Helicobacter delphinicola infection and the risk of gastric disease in common bottlenose dolphin.

Gastritis and gastric ulcers are well-recognized conditions in cetaceans; bacteria of the genus Helicobacter are considered the primary cause of these diseases. Dolphins have been shown to be susceptible to infection by at least 2 gastric species of Helicobacter, H. cetorum and H. delphinicola, both of which are closely related to the human pathogen H. pylori. In the present study, we evaluated the carriage rate and relationship to gastric disease of H. cetorum and H. delphinicola, based on a study population of 82 dolphins maintained at 21 facilities in Japan. Of these 82 dolphins, 79 (96.3%) and 45 (54.9%) were positive for H. cetorum and H. delphinicola, respectively; H. delphinicola infection was significantly associated with chronic gastric diseases (odds rate: 5.9; 95% CI: 2.1-16.9), but no such association was detected for H. cetorum. Of the 21 facilities, 20 (95%) and 11 (55%) housed H. cetorum- and H. delphinicola-positive dolphins, respectively, and our study suggested that the transmission between dolphins occurs quickly within pools. These findings indicate that methods will need to be established to prevent the transmission of Helicobacter infections within facilities housing dolphins.

A New Enzyme-linked Immunosorbent Assay for Serological Diagnosis of Seal Parapoxvirus Infection in Marine Mammals.

Introduction: Seal parapoxvirus (SPPV) infection has been reported among pinnipeds in aquaria in Japan; however, its seroprevalence is unknown. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed for serological diagnosis of SPPV infection. Material and Methods: The gene encoding the major envelope protein of SPPV was cloned into the eukaryotic expression vector pAcGFP1-N1, which encodes the green fluorescence protein (GFP), thereby producing a fusion protein (Env-GFP). Parental and cloned vector DNA was independently transfected into cultured seal cells for the expression of GFP and Env-GFP. The wells of an ELISA plate were coated with either GFP- or Env-GFP-transfected cell lysates. The light absorbance of each serum sample was adjusted by subtracting the absorbance of GFP-coated wells from that of Env-GFP-coated wells. Sera from two spotted seals (Phoca largha), six beluga whales (Delphinapterus leucas), three Pacific white-sided dolphins (Lagenorhynchus obliquidens), and ten bottlenose dolphins (Tursiops truncatus) from an aquarium in Japan were examined using the ELISA. Results: Positive reactions were not observed, except in one preserved sample collected ten years ago from a naturally SPPV-infected spotted seal. Conclusion: The established ELISA could be useful in screening marine mammal sera for anti-SPPV antibodies.

Helicobacter delphinicola sp. nov., isolated from common bottlenose dolphins Tursiops truncatus with gastric diseases.

Gastritis and gastric ulcers are well-recognized symptoms in cetaceans, and the genus Helicobacter is considered as the main cause. In this study, we examined the gastric fluid of captive common bottlenose dolphins Tursiops truncatus with gastric diseases in order to isolate the organisms responsible for diagnosis and treatment. Four Gram-negative, rod-shaped isolates (TSBT, TSH1, TSZ, and TSH3) with tightly coiled spirals with 2-4 turns and 2-6 bipolar, sheathed flagella, were obtained from gastric fluids of common bottlenose dolphins with gastric diseases. Phylogenetic analysis, based on 16S rRNA, atpA, and 60 kDa heat-shock protein (hsp60) genes, demonstrated that these isolates form a novel lineage within the genus Helicobacter. Analyses of 16S rRNA, atpA, and hsp60 gene sequences showed that isolate TSBT was most closely related to H. cetorum MIT99-5656T (98.5% similarity), H. pylori ATCC 43504T (76.7% similarity), and H. pylori ATCC 43504T (78.0% similarity), respectively. Type strains of Helicobacter showing resistance to 2% NaCl have not been reported previously; however, these novel isolates were resistant to 2% NaCl. Culture supernatant of some isolates induced intracellular vacuolization in mammalian cultured cells. These data, together with the different morphological and biochemical characteristics of the isolates, reveal that these isolates represent a novel species for which we propose the name Helicobacter delphinicola sp. nov. with type strain TSBT (= JCM 32789T = TSD-183T). Future studies will confirm whether H. delphinicola plays a role in lesion etiopathogenesis in cetaceans.

Placental and breastmilk transfer of voriconazole to offspring from pregnant and lactating bottlenose dolphins (Tursiops truncatus).

Fungal pneumonia is a common disease in bottlenose dolphins (Tursiops truncatus), including pregnant and lactating ones. Voriconazole (VRCZ) is commonly used to treat respiratory fungal infections in this species; however, it is unknown whether VRCZ is transferred via the placenta and breastmilk and whether its usage is safe in pregnant and lactating dolphins. We measured VRCZ concentrations in breastmilk and dams', umbilical cord, and calves' plasma samples from four dam-calf dolphin pairs in the Port of Nagoya Public Aquarium, Japan, treated with or without VRCZ. Three pregnant and/or lactating dams were administered VRCZ (loading dose 1.5-2.3 mg/kg, for 3 days; maintenance dose 1.5-3.1 mg/kg, every 5-18 days), twice daily, orally, without side effects in dams or calves. VRCZ was detected in two dams' umbilical cord plasma (0.14 and 2.35 µg/ml) and in one calf's plasma (0.18 µg/ml), collected immediately after birth. Further, VRCZ was detected in breastmilk samples (maximum 13.45 µg/ml) from three VRCZ-administered dams and in plasma from three calves (maximum 7.54 µg/ml) given or nursed from VRCZ-administered dams' breastmilk. The calves' plasma VRCZ concentrations varied, depending on the amount of breastmilk and food consumed. VRCZ concentrations were higher in breastmilk samples than in dams' plasma. To our knowledge, this is the first report on placental and breastmilk VRCZ transfer to offspring in bottlenose dolphins. During VRCZ medication in pregnant and lactating bottlenose dolphins, it is crucial to monitor plasma VRCZ concentrations and any side effects in dams as well as in their calves.

First isolation of voriconazole-resistant Candida albicans, C. tropicalis, and Aspergillus niger from the blowholes of bottlenose dolphins (Tursiops truncatus).

Pulmonary mycosis is a fungal disease that commonly affects bottlenose dolphins (Tursiops truncatus) and is generally treated by the oral administration of azoles, such as itraconazole (ITZ) and voriconazole (VRZ). However, antifungal susceptibility testing of clinical isolates has not been well performed as a routine clinical examination in aquaria. In this study, we collected fungal species from the blowholes of 14 bottlenose dolphins, of which 12 were treated with ITZ or VRZ. All dolphins were housed in the Port of Nagoya Public Aquarium. The fungal species Candida albicans, C. tropicalis, C. glabrata, Aspergillus fumigatus, and A. niger were isolated. E-tests were performed to determine the minimum inhibitory concentrations (MICs) of ITZ and VRZ on these isolates. VRZ-resistant C. tropicalis (MIC: >32 µg/ml) and A. niger (MIC: >32 µg/ml) were isolated from three dolphins treated with ITZ or VRZ. Additionally, azole-resistant isolates of C. albicans and C. glabrata were collected from two dolphins that had never received azole therapy. To the best of our knowledge, our study is the first to report the isolation of VRZ-resistant C. albicans, C. tropicalis, and A. niger from the blowholes of bottlenose dolphins. Thus, antifungal susceptibility testing is a crucial strategy for selecting antifungal agents to treat respiratory fungal infections in bottlenose dolphins in aquaria.

Leukopenia induced by micafungin in a bottlenose dolphin (Tursiops truncatus): a case report.

A bottlenose dolphin (Tursiops truncatus) housed in the Port of Nagoya Public Aquarium (PNPA) presented with symptomatic pneumonia caused by Aspergillus fumigatus. The dolphin was treated with micafungin. On days 2 and 11 after the first administration of micafungin, results from a physical examination and laboratory test indicated a decline of body temperature (BT) and leukopenia, with lowest BT, white blood cells (WBCs), and segmented neutrophils (SEGs) of 34.2°C, 600 cells/µl, and 67 cells/µl, respectively. BT, WBCs, and SEGs returned to normal range after administration of granulocyte colony stimulating factor (G-CSF). To the best of our knowledge, this is the first report of micafungin-induced decline of BT and leukopenia that was successfully treated with G-CSF in a bottlenose dolphin.

Plasma metabolomic analysis in mature female common bottlenose dolphins: profiling the characteristics of metabolites after overnight fasting by comparison with data in beagle dogs.

The present study was aimed at determining the characteristics of plasma metabolites in bottlenose dolphins to provide a greater understanding of their metabolism and to obtain information for the health management of cetaceans. Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) and liquid chromatograph-time-of-flight mass spectrometry (LC-TOFMS) were conducted on plasma samples after overnight fasting from three common bottlenose dolphins as well as three beagle dogs (representative terrestrial carnivores) for comparison. In total, 257 and 227 plasma metabolites were identified in the dolphins and the dogs, respectively. Although a small number of animals were used for each species, the heatmap patterns, a principal component analysis and a cluster analysis confirmed that the composition of metabolites could be segregated from each other. Of 257 compounds detected in dolphin plasma, 24 compounds including branched amino acids, creatinine, urea, and methylhistidine were more abundant than in dogs; 26 compounds including long-chained acyl-carnitines and fatty acids, astaxanthin, and pantothenic acid were detected only in dolphins. In contrast, 25 compounds containing lactic acid and glycerol 3-phosphate were lower in dolphins compared to dogs. These data imply active protein metabolism, differences in usage of lipids, a unique urea cycle, and a low activity of the glycolytic pathway in dolphins.

Environmental isolates of fungi from aquarium pools housing killer whales (Orcinus orca).

Systemic mycoses in killer whales (Orcinus orca) are rare diseases, but have been reported. Two killer whales died by fungal infections at the Port of Nagoya Public Aquarium in Japan. In this study, the fungal flora of the pool environment at the aquarium was characterized. Alternaria spp., Aspergillus spp. (A. fumigatus, A. niger, A. versicolor), Fusarium spp. and Penicillium spp. were isolated from the air and the pool surroundings. The other isolates were identified as fungal species non-pathogenic for mammals. However, the species of fungi isolated from the environmental samples in this study were not the same as those isolated from the cases of disease in killer whales previously reported.

Molecular analysis of parapoxvirus from a spotted seal Phoca largha in Japan.

A spotted seal Phoca largha with nodular and scab lesions on the whole body was brought to an aquarium in Nagoya, Japan. We extracted DNA from the lesions and used the polymerase chain reaction (PCR) method for detecting orthopoxvirus and parapoxvirus DNA. Parapoxvirus but not orthopoxvirus DNA was detected. The partial nucleotide sequence of the envelope gene was determined from the PCR product, and the sequence was seen to be closely related to 2 parapoxvirus strains from spotted seals in Alaska, showing 100% identity at the amino acid level, with one nucleotide substitution. Virus-neutralizing (VN) antibody against canine distemper virus (CDV) was not detected in the serum, indicating that this individual was not infected with CDV or phocine distemper virus (PDV), which both have a high mortality rate for marine mammals. These results suggest that the lesions were caused by infection with pinniped parapoxvirus, and that the viruses spread and are maintained within the habitat range or populations of spotted seals from the Bering Sea to the Japan Sea. This is the first report of molecular analysis of parapoxvirus in marine mammals in Japan.

Dogs as sentinels for human infection with Japanese encephalitis virus.

Because serosurveys of Japanese encephalitis virus (JEV) among wild animals and pigs may not accurately reflect risk for humans in urban/residential areas, we examined seroprevalence among dogs and cats. We found that JEV-infected mosquitoes have spread throughout Japan and that dogs, but not cats, might be good sentinels for monitoring JEV infection in urban/residential areas.

Detection of antibodies against Japanese encephalitis virus in raccoons, raccoon dogs and wild boars in Japan.

Japanese encephalitis virus (JEV) infects numerous animal species including humans, horses and pigs. In this study, antibodies against JEV in feral raccoons (Procyon lotor), wild boars (Sus scrofa) and raccoon dogs (Nyctereutes procyonoides) in Japan were examined. The results showed that 40.7% (22 out of 54), 64.5% (40 out of 62), 69.1% (47 out of 68) and 0% (0 out of 20) of raccoons in Hyogo, Osaka, Wakayama and Hokkaido, respectively, had virus-neutralizing antibodies against JEV. In addition, 83.3% (30 out of 36) of wild boars and 63.2% (12 out of 19) of raccoon dogs in Wakayama were seropositive for JEV. There were no significant differences in seroprevalence of JEV between males and females or between adults and juveniles in these wild animals. JEV seroprevalence was compared between 37 raccoons and 30 wild boars captured in a limited period (November 2007 to February 2008), and we found that wild boars (86.7%) were significantly more seropositive for JEV antibody than raccoons (59.5%). In conclusion, JEV was prevalent in wild mammals, indicating that the possibility of JEV infection in humans may still be high in Japan. In addition, these wild animals may be good sentinels to estimate JEV infection risk in residents, as they live near humans and are not vaccinated.

Establishment of canine and feline cells expressing canine signaling lymphocyte activation molecule for canine distemper virus study.

Signaling lymphocyte activation molecule (SLAM) is one of the receptors for canine distemper virus (CDV). In this study, canine and feline cells expressing canine SLAM, designated A-72/cSLAM and CRFK/cSLAM, were established for the in vitro study of canine distemper. Recent CDV isolates, KDK-1 and 246, which belong to genotypes Asia/H1 and Asia/H2, respectively, rapidly grew and produced distinct syncytia in both the SLAM-expressing cells. The virus-neutralizing (VN) test was successfully performed using these cells, and the results indicated that sera from dogs experimentally infected with KDK-1 had higher VN titers for homologous strain KDK-1 than for heterologous strain 246 and the vaccine Onderstepoort. These newly established cells expressing canine SLAM would help virological and serological analyses of canine distemper.