus orca), and pacific white-sided dolphins (Lagenorhynchus obliquidens).

During a 10-yr period, a killer whale (Orcinus orca), two Pacific white-sided dolphins (Lagenorhynchus obliquidens), and two bottlenose dolphins (Tursiops truncatus), all housed at SeaWorld of Texas from 1991 to 2001, were infected with fungi from the class Zygomycetes. In four out of five cases, the fungi were identified as either Saksenaea vasiformis or Apophysomyces elegans. All fungi in the class Zygomycetes aggressively invade the vascular system. Death occurred within 23 days after the initial clinical signs. The primary site of infection involved the s.c. tissue and skeletal musculature. In one case, infection originated in the placenta and uterus of a periparturient animal. All cases exhibited systemic spread of the organisms, including two to the central nervous system. The fifth and most recent case, a bottlenose dolphin, was treated with liposomal nystatin, an antifungal formulation with reduced nephrotoxicity. This animal initially responded to therapy; however, 14 days after cessation of therapy, fungal growth reoccurred. Thus, the animal was euthanatized 39 days after the initial clinical signs. This drug represents a promising treatment option if combined with early disease detection and aggressive tissue resection.     

Escherichia coli septicemia associated with lack of maternally acquired immunity in a bottlenose dolphin calf

Stillbirth and neonatal mortality are substantial problems in captive bottlenose dolphins (Tursiops truncatus). The cause of these problems often is unknown. We report a case of Escherichia coli septicemia in a male 3-day-old bottlenose dolphin calf. Lesions included omphalitis, synovitis, and hepatic necrosis associated with the presence of Gram-negative bacilli. E. coli was isolated in pure culture from multiple organs. A serum gammaglobulin level of 1.5 g/L indicated a lack of maternally acquired immunity. The observed failure to nurse may have resulted from brain injury due to perinatal asphyxia. Evidence for perinatal asphyxia was the diffuse presence of a moderate amount of meconium in the lungs.     

Paternity determination of captive bottlenose dolphins (Tursiops truncatus) using microsatellite DNA analysis

We applied previously published PCR primer pairs to amplify alleles at three polymorphic microsatellite loci to determine the genetic relationship of 6 bottlenose dolphins (Tursiops truncatus) that were living together in a Japanese aquarium. The three microsatellite loci were sufficient to determine the haplotype relationships of the six dolphins, which represented three different generations. It was confirmed that this genotyping method is simple and economical for assessing, establishing and maintaining genetic diversity in captive populations and will become a very effective technique for ex situ conservation in aquariums and zoos.     

Congenital diffuse hyperplastic goiter associated with perinatal mortality in 11 captive-born bottlenose dolphins (Tursiops truncatus).

Diffuse hyperplastic goiter was diagnosed by histopathology in 11 perinatal bottlenose dolphins (Tursiops truncatus) that died at four separate zoos and aquaria. Thyroid morphology of these animals was compared with the histologically normal thyroids of two stranded wild bottlenose dolphin calves, a neonate and a 2-mo-old calf. Histologic changes included reduced follicular luminal diameter, markedly reduced or absent luminal colloid, hypertrophy of follicular epithelium, and follicular dysplasia. The etiology of the thyroid gland lesion was not identified. Cause of death was not determined for most of these animals, but they were presumed to have died from metabolic derangements associated with the thyroid lesion, drowning, or dystocia.     

Histopathological and immunohistochemical studies on cetaceans found stranded on the coast of Italy between 1990 and 1997

Detailed histopathological and immunohistochemical studies were carried out on 24 cetaceans, 17 of which were striped dolphins (Stenella coeruleoalba), four bottlenose dolphins (Tursiops truncatus) and three Risso's dolphins (Grampus griseus), all found stranded on the Italian coast between 1990 and 1997. The most frequently detected lesions were chronic pneumonia (73.7% of the examined lungs), focal chronic pancreatitis (71.4%), non-purulent encephalitis (50%), chronic hepatitis (42.1%), and chronic focal interstitial nephritis (31.2%). The skin and the subcutaneous panniculus were often (33.3%) affected by parasitic diseases (Phyllobothrium delphini and Pennella sp.). An appreciable percentage of animals showed lymphoid depletion in their lymphatic organs (47.2%), as well as a high rate of parasitic infestations in their alimentary tracts (25%).     

An insight into the epidemiology of dolphin morbillivirus worldwide

Serum samples from 288 cetaceans representing 25 species and originating from 11 different countries were collected between 1995 and 1999 and examined for the presence of dolphin morbillivirus (DMV)-specific antibodies by an indirect ELISA (iELISA) (N = 267) or a plaque reduction assay (N = 21). A total of 35 odontocetes were seropositive: three harbour porpoises (Phocoena phocoena) and a common dolphin (Delphinus delphis) from the Northeastern (NE) Atlantic, a bottlenose dolphin (Tursiops truncatus) from Kent (England), three striped dolphins (Stenella coeruleoalba), two Risso's dolphins (Grampus griseus) and a bottlenose dolphin from the Mediterranean Sea, one common dolphin from the Southwest (SW) Indian Ocean, three Fraser's dolphins (Lagenodelphis hosei) from the SW Atlantic, 18 long-finned pilot whales (Globicephala melas) and a bottlenose dolphin from the SW Pacific as well as a captive bottlenose dolphin (Tursiops aduncus) originally from Taiwan. The presence of morbillivirus antibodies in 17 of these animals was further examined in other iELISAs and virus neutralization tests. Our results indicate that DMV infects cetaceans worldwide. This is the first report of DMV-seropositive animals from the SW Indian, SW Atlantic and West Pacific Oceans. Prevalence of DMV-seropositives was 85.7% in 21 pilot whales from the SW Pacific and both sexually mature and immature individuals were infected. This indicates that DMV is endemic in these animals. The same situation may occur among Fraser's dolphins from the SW Atlantic. The prevalence of DMV-seropositives was 5.26% and 5.36% in 19 common dolphins and 56 harbour porpoise from the NE Atlantic, respectively, and 18.75% in 16 striped dolphins from the Mediterranean. Prevalence varied significantly with sexual maturity in harbour porpoises and striped dolphins; all DMV-seropositives being mature animals. The prevalence of seropositive harbour porpoise and striped dolphins appeared to have decreased since previous studies. These data suggest that DMV is not endemic within these populations, that they are losing their humoral immunity against the virus and that they may be vulnerable to new epidemics.     

Comparative histological study of the mammalian facial nucleus

We performed comparative Nissl, Klüver-Barrera and Golgi staining studies of the mammalian facial nucleus to classify the morphologically distinct subdivisions and the neuronal types in the rat, rabbit, ferret, Japanese monkey (Macaca fuscata), pig, horse, Risso's dolphin (Grampus griseus), and bottlenose dolphin (Tursiops truncatus). The medial subnucleus was observed in all examined species; however, that of the Risso's and bottlenose dolphins was a poorly-developed structure comprised of scattered neurons. The medial subnuclei of terrestrial mammals were well-developed cytoarchitectonic structures, usually a rounded column comprised of densely clustered neurons. Intermediate and lateral subnuclei were found in all studied mammals, with differences in columnar shape and neuronal types from species to species. The dorsolateral subnucleus was detected in all mammals but the Japanese monkey, whose facial neurons converged into the intermediate subnucleus. The dorsolateral subnuclei of the two dolphin species studied were expanded subdivisions comprised of densely clustered cells. The ventromedial subnuclei of the ferret, pig, and horse were richly-developed columns comprised of large multipolar neurons. Pig and horse facial nuclei contained another ventral cluster, the ventrolateral subnucleus. The facial nuclei of the Japanese monkey and the bottlenose dolphin were similar in their ventral subnuclear organization. Our findings show species-specific subnuclear organization and distribution patterns of distinct types of neurons within morphological discrete subdivisions, reflecting functional differences.     

Phylogenetic analysis of marine mammal herpesviruses

Five novel DNA-dependent DNA polymerase (Dpol) herpesviral sequences were generated using nested consensus polymerase chain reaction (PCR) in clinical samples from a harbor seal (Phoca vitulina), bottlenose dolphin (Tursiops truncatus), orca (Orcinus orca), California sea lion (Zalophus californianus), and a Phocid herpesvirus 2 (PhHV-2) isolate from a harbor seal (used as positive control). These novel sequences and other representative herpesvirus sequences were included in Bayesian and Maximum Likelihood analyses to illustrate the phylogeny of herpesviruses amongst the marine mammal host species and in comparison to those of other animals. All 19 novel and known marine mammal herpesviruses included in the analyses aligned with members of the Alphaherpesvirinae or Gammaherpesvirinae subfamilies. The novel harbor seal herpesvirus clustered with members of the Macavirus genus, subfamily Gammaherpesvirinae. The novel bottlenose dolphin herpesvirus clustered together in a monophyletic group with another delphinid alphaherpesvirus but could not be associated with an established genus. The orca herpesvirus also clustered with a delphinid alphaherpesvirus and formed a separate clade. The sea lion herpesvirus clustered with PhHV-2. PhHV-1 clustered with varicelloviruses and PhHV-2 clustered strongly in the Gammaherpesvirinae genus Percavirus. All cetacean gammaherpesviruses formed a monophyletic clade and could not be associated with an established gammaherpesviral genus.