Detection of Helicobacter and Campylobacter spp. from the aquatic environment of marine mammals

The mechanism by which Helicobacter species are transmitted remains unclear. To examine the possible role of environmental transmission in marine mammals, we sought the presence of Helicobacter spp. and non-Helicobacter bacteria within the order Campylobacterales in water from the aquatic environment of marine mammals, and in fish otoliths regurgitated by dolphins. Water was collected from six pools, two inhabited by dolphins and four inhabited by seals. Regurgitated otoliths were collected from the bottom of dolphins' pools. Samples were evaluated by culture, PCR and DNA sequence analysis. Sequences from dolphins' water and from regurgitated otoliths clustered with 99.8-100% homology with sequences from gastric fluids, dental plaque and saliva from dolphins living in those pools, and with 99.5% homology with H. cetorum. Sequences from seals' water clustered with 99.5% homology with a sequence amplified from a Northern sea lion (AY203900). Control PCR on source water for the pools and from otoliths dissected from feeder fish were negative. The findings of Helicobacter spp. DNA in the aquatic environment suggests that contaminated water from regurgitated fish otoliths and perhaps other tissues may play a role in Helicobacter transmission among marine mammals.     

Helicobacter spp. from gastric biopsies of stranded South American fur seals (Arctocephalus australis)

Gastrointestinal lesions with uncertain etiology have been widely described among pinnipeds. The aim of our study was to investigate the presence of Helicobacter spp. in the gastric mucosa of South American fur seals (Arctocephalusaustralis). Gastric biopsies from thirteen seals, stranded on the shores of the Southwestern Atlantic Ocean in Argentina, were evaluated for the presence of Helicobacter spp. by PCR and DNA sequence analysis. Six gastric biopsies were positive for Helicobacter spp. Pairwise sequence comparisons showed less than 95% identity to novel Helicobacter spp. described from pinnipeds from North America and Australia. However, phylogenetic analysis revealed that the South American fur seal sequences clustered with 99-100% homology with H. cetorum, a species isolated from dolphins and whales. The presence of H. cetorum in pinnipeds, if confirmed by its isolation from the gastric mucosa of these mammals, demonstrates the wide host range of this bacterium in the marine environment.     

Gastritis and intestinal metaplasia in Syrian hamsters infected with Helicobacter aurati and two other microaerobes

Chronic gastritis and intestinal metaplasia associated with naturally occurring colonization by Helicobacter aurati and two other microaerobic species were observed in Syrian hamsters. Thirty-five hamsters, between 7 and 12 months of age, were evaluated from two research and three commercial facilities. Microaerobic bacteria were cultured from the hamster stomachs. These bacteria included H. aurati, a fusiform, urease-positive species; a second novel helical, urease-negative Helicobacter sp.; as well as a smaller, urease-negative Campylobacter sp. Southern blot analysis detected Helicobacter spp. DNA in the gastric tissues of all 35 hamsters; 15 hamsters also had Campylobacter sp. DNA in their gastric tissues. When examined by light microscopy, argyrophilic bacteria consistent with H. aurati or the second Helicobacter sp. were present in antral sections of 12 out of the 15 hamsters where bacteria were seen, while 9 out of the 15 hamsters had bacteria resembling the Campylobacter sp. The presence of Helicobacter spp. but not the presence of Campylobacter sp. was significantly correlated to gastritis severity (P < 0.0001 for Helicobacter spp., P = 0.6025 for Campylobacter sp.) and intestinal metaplasia, as measured by numbers of goblet cells (P = 0.0239 for Helicobacter spp., P = 0.5525 for Campylobacter sp.). Severely affected hamsters also had Giardia sp. within their metaplastic gastric pits. Hamsters with naturally occurring helicobacter-associated gastritis provide a model for studying the development of intestinal metaplasia and gastric giardiasis in H. pylori-infected humans.     

Detection of Helicobacter spp. DNA in the oral cavity of dogs

The mode of acquisition of gastric Helicobacter spp. infection in dogs has not been determined. It is suspected that oral-oral and faecal-oral transmission may be involved. The present study sought to determine if Helicobacter spp. DNA is present in the oral cavity of healthy and vomiting dogs. Thirty-eight pet dogs (27 vomiting and 11 clinically healthy) were studied. The presence of Helicobacter spp. was determined by single and nested PCR evaluation of DNA extracted from saliva, dental plaque and gastric biopsy samples. Helicobacter spp. DNA was detected by nested PCR in 36 (94.7%) gastric biopsies, 17 (44.7%) dental plaque and 19 (50%) saliva samples out of the 38 dogs examined. Overall 27 (71.1%) dogs screened by nested PCR were found to harbour Helicobacter spp. DNA in the oral cavity (dental plaque and/or saliva). There was no significant difference in the prevalence of Helicobacter spp. DNA in the oral cavity of vomiting and healthy dogs, and the time from vomiting to oral sampling did not have significant impact. This study confirms the high prevalence of gastric Helicobacter spp. infection in dogs, and reveals that Helicobacter spp. DNA is detectable in the oral cavity of over 70% of dogs. These findings support the possibility of oral-oral transmission between dogs and that the canine oral cavity may act as source of non-pylori Helicobacter spp. infection for humans.     

Bartonella species detection in captive, stranded and free-ranging cetaceans

We present prevalence of Bartonella spp. for multiple cohorts of wild and captive cetaceans. One hundred and six cetaceans including 86 bottlenose dolphins (71 free-ranging, 14 captive in a facility with a dolphin experiencing debility of unknown origin, 1 stranded), 11 striped dolphins, 4 harbor porpoises, 3 Risso's dolphins, 1 dwarf sperm whale and 1 pygmy sperm whale (all stranded) were sampled. Whole blood (n = 95 live animals) and tissues (n = 15 freshly dead animals) were screened by PCR (n = 106 animals), PCR of enrichment cultures (n = 50 animals), and subcultures (n = 50 animals). Bartonella spp. were detected from 17 cetaceans, including 12 by direct extraction PCR of blood or tissues, 6 by PCR of enrichment cultures, and 4 by subculture isolation. Bartonella spp. were more commonly detected from the captive (6/14, 43%) than from free-ranging (2/71, 2.8%) bottlenose dolphins, and were commonly detected from the stranded animals (9/21, 43%; 3/11 striped dolphins, 3/4 harbor porpoises, 2/3 Risso's dolphins, 1/1 pygmy sperm whale, 0/1 dwarf sperm whale, 0/1 bottlenose dolphin). Sequencing identified a Bartonella spp. most similar to B. henselae San Antonio 2 in eight cases (4 bottlenose dolphins, 2 striped dolphins, 2 harbor porpoises), B. henselae Houston 1 in three cases (2 Risso's dolphins, 1 harbor porpoise), and untyped in six cases (4 bottlenose dolphins, 1 striped dolphin, 1 pygmy sperm whale). Although disease causation has not been established, Bartonella species were detected more commonly from cetaceans that were overtly debilitated or were cohabiting in captivity with a debilitated animal than from free-ranging animals. The detection of Bartonella spp. from cetaceans may be of pathophysiological concern.     

Helicobacter spp. from captive bottlenose dolphins (Tursiops spp.) and polar bears (Ursus maritimus)

The gastric fluid of six bottlenose dolphins and the faeces of four polar bears from the same oceanarium were examined for the presence of Helicobacter. As detected by PCR, all dolphins and 8/12 samples collected from polar bears were positive for Helicobacter. Novel sequence types were identified in samples collected from these animals of which several were unique to either the dolphins or the polar bears. At least one sequence type was, however, detected in both animal taxa. In addition, a sequence type from a dolphin shared a 98.2-100% identity to sequences from other Helicobacter species from harp seals, sea otters and sea lions. This study reports on the occurrence of novel Helicobacter sequence types in polar bears and dolphins and demonstrates the broad-host range of some species within these animals.     

Are goats naturally resistant to gastric Helicobacter infection?

Gastric Helicobacter species are widespread and have been reported in wild and domestic mammals of different dietary habits such as humans, dogs, cats, macaques, mice, cheetahs, ferrets, swine and cattle. All have been associated with gastric pathologies. Recently, gastric Helicobacter species were shown to be widespread in cattle and swine in Europe, and there is a report of Helicobacter pylori in sheep in Italy. However, there are no reports of Helicobacter infection in the goat, another important domestic animal of human consumption. The aim of our study was to assess whether Helicobacter abomasal infection was common in goats slaughtered for human consumption. Infection was detected through PCR analysis of DNA extracted from gastric biopsies, using genus- and species-specific primers. Bovine and porcine gastric samples were also analyzed as positive controls. None of the 70 goats were positive for Helicobacter spp.; however, Candidatus Helicobacter bovis and Candidatus Helicobacter suis were detected in 85% of the bovine and 45% of the porcine samples, respectively. We discuss the possibility that goats may exhibit natural resistance to abomasal infection by Helicobacter spp.     

Early divergence and differential population histories of the Indo-Pacific humpback dolphin in the Pacific and Indian Oceans

The currently recognized Indo-Pacific humpback dolphin occurs in estuaries and surrounding shallow waters from the South China Sea to the Asian coast of the Indian Ocean. However, a recent study suggested that the humpback dolphin from the Bay of Bengal may represent a distinct phylogenetic species. In this study, we sequenced 915-bp mtDNA segments from five geographic populations in both Chinese and Thai waters; together with previously published sequences, these data revealed that the ancestral Indo-Pacific humpback dolphin might have split during the transition from the Oligocene to Miocene (23.45 Mya, 95% HPD: 16.65–26.55 Mya), and then dispersed along the Pacific and Indian Ocean coasts of Asia. Genetic differentiation was detected between most of the examined populations, except for only a few pairwise populations in the northern South China Sea. Genetic differentiation/distance between the humpback dolphins from the northern and southern South China Sea met the sub-species threshold value proposed for marine mammals, whereas that between the humpback dolphins in the Pacific and the Indian Ocean was above the species threshold. Bayesian inference of historic gene flow indicated low but constant northward gene flow along the Indian Ocean coast; however, there was a recent abrupt increase in gene flow in the Pacific region, likely due to the shortening coastline at the low stand of sea level. Our results revealed that the current taxonomic classification of Indo-Pacific humpback dolphins may not reflect their phylogeography.     

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.     

Measurement of serum immunoglobulin concentration in killer whales and sea otters by radial immunodiffusion

Killer whales and sea otters maintained in captivity are the subjects of routine health monitoring programs, and interest in immunologic studies in sea otters has been rising recently in response to potential impacts from infectious disease and environmental pollution on the threatened southern sea otter population. Development of species-specific reagents for immunologic studies in these two marine mammals is currently in its infancy. In this study, killer whale and sea otter immunoglobulin-specific polyclonal antibodies were generated, and used to develop tests for serum Ig concentration in the killer whale (Orcinus orca) and the southern (Enhydra lutris nereis) and northern sea otter (Enhydra lutris lutris). Killer whale serum IgG was purified using caprylic acid/ammonium sulfate precipitation. Sea otter plasma IgG was purified using protein-A-agarose. Polyclonal anti-Ig antisera were produced in rabbits, and specificity confirmed by immunoelectrophoresis. Radial immunodiffusion was used to measure Ig concentration in serum or plasma samples derived from 21 captive killer whales, 18 wild and 4 captive southern sea otters and 15 wild and 4 captive northern sea otters grouped by age. Mean killer whale serum Ig concentration (+/-95% confidence interval) ranged from 15.04 +/- 3.97 g/l for animals aged 0-5 years to 26.65 +/- 9.8 g/l for animals aged >10 years. Mean sea otter serum Ig concentration (+/-95% confidence interval) ranged from 28.39 +/- 11.00 g/l for southern sub-adults to 32.76 +/- 11.58 g/l for southern adults. No significant difference in serum Ig concentration was found between southern and northern sea otters. Serum Ig concentrations in two northern sea otter pups were low compared to those of adult sea otters. The two serum Ig quantitation assays produced were highly specific and reproducible and will be useful additions to the limited number of tests available for immune function in these marine mammal species.     

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.     

Human Pathogens in Marine Mammal Meat – A Northern Perspective

Only a few countries worldwide hunt seals and whales commercially. In Norway, hooded and harp seals and minke whales are commercially harvested, and coastal seals (harbour and grey seals) are hunted as game. Marine mammal meat is sold to the public and thus included in general microbiological meat control regulations. Slaughtering and dressing of marine mammals are performed in the open air on deck, and many factors on board sealing or whaling vessels may affect meat quality, such as the ice used for cooling whale meat and the seawater used for cleaning, storage of whale meat in the open air until ambient temperature is reached, and the hygienic conditions of equipment, decks, and other surfaces. Based on existing reports, it appears that meat of seal and whale does not usually represent a microbiological hazard to consumers in Norway, because human disease has not been associated with consumption of such foods. However, as hygienic control on marine mammal meat is ad hoc, mainly based on spot‐testing, and addresses very few human pathogens, this conclusion may be premature. Additionally, few data from surveys or systematic quality control screenings have been published. This review examines the occurrence of potential human pathogens in marine mammals, as well as critical points for contamination of meat during the slaughter, dressing, cooling, storage and processing of meat. Some zoonotic agents are of particular relevance as foodborne pathogens, such as Trichinella spp., Toxoplasma gondii, Salmonella and Leptospira spp. In addition, Mycoplasma spp. parapoxvirus and Mycobacterium spp. constitute occupational risks during handling of marine mammals and marine mammal products. Adequate training in hygienic procedures is necessary to minimize the risk of contamination on board, and acquiring further data is essential for obtaining a realistic assessment of the microbiological risk to humans from consuming marine mammal meat.     

Occurrence and molecular analysis of Campylobacter in wildlife on livestock farms

Wildlife harbor a variety of Campylobacter spp. and may play a significant role in the transmission of Campylobacter to livestock. Although studies have been conducted on wildlife-associated Campylobacter isolates from farms in other countries, there are little data available for livestock farms in the United States. In addition, the critical questions of whether wildlife harbor Campylobacter that is pathogenic to ruminants and/or antibiotic-resistant Campylobacter have yet to be addressed. We captured wild small mammals (n=142) and small birds (n=188) at livestock farms in central Iowa and sampled them for thermophilic Campylobacter during autumn 2009, spring 2010, and autumn 2010. Overall prevalence was 4.79%, with isolates found only in wild birds. Molecular typing revealed four multilocus sequence types (STs), three of which are novel. The remaining ST (ST-806) was found in two house sparrows and is an ST previously associated with ruminant abortion cases. Further analysis of ST-806 wild bird and ruminant abortion isolates by pulsed-field gel electrophoresis, resistance gene location, and antibiotic susceptibility tests indicated that the isolates are nearly identical. This is the first account of isolation of Campylobacter types from wild birds that are known to be pathogenic to ruminants. Furthermore, these same two wild bird isolates are resistant to the antibiotic fluoroquinolone. Our results indicate there is an overall low prevalence of Campylobacter in selected wildlife in Iowa, but suggest that wildlife may play a role in the epidemiology of pathogenic Campylobacter for domestic livestock, and may also serve as a reservoir for antibiotic-resistant Campylobacter.     

Immunology of whales and dolphins

The increasing disease susceptibility in different whale and dolphin populations has led to speculation about a possible negative influence of environmental contaminants on the immune system and therefore on the health status of marine mammals. Despite current efforts in the immunology of marine mammals several aspects of immune functions in aquatic mammals remain unknown. However, assays for evaluating cellular immune responses, such as lymphocyte proliferation, respiratory burst as well as phagocytic and cytotoxic activity of leukocytes and humoral immune responses have been established for different cetacean species. Additionally, immunological and molecular techniques enable the detection and quantification of pro- and anti-inflammatory cytokines in lymphoid cells during inflammation or immune responses, respectively. Different T and B cell subsets as well as antigen-presenting cells can be detected by flow cytometry and immunohistochemistry. Despite great homologies between marine and terrestrial mammal lymphoid organs, some unique anatomical structures, particularly the complex lymphoepithelial laryngeal glands in cetaceans represent an adaptation to the marine environment. Additionally, physiological changes, such as age-related thymic atrophy and cystic degeneration of the “anal tonsil” of whales have to be taken into account when investigating these lymphoid structures. Systemic morbillivirus infections lead to fatalities in cetaceans associated with generalized lymphoid depletion. Similarly, chronic diseases and starvation are associated with a loss of functional lymphoid cells and decreased resistance against opportunistic infections. There is growing evidence for an immunotoxic effect of different environmental contaminants in whales and dolphins, as demonstrated in field studies. Furthermore, immunomodulatory properties of different persistent xenobiotics have been confirmed in cetacean lymphoid cells in vitro as well as in animal models in vivo. However, species-specific differences of the immune system and detoxification of xenobiotics between cetaceans and laboratory rodents have to be considered when interpreting these toxicological data for risk assessment in whales and dolphins.     

A review of Brucella infection in marine mammals, with special emphasis on Brucella pinnipedialis in the hooded seal (Cystophora cristata)

ABSTRACT: Brucella spp. were isolated from marine mammals for the first time in 1994. Two novel species were later included in the genus; Brucella ceti and Brucella pinnipedialis, with cetaceans and seals as their preferred hosts, respectively. Brucella spp. have since been isolated from a variety of marine mammals. Pathological changes, including lesions of the reproductive organs and associated abortions, have only been registered in cetaceans. The zoonotic potential differs among the marine mammal Brucella strains. Many techniques, both classical typing and molecular microbiology, have been utilised for characterisation of the marine mammal Brucella spp. and the change from the band-based approaches to the sequence-based approaches has greatly increased our knowledge about these strains. Several clusters have been identified within the B. ceti and B. pinnipedialis species, and multiple studies have shown that the hooded seal isolates differ from other pinniped isolates. We describe how different molecular methods have contributed to species identification and differentiation of B. ceti and B. pinnipedialis, with special emphasis on the hooded seal isolates. We further discuss the potential role of B. pinnipedialis for the declining Northwest Atlantic hooded seal population.     

Deep granulomatous dermatitis of the fin caused by Fusarium solani in a false killer whale (Pseudorca crassidens)

A 10-year-old female false killer whale (Pseudorca crassidens) developed skin lesions in the left breast fin. Histopathologically, the lesions consisted of multiple granulomas spread diffusely into the deep dermis and bone; characteristically, each granuloma had septate, branching fungal hyphae and chlamydospores surrounded by eosinophilic Splendore-Hoeppli materials. Macrophages, epithelioid cells and multinucleated giant cells in the granulomas reacted mainly to anti-SRA-E5 antibody against human macrophage scavenger receptor type I. Fusarium solani was isolated and its gene was detected from the skin samples. Mycotic skin lesions by Fusarium spp. reported so far in marine mammals were regarded as superficial dermatitis; therefore, the present case is very uncommon in that the lesions spread deeper into the skin.     

Molecular cloning and phylogenetic analysis of beluga whale (Delphinapterus leucas) and grey seal (Halichoerus grypus) interleukin 2

Interleukin 2 (IL-2) is a lymphokine produced by activated T helper lymphocytes which exerts immunoregulatory effects on a variety of immune cells, including T cells, activated B cells, natural killer cells, and lymphokine-activated killer cells. In this study, we cloned and determined the entire beluga whale (Delphinapterus leucas) and grey seal (Halichoerus grypus) IL-2-encoding cDNA sequences, and analysed their genetic relationships with those from several mammalian species obtained from the Genbank Database. The encoding nucleic acid sequences of beluga whale and grey seal IL-2 were 465 and 468 bp in length, respectively. The identity levels of IL-2 nucleic and deduced amino acid sequences from the beluga whale and grey seal with those from the other mammalian species, ranged from 59.9% to 89.5%, and 52.9% to 77.3%, and from 61.1% to 93.2%, and 58.7% to 88.4%, respectively. Phylogenetic analysis based on both nucleic and amino acid sequences showed that the beluga whale IL-2 was closely related to that of the ruminant species, which includes the bovine, while the grey seal IL-2 was closely related to that of the canine.     

The relationship between the presence of Helicobacter pylori, Clostridium perfringens type A, Campylobacter spp, or fungi and fatal abomasal ulcers in unweaned beef calves.

A case-control study involving 30 unweaned beef calves was conducted to determine whether specific species of bacteria or fungi were associated with fatal abomasal ulcer formation. Special microbiological and histological techniques were used to detect Clostridium perfringens type A, Helicobacter pylori, or Campylobacter spp. It has been speculated that these bacteria are potential ulcerogenic agents of unweaned beef calves. Calves were recruited for the study at necropsy, with those dying of either a perforating or a hemorrhagic ulcer representing the cases, and calves of a similar age dying of a disease unrelated to the abomasum representing the controls. Helicobacter pylori was not visualized in or cultured from any of the abomasal tissue samples. Clostridium perfringens type A was isolated from 78.6% of the cases and 75% of the controls. These isolates were further dichotomized into "heavy" and "light" growth; no significant association was found between ulcers and the amount of growth. A light growth of Campylobacter spp. was recovered from 3 cases and 3 controls. There was no compelling evidence to suggest that Clostridium perfringens type A, Helicobacter pylori, or Campylobacter spp. were involved in ulcer formation.