Takao Segawa

Molecular cloning and expression of bottlenose dolphin CD34.

In terrestrial mammals, the surface molecule CD34 is used as a marker to identify hematopoietic progenitor cells. To clarify whether CD34 expression can be used to confirm the undifferentiated state of hematopoietic-like cells isolated from the bone marrow of bottlenose dolphin, Tursiops truncates, we determined in this study the sequence of dolphin CD34 cDNA and analyzed its mRNA expression. Dolphin CD34 cDNA can be expressed as two forms, one that encodes a full-length version and a variant, truncated version of the gene. Both forms were detected in bone marrow mononuclear cells and in various tissues using RT-PCR. The truncated form was not detected in peripheral blood mononuclear cells, and neither form was detected in polymorphonuclear leukocytes. This is the first report on CD34 in marine mammals and our results suggest that dolphin CD34 may be a useful marker to identify hematopoietic progenitor cells.

Bone marrow biopsy from the flipper of a dolphin.

To find macroscopically palpable bone marrow cavities in dolphins is difficult because of their extremely retrogressive limbs and pelvis and because they do not contain abundant modular cavities (as in terrestrial mammals) that can serve as sites for bone marrow biopsies. Three-dimensional computed tomography analysis of dolphin skeletons suggests that bone marrow could be harvested from the humerus and radius. In this report, post-mortem paracentesis of the humerus from a captive rough-toothed dolphin using a biopsy needle provided a marrow preparation containing myelocytes, erythroblasts and megakaryocytes. This type of bone marrow collection from the flipper might be useful for clinical diagnostic work in cetaceans.

Two isoforms of aquaporin 2 responsive to hypertonic stress in the bottlenose dolphin.

ABSTRACT This study investigated the expression of aquaporin 2 (AQP2) and its newly found alternatively spliced isoform (alternative AQP2) and the functions of these AQP2 isoforms in the cellular hyperosmotic tolerance in the bottlenose dolphin, Tursiops truncatus. mRNA sequencing revealed that alternative AQP2 lacks the fourth exon and instead has a longer third exon that includes a part of the original third intron. The portion of the third intron, now part of the coding region of alternative AQP2, is highly conserved among many species of the order Cetacea but not among terrestrial mammals. Semi-quantitative PCR revealed that AQP2 was expressed only in the kidney, similar to terrestrial mammals. In contrast, alternative AQP2 was expressed in all organs examined, with strong expression in the kidney. In cultured renal cells, expression of both AQP2 isoforms was upregulated by the addition to the medium of NaCl but not by the addition of mannitol, indicating that the expression of both isoforms is induced by hypersalinity. Treatment with small interfering RNA for both isoforms resulted in a decrease in cell viability in hypertonic medium (500 mOsm kg−1) when compared with controls. These findings indicate that the expression of alternatively spliced AQP2 is ubiquitous in cetacean species, and it may be one of the molecules important for cellular osmotic tolerance throughout the body. Summary: Expression of alternatively splices AQP2 is ubiquitous in cetaceans, and it may be one of the molecules important for cellular osmotic tolerance throughout the body.

Characterization of the circulating serum amyloid A in bottlenose dolphins

Several isoforms of serum amyloid A (SAA) have been identified so far and because the plasma concentration of it increases dramatically, it is used as an indicator of inflammation in animals. In many terrestrial mammals, the circulating isoforms are SAA1 and SAA2, which are synthesized in the liver. Extra-hepatically synthesized SAA3, however, is a predominantly local SAA isoform with a characteristic N-terminal TFLK motif and a highly alkaline isoelectric point (pI). The aim of this study was to characterize the circulating SAA isoforms in bottlenose dolphins (dSAA) by determining the deduced amino acid sequence isolated from liver and the pI of plasma from healthy dolphins and those with inflammation. The deduced amino acid sequences of dSAA showed characteristics of SAA3 with an N-terminal TFLK motif, a predicted alkaline pI and were phylogenetically clustered with the SAA3 group rather than the SAA1 and SAA2 groups. Various tissues contained dSAA mRNA with the highest levels being detected in the liver. Isoelectric focusing and western blot analysis showed that one highly alkaline SAA was markedly detected in plasma obtained from dolphins affected by inflammation. These results suggest that, unlike other mammals, the circulating SAA in dolphins exhibits SAA3 properties, as is the case in pigs.

Establishing conditions for the storage and elution of rabies virus RNA using FTA® cards

The Flinders Technology Associates filter paper cards (FTA® cards) can be used to store nucleic acid from various samples and are easily portable. However, RNA is physicochemically unstable compared with DNA, and appropriate methods have not been established for storage and extraction of RNA from FTA® cards. The present study investigated the optimum conditions for storage and elution of viral RNA (vRNA) using rabies virus (RABV) applied to FTA® cards. When TE buffer was used, the elution rates of vRNA increased with the length of the elution time. When the cards were stored at −80°C or −20°C, vRNA was stable over 3 months. Degradation of vRNAs occurred following storage at 4°C and room temperature, suggesting that RNA should be extracted from cards as soon as possible if no freezer is available. When we tried to amplify vRNA from RABV-infected animal brains applied to FTA® cards and stored at −80°C for 6 months, we did not detect any amplified products with the primer set for 964 bp of RABV N gene. However, we were able to detect amplified products by increasing the elution time of vRNA from FTA® cards from 30 min to 24 hr or by changing the primer sets to amplify 290 bp of N gene. Thus, we recommend extending the elution time for damaged or low concentration samples in FTA® cards.

Easy-to-use rapid gene amplification method for direct detection of RNA and DNA viruses in sera and feces from various animals.

The development of rapid and simple gene amplification tests is required for detection of pathogens to prevent transmission of infectious diseases between animals or from animals to humans. An easy-to-use rapid gene amplification method that can directly detect RNA and DNA viruses in clinical samples was developed. This method is based on combining loop-mediated isothermal amplification (LAMP) or reverse transcription-LAMP (RT-LAMP) and RNA GEM Tissue, a thermophilic enzyme that extracts nucleic acid by quickly digesting proteins and ribonucleases. The authors named these methods GEM LAMP and GEM RT-LAMP. These methods were able to detect viral DNA and RNA within 70 min in a single tube using only a water bath. The detection capacities were 10-100-fold more sensitive than those of previously established LAMP and RT-LAMP methods. The GEM LAMP and GEM RT-LAMP methods were used to detect macroscopically the presence of DNA and RNA viruses in sera or fecal samples from cattle, pigs, horses, dolphins, penguins, and sea lions using SYBR green I. The GEM LAMP and GEM RT-LAMP methods thus have considerable versatility as tools for detecting pathogens and are applicable to basic human and veterinary medicine, environmental hygiene, and point-of-care-testing.

Isolation of a phylogenetically distinct rabies virus from a tufted capuchin monkey (Cebus apella) in Brazil

A rabies virus isolate (BRmk1358 strain) was discovered from a rabid tufted capuchin monkey in Brazil. The present study determined the nucleotide sequence of the BRmk1358 strain and compared with the rabies viruses isolated from marmosets and other animals in the Americas. Phylogenetic analyses showed that the BRmk1358 strain formed a lineage distant from that of marmoset rabies virus within the Chiroptera-related rabies virus cluster. This result suggests that the source of rabies infection in the tufted capuchin monkey may have been bat, and that they have a risk to act as rabies reservoir in Brazil.

Cloning and characterization of bottlenose dolphin (Tursiops truncatus) interleukin-10.

The function of cytokines in cetaceans has so far only been determined for the proinflammatory cytokines. In this study, we cloned bottlenose dolphin (Tursiops truncatus) interleukin-10 (IL-10) cDNA from concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC), and investigated the mRNA expression levels in various tissues and the bioactivity of recombinant dolphin (rd) IL-10. The gene encodes a polypeptide of 178 amino acids which encompasses the mature protein sequence of 158 amino acids. Quantitative expression analysis of dolphin IL-10 revealed that the highest mRNA levels are found in the spleen. To assess its function, rdIL-10 was produced in human embryonic kidney 293 cells and its bioactivity was demonstrated through IL-10-induced inhibition of proinflammatory cytokine mRNA expression IFN-γ, TNF-α, and IL-2 of Con A-stimulated PBMC. These results indicated that the structure and function of bottlenose dolphin IL-10 is similar to that of other animals. This is the first report of the characterization of an anti-inflammatory cytokine in cetaceans.

Identification and Expression Profiles of microRNA in Dolphin

Recently, microRNAs (miRNAs) are focused on the role of biomarker because they are stable in serum and plasma, and some of them express in the specific organs and increase with the organ injury. Thus miRNAs may be very useful as biomarkers for monitoring the health and condition of dolphins and for detecting disorders in aquariums. Here, a small RNA library was made from dolphin lung, liver and spleen, and miRNA expression patterns were then determined for 15 different tissues. We identified 62 conserved miRNA homologs in the dolphin small RNA library and found high expression miRNAs in specific tissues: miR-125b and miR-221 were highly expressed in brain, miR-23b in heart, miR-199a and miR-223 in lung, and miR-122-5p in liver. Some of these tissue-enriched miRNAs may be useful as specific and sensitive diagnostic blood biomarkers for organ injury in dolphins.

Molecular characterization and validation of commercially available methods for haptoglobin measurement in bottlenose dolphin

Haptoglobin (Hp) is a positive acute-phase protein and a valuable marker of inflammation in both human and veterinary medicine. The aim of this study was to validate the molecular characterization of Hp in dolphins and to validate commercially available Hp measurement methods such as Hp-ELISA (originally designed for pigs) and Hp-hemoglobin (Hb) binding assay. The dolphin Hp (dHp) amino acid sequence appeared most similar to pig Hp by sequence homology and phylogenetic clustering. Amino acid sequence analysis revealed that dHp comprises the Hp1 form of α1 and β chains. The anti-pig Hp antibody cross-reacted with both recombinant dHp, expressed by Escherichia coli, and dHp from serum. The intra- and inter-assay levels of imprecision of pig Hp-ELISA and the Hp-Hb binding assay were found to be tolerable for the determination of Hp in dolphin, and there was no significant discrepancy between the two determination methods. The ability of the assay to differentiate between healthy and inflammation groups was investigated, and a significant increase in Hp concentration was detected in inflammatory conditions. Thus, Hp is a useful inflammation marker for dolphin, and the Hp concentration in dolphin serum samples can be reliably measured using commercially available pig Hp-ELISA and Hp-Hb binding assay.