Evidence of Helicobacter sp. in dental plaque of captive dolphins (Tursiops gephyreus)

Gastrointestinal lesions have been extensively reported in wild and captive marine mammals. However, their etiology remains unclear. In humans and other animals, chronic gastritis and peptic ulcers have been associated with Helicobacter sp. Therefore, the aim of our study was to investigate the presence of Helicobacter sp. in the gastric juice, dental plaque, and saliva of marine mammals living in a controlled environment. Five dolphins (Tursiops gephyreus), one killer whale (Orcinus orca), one false killer whale (Pseudorca crassidens), three sea lions (Otaria flavescens), two elephant seals (Mirounga leonina), and two fur seals (Arctocephalus australis) were studied. Saliva, dental plaque, and gastric juice samples were examined for Helicobacter sp. using polymerase chain reaction. None of the gastric juice or saliva samples were positive for Helicobacter sp. However, Helicobacter sp. DNA was detected in dental plaque from two dolphins, suggesting the oral cavity might be a reservoir of this bacterium.     

Molecular and morphometric evidence for separate species of Uncinaria (Nematoda: Ancylostomatidae) in California sea lions and northern fur seals: hypothesis testing supplants verification

California sea lions (Zalophus californianus) and northern fur seals (Callorhinus ursinus) are each believed to host distinct hookworm species (Uncinaria spp.). However, a recent morphometric analysis suggested that a single species parasitizes multiple pinniped hosts, and that the observed differences are host-induced. To explore the systematics of these hookworms and test these competing hypotheses, we obtained nucleotide sequences of nuclear ribosomal DNA (D2/D3 28S, D18/D19 28S, and internal transcribed spacer [ITS] regions) from 20 individual hookworms parasitizing California sea lion and northern fur seal pups where their breeding grounds are sympatric. Five individuals from an allopatric population of California sea lions were also sampled for ITS-1 and D18/D19 28S sequences. The 28S D2/D3 sequences showed no diagnostic differences among hookworms sampled from individual sea lions and fur seals, whereas the 28S D18/D19 sequences had one derived (apomorphic) character demarcating hookworms from northern fur seals. ITS sequences were variable for 7 characters, with 4 derived (apomorphic) states in ITS-1 demarcating hookworms from California sea lions. Multivariate analysis of morphometric data also revealed significant differences between nematodes representing these 2 host-associated lineages. These results indicate that these hookworms represent 2 species that are not distributed indiscriminately between these host species, but instead exhibit host fidelity, evolving independently with each respective host species. This evolutionary approach to analyzing sequence data for species delimitation is contrasted with similarity-based methods that have been applied to numerous diagnostic studies of nematode parasites.     

Serum chemistry and antibodies against pathogens in antarctic fur seals, Weddell seals, crabeater seals, and Ross seals

Information on health parameters, such as antibody prevalences and serum chemistry that can reveal exposure to pathogens, disease, and abnormal physiologic conditions, is scarce for Antarctic seal species. Serum samples from Antarctic fur seals (Arctocephalus gazella, n=88) from Bouvet?ya (2000-2001 and 2001-2002), and from Weddell seals (Leptonychotes weddellii, n=20), Ross seals (Ommatophoca rossii, n=20), and crabeater seals (Lobodon carcinophagus, n=9) from the pack-ice off Queen Maud Land, Antarctica (2001) were analyzed for enzyme activity, and concentrations of protein, metabolites, minerals, and cortisol. Adult Antarctic fur seal males had elevated levels of total protein (range 64-99 g/l) compared to adult females and pups (range 52-79 g/l). Antarctic fur seals had higher enzyme activities of creatine kinase, lactate dehydrogenase, and amylase, compared to Weddell, Ross, and crabeater seals. Antibodies against Brucella spp. were detected in Weddell seals (37%), Ross seals (5%), and crabeater seals (11%), but not in Antarctic fur seals. Antibodies against phocine herpesvirus 1 were detected in all species examined (Antarctic fur seals, 58%; Weddell seals, 100%; Ross seals, 15%; and crabeater seals, 44%). No antibodies against Trichinella spp., Toxoplasma, or phocine distemper virus (PDV) were detected (Antarctic fur seals were not tested for PDV antibodies). Antarctic seals are challenged by reduced sea ice and increasing temperatures due to climate change, and increased anthropogenic activity can introduce new pathogens to these vulnerable ecosystems and represent a threat for these animals. Our data provide a baseline for future monitoring of health parameters of these Antarctic seal species, for tracking the impact of environmental, climatic, and anthropogenic changes in Antarctica over time.     

Isolation and characterization of novel Helicobacter spp. from the gastric mucosa of harp seals Phoca groenlandica

Since the recent discovery of Helicobacter cetorum in cetaceans and its role in the development of gastritis, speculation has existed as to whether pinnipeds have Helicobacter spp. associated gastritis and peptic ulcer disease. The gastric mucosa of 4 stranded harp seals Phoca groenlandica from the Massachusetts coastline were assessed for Helicobacter spp. by culture and PCR. We cultured 2 novel Helicobacter spp. from the pyloric antrum of 1 of the 4 harp seals studied, and identified these by PCR in 2 of the 4 seals. Both gram-negative bacterial isolates were catalase- and oxidase-positive. However, a fusiform helicobacter with flexispira morphology was urease-positive, and a spiral-shaped helicobacter was urease-negative. Slender, spiral and fusiform-shaped bacteria were detected in the gastric mucosa by the Warthin-Starry stain. Histopathologic analysis revealed mild diffuse lymphoplasmacytic gastritis within the superficial mucosa of the pyloric antrum of both infected seals. The 2 bacterial isolates were classified by 16S rRNA analysis; they clustered with other enteric helicobacters and represent 2 novel Helicobacter spp. The urease-negative bacterial isolate clustered with H. canis and the urease-positive isolate clustered with an isolate from a sea lion and isolates from sea otters. This cluster of pinniped isolates has 97 % similarity to a number of Helicobacter species, but appears to be most closely related to other helicobacters with flexispira morphology. These findings suggest that the novel Helicobacter spp. may play a role in the etiopathogenesis of gastrointestinal diseases in pinnipeds. To our knowledge, this represents the first isolation and characterization of a novel Helicobacter spp. from pinnipeds.     

Novel and canine genotypes of Giardia duodenalis in harbor seals ( Phoca vitulina richardsi)

Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.     

Prey capture and processing behaviors vary with prey size and shape in Australian and subantarctic fur seals

When hunting at sea, pinnipeds should adapt their foraging behaviors to suit the prey they are targeting. We performed captive feeding trials with two species of otariid seal, Australian fur seals (Arctocephalus pusillus doriferus) and subantarctic fur seals (Arctocephalus tropicalis). This allowed us to record detailed observations of how their foraging behaviors vary when presented with prey items that cover the full range of body shapes and sizes encountered in the wild. Small prey were captured using suction alone, while larger prey items were caught in the teeth using raptorial biting. Small fish and long skinny prey items could then be swallowed whole or processed by shaking, while all prey items with body depths greater than 7.5 cm were processed by shaking at the water's surface. This matched opportunistic observations of feeding in wild Australian fur seals. Use of “shake feeding” as the main prey processing tactic also matches predictions that this method would be one of the only tactics available to aquatic tetrapods that are unable to secure prey using their forelimbs.     

Metagenomic Survey of Viral Diversity Obtained from Feces of Subantarctic and South American Fur Seals

The Brazilian South coast seasonally hosts numerous marine species, observed particularly during winter months. Some animals, including fur seals, are found dead or debilitated along the shore and may harbor potential pathogens within their microbiota. In the present study, a metagenomic approach was performed to evaluate the viral diversity in feces of fur seals found deceased along the coast of the state of Rio Grande do Sul. The fecal virome of two fur seal species was characterized: the South American fur seal (Arctocephalus australis) and the Subantarctic fur seal (Arctocephalus tropicalis). Fecal samples from 10 specimens (A. australis, n = 5; A. tropicalis, n = 5) were collected and viral particles were purified, extracted and amplified with a random PCR. The products were sequenced through Ion Torrent and Illumina platforms and assembled reads were submitted to BLASTx searches. Both viromes were dominated by bacteriophages and included a number of potentially novel virus genomes. Sequences of picobirnaviruses, picornaviruses and a hepevirus-like were identified in A. australis. A rotavirus related to group C, a novel member of the Sakobuvirus and a sapovirus very similar to California sea lion sapovirus 1 were found in A. tropicalis. Additionally, sequences of members of the Anelloviridae and Parvoviridae families were detected in both fur seal species. This is the first metagenomic study to screen the fecal virome of fur seals, contributing to a better understanding of the complexity of the viral community present in the intestinal microbiota of these animals.     

Prevalence of Cryptosporidium spp. and Giardia spp. in five marine mammal species

Cryptosporidium spp. and Giardia spp. are protozoan parasites that are often associated with severe diarrheal disease in a variety of mammals. Although these parasites have been extensively studied in terrestrial ecosystems, little is known about either parasite in the marine environment. Therefore, the objective of this study was to determine the prevalence of both Cryptosporidium spp. and Giardia spp. in 5 marine mammal species. Fecal samples were collected from 39 bowhead whales (Balaena mysticetus), 49 North Atlantic right whales (Eubalaena glacialis), 31 ringed seals (Phoca hispida), 22 bearded seals (Erignathus barbatus), and 18 beluga whales (Delphinapterus leucas) between 1998 and 2003. Using an immunofluorescent assay, parasites were detected in the feces of bowhead whales, right whales, and ringed seals, while neither parasite was detected in samples from bearded seals or beluga whales. Overall, prevalences were highest in ringed seals (Cryptosporidium spp., 22.6%; Giardia spp., 64.5%) and right whales (Cryptosporidium spp., 24.5%; Giardia spp., 71.4%) and lowest in bowhead whales (Cryptosporidium spp., 5.1%; Giardia spp., 33.3%). To our knowledge, this is the first report of Cryptosporidium spp. and Giardia spp. in either whale species and of Cryptosporidium spp. in the ringed seal.     

CAN NEAR INFRARED SPECTROSCOPY BE USED TO IMPROVE ASSESSMENT OF MARINE MAMMAL DIETS VIA FECAL ANALYSIS?

We investigated the utility of Near Infrared Spectroscopy as a means to quantify the diet of seals via analysis of feces. A pilot study showed that we could accurately determine the proportion of each species in a mixture of flesh of two species of fish and one squid. Having done so, we then assessed whether the same capacity existed for fecal material. Again we used two species of fish and one squid as the diet components offered to two captive seals (a California sea lion and an Australian fur seal). Five of the six calibrations could accurately and precisely quantify how much of a given diet component the seal had eaten the previous day from an NIR scan of the feces. NIR spectroscopy is theoretically a viable way to quantify seal diets. We discuss the logistical requirements for development of calibration equations for application to a field study. These may be prohibitive in many cases, but in others may be offset by the particular circumstances or objectives of the study. Adoption of NIRS may confer significant benefits in these circumstances.     

Molecular Detection of Circovirus and Adenovirus in Feces of Fur Seals (<Emphasis Type="Italic">Arctocephalus</Emphasis> spp.)

In some regions, little is known about exposure to viruses in coastal marine mammals. The present study aimed to detect viral RNA or DNA in 23 free-ranging fur seals on the northern coastline of Rio Grande do Sul State, Brazil. Polymerase chain reaction was used to detect nucleic acids of circoviruses, adenoviruses, morbilliviruses, vesiviruses, and coronaviruses in the feces from twenty-one South American fur seals (Arctocephalus australis) and two Subantarctic fur seals (A. tropicalis). Adenovirus DNA fragments were detected in two South American fur seals; nucleotide sequences of these fragments revealed a high degree of similarity to human adenovirus type C. Circovirus DNA fragments were detected in six animals of the same species. Two were phylogenetically similar to the Circovirus genus, whereas the other four nucleotide fragments showed no similarity to any of the known genera within the family Circoviridae. RNA fragments indicating the presence of coronavirus, vesivirus, and morbillivirus were not detected. These findings suggest that adenoviruses and circoviruses are circulating in fur seal populations found along the coast of Rio Grande do Sul State, Brazil.     

Survey and comparison of major intestinal flora in captive and wild ring-tailed lemur (Lemur catta) populations

A survey to identify the major intestinal species of aerobic bacteria, protozoa and helminths was conducted on captive and wild populations of ring-tailed lemurs (Lemur catta). Samples were collected from 50 captive lemurs at 11 zoological institutions in the United States. In Madagascar, 98 aerobic bacteria samples and 99 parasite samples were collected from eight sites chosen to cover a variety of populations across the species range. Identical collection, preservation and lab techniques were used for captive and wild populations. The predominant types of aerobic bacteria flora were identified via five separate tests. The tests for parasites conducted included flotation, sedimentation and FA/GC. Twenty-seven bacteria unique to either the captive or wild populations were cultured with eight of these being statistically significantly different. Fourteen bacteria common to both populations were cultured, of which six differed significantly. Entamoeba coli was the only parasite common to both the captive and wild populations. Giardia spp., Isospora spp., strongyles-type ova, Entamoeba spp. and Entamoeba polecki were found only in captive samples. Cryptosporidium, Balantidium coli, pinworm-type ova, and two fluke-like ova were seen only in wild samples. In addition, samples were compared for both bacteria and parasites from three unique field sites in Madagascar. In this three-site comparison, six types of bacteria were statistically significantly different. No significant differences regarding parasites were seen. Significant differences were found between the captive and wild populations, whereas fewer differences were found between sites within Madagascar. Although we isolated Campylobacter and Giardia, all animals appeared clinically healthy.     

PCR-denaturing gradient gel electrophoresis and two feces antigen tests for detection of Helicobacter pylori in mice

PCR-denaturing Gradient Gel Electrophoresis (PCR-DGGE), a method suitable for the detection of microbial species in complex ecosystems, was evaluated for the detection and identification of Helicobacter spp. in feces and stomach tissue of mice. Two commercially available stool antigen tests for clinical diagnostics in humans were also evaluated in the C57Bl/6 mouse model of H. pylori infection. PCR-DGGE detected only Helicobacter ganmani in feces from H. pylori-infected as well as control animals, whereas in stomach specimens it demonstrated the presence of H. pylori in challenged and H. ganmani in control animals. Hence, the method detected DNA only of the predominant Helicobacter spp., which was also shown in cell dilution experiments. The Amplified IDEIA Hp StAR feces antigen test detected H. pylori in feces from all infected animals and generated no false-positive results, whereas the Premier Platinum HpSA-test also detected H. pylori in all infected animals but generated false-positive or equivocal results in 50% of the control animals. Premier Platinum HpSA, as opposed to Hp StAR, cross-reacted with non-pylori Helicobacter spp. in vitro.     

Tuberculosis in a captive colony of pinnipeds

Tuberculosis was diagnosed in 10 of 16 otariid seals upon post mortem examination. The species involved were New Zealand fur seals (Arctocephalus forsteri), Australian sea lions (Neophoca cinerea) and an Australian fur seal (Arctocephalus pusillus doriferus). Five seals died, four as a direct result of mycobacterial infection. One seal died of unrelated disease. The remaining 10 animals were subsequently tuberculin tested and then killed and necropsied. Tuberculous lesions were seen in five. Gross pathological changes were most commonly seen in the respiratory system. However, a generalized infection, a case with lesions confined to the liver and draining lymph nodes, and a case with tuberculous meningitis also were seen. Histological lesions were characterized by spindle cell proliferation and necrosis without mineralization or giant cell formation. The mycobacteria isolated was identified as belonging to the Mycobacterium tuberculosis complex but it appeared to be unique. Intradermal tuberculin testing showed promise as a diagnostic aid; however, the results were not statistically significant. Circumstances suggest that the initial infection was present when the seals were captured from the wild.     

Photoperiod and reproduction in captive female Northern fur seals*

The reproductive cycles of captive female Northern fur seals (Callorhinus ursinus) were unaffected over two consecutive years by a photoperiod that differed substantially from that of the natural range. Breeding was observed in July and August, or at the same time as in the wild. Growth was comparable in captive and wild fetuses, indicating a similar timing of delayed implantation of the blastocysts.     

Ubiquitous Hepatocystis infections, but no evidence of Plasmodium falciparum-like malaria parasites in wild greater spot-nosed monkeys (Cercopithecus nictitans)

Western gorillas (Gorilla gorilla) have been identified as the natural reservoir of the parasites that were the immediate precursor of Plasmodium falciparum infecting humans. Recently, a P. falciparum-like sequence was reported in a sample from a captive greater spot-nosed monkey (Cercopithecus nictitans), and was taken to indicate that this species may also be a natural reservoir for P. falciparum-related parasites. To test this hypothesis we screened blood samples from 292 wild C. nictitans monkeys that had been hunted for bushmeat in Cameroon. We detected Hepatocystis spp. in 49% of the samples, as well as one sequence from a clade of Plasmodium spp. previously found in birds, lizards and bats. However, none of the 292 wild C. nictitans harbored P. falciparum-like parasites.     

Isolation of Helicobacter strains from wild bird and swine feces.

We report the first isolations of Helicobacter strains from wild birds and swine. Genus-specific oligonucleotide probes identified nine Cape Cod isolates from gull, tern, house sparrow, and pig feces as Helicobacter spp. and not Campylobacter spp. Antibiotic sensitivity and urease tests distinguished three phenotypes. Strains examined rapidly lost culturability under simulated natural conditions.     

Differentiating between Steller sea lion (Eumetopias jubatus) and northern fur seal (Callorhinus ursinus) scats through analysis of faecal DNA

We describe a method to determine the species of pinniped from faeces collected from sympatric Steller sea lion (Eumetopias jubatus) and northern fur seal (Callorhinus ursinus) rookeries using newly developed species-specific primers that amplify a 667-669-base pair segment from the mitochondrial DNA (mtDNA) cytochrome B (cytB) gene region. The primers yielded the correct species in 100% of tissue samples from 10 known animals and 100% of faecal samples from 13 known animals. Species could be identified unequivocally for 87.7% of faecal samples from 122 unknown individuals. The ability to differentiate between scats of sympatrically breeding Steller sea lions and northern fur seals will contribute to the range-wide knowledge of the foraging strategies of both species as well as allow researchers to examine the niche partitioning and potential resource competition between the two predators.     

Epidemiology and potential land-sea transfer of enteric bacteria from terrestrial to marine species in the Monterey Bay Region of California

Marine mammals are at risk for infection by fecal-associated zoonotic pathogens when they swim and feed in polluted nearshore marine waters. Because of their tendency to consume 25-30% of their body weight per day in coastal filter-feeding invertebrates, southern sea otters (Enhydra lutris nereis) can act as sentinels of marine ecosystem health in California. Feces from domestic and wildlife species were tested to determine prevalence, potential virulence, and diversity of selected opportunistic enteric bacterial pathogens in the Monterey Bay region. We hypothesized that if sea otters are sentinels of coastal health, and fecal pollution flows from land to sea, then sea otters and terrestrial animals might share the same enteric bacterial species and strains. Twenty-eight percent of fecal samples tested during 2007-2010 were positive for one or more potential pathogens. Campylobacter spp. were isolated most frequently, with an overall prevalence of 11%, followed by Vibrio cholerae (9%), Salmonella spp. (6%), V. parahaemolyticus (5%), and V. alginolyticus (3%). Sea otters were found positive for all target bacteria, exhibiting similar prevalences for Campylobacter and Salmonella spp. but greater prevalences for Vibrio spp. when compared to terrestrial animals. Fifteen Salmonella serotypes were detected, 11 of which were isolated from opossums. This is the first report of sea otter infection by S. enterica Heidelberg, a serotype also associated with human clinical disease. Similar strains of S. enterica Typhimurium were identified in otters, opossums, and gulls, suggesting the possibility of land-sea transfer of enteric bacterial pathogens from terrestrial sources to sea otters.     

Broad thermal capacity facilitates the primarily pelagic existence of northern fur seals (Callorhinus ursinus)

Thermoregulatory capacity may constrain the distribution of marine mammals despite having anatomical and physiological adaptations to compensate for the thermal challenges of an aquatic lifestyle. We tested whether subadult female northern fur seals (Callorhinus ursinus) experience increased thermoregulatory costs in water temperatures potentially encountered during their annual migration in the Bering Sea and North Pacific Ocean. Metabolic rates were measured seasonally in 6 captive female northern fur seals (2.75–3.5 yr old) in ambient air and controlled water temperatures of 2°C, 10°C, and 18°C. Rates of oxygen consumption in ambient air (1°C–18°C) were not related to environmental temperature except below 2.5°C (winter only). However, metabolism was significantly higher during the fall seasonal trials (September–October) compared to other times of year, perhaps due to the costs of molting. The fur seals appeared thermally neutral in all seasons for all water temperatures tested (2°C–18°C) except during the summer when metabolic rates were higher in the 2°C water. Comparing this broad thermal neutral zone to the average sea surface temperatures potentially encountered during annual migrations indicates wild fur seals can likely exploit a large geographic area without added thermal metabolic costs.     

Individual Foraging Strategies Reveal Niche Overlap between Endangered Galapagos Pinnipeds

Most competition studies between species are conducted from a population-level approach. Few studies have examined inter-specific competition in conjunction with intra-specific competition, with an individual-based approach. To our knowledge, none has been conducted on marine top predators. Sympatric Galapagos fur seals (Arctocephalus galapagoensis) and sea lions (Zalophus wollebaeki) share similar geographic habitats and potentially compete. We studied their foraging niche overlap at Cabo Douglas, Fernandina Island from simultaneously collected dive and movement data to examine spatial and temporal inter- and intra-specific competition. Sea lions exhibited 3 foraging strategies (shallow, intermediate and deep) indicating intra-specific competition. Fur seals exhibited one foraging strategy, diving predominantly at night, between 0–80 m depth and mostly at 19–22 h. Most sea lion dives also occurred at night (63%), between 0–40 m, within fur seals'' diving depth range. 34% of sea lions night dives occurred at 19–22 h, when fur seals dived the most, but most of them occurred at dawn and dusk, when fur seals exhibited the least amount of dives. Fur seals and sea lions foraging behavior overlapped at 19 and 21 h between 0–30 m depths. Sea lions from the deep diving strategy exhibited the greatest foraging overlap with fur seals, in time (19 h), depth during overlapping time (21–24 m), and foraging range (37.7%). Fur seals foraging range was larger. Cabo Douglas northwest coastal area, region of highest diving density, is a foraging “hot spot” for both species. Fur seals and sea lions foraging niche overlap occurred, but segregation also occurred; fur seals primarily dived at night, while sea lions exhibited night and day diving. Both species exploited depths and areas exclusive to their species. Niche breadth generally increases with environmental uncertainty and decreased productivity. Potential competition between these species could be greater during warmer periods when prey availability is reduced.