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A novel trypanoplasm-like flagellate Jarrellia atramenti n. g., n. sp. (Kinetoplastida: Bodonidae) and ciliates from the blowhole of a stranded pygmy sperm whale Kogia breviceps (Physeteridae): morphology, life cycle and potential pathogenicity.

The successful 6 mo rehabilitation of a stranded juvenile pygmy sperm whale Kogia breviceps afforded the opportunity to study the poorly known protozoan fauna of the upper respiratory tract of cetaceans. Mucus samples were collected by holding either a petri dish or glass slides over the blowhole for 3 to 5 exhalations; preparations were examined as wet mounts, and then stained with Wrights-Giemsa or Gram stain. Blood smears were stained with Wrights-Giemsa. Unidentified spindle-shaped and unidentified broad ciliates, reported from the blowhole of the pygmy sperm whale for the first time, were seen only initially, while yeast-like organisms and bacteria were seen intermittently. Epithelial cells and white blood cells were often present in the blowhole mucus, but red blood cells were never seen. A novel trypanoplasm-like bodonid kinetoplastid biflagellate (Order Kinetoplastida) was commonly encountered in the blowhole mucus, but never in the blood. Both mature flagellates and those undergoing longitudinal binary fission were present. The elongate flagellate had a long whiplash anterior flagellum; the recurrent flagellum was attached along at least two-thirds of the body length, forming a prominent undulating membrane, and the trailing portion was short. The kinetoplast was irregularly fragmented. The flagellates were either free-swimming, or attached to host material via the free portion of the posterior flagellum. The prominent undulating membrane was characteristic of Trypanoplasma, while the fragmented kinetoplast was characteristic of some species of Cryptobia. For the novel bodonid kinetoplastid, with its unique combination of morphological features (prominent undulating membrane and fragmented kinetoplast), we propose the creation of a new genus Jarrellia. We believe this to be the first published description of a flagellate from a marine mammal, and among the first reports of a trypanoplasm-like flagellate from a warm-blooded host. We expect that a diversity of flagellates and ciliates are commonly present in the blowhole of cetaceans. Future studies on the identity of the protozoans and the health of their cetacean hosts, which are readily studied in captivity, are necessary to establish their status as commensals or parasites.

Animals↗

An investigation of North Atlantic whales for trichinosis.

In 1971, 81 finback (Balaenoptera physalus), 119 sei (B. borealis), two piked (B. acutorostrata), 15 humpback (Megaptera novaeangliae) and one sperm whale (Physeter catodon) taken in the North Atlantic off the Newfoundland and Nova Scotia coasts were examined for the presence of Trichinella spiralis. All 218 whales were negative for trichinosis.

Animals↗

Reported causes of death of captive killer whales (Orcinus orca).

Inquiries were made to all oceanaria that maintain killer whales in North America. Causes of death determined at necropsy included mediastinal abscesses, pyometra, pneumonia, influenza, salmonellosis, nephritis, Chediak-Higashi syndrome, fungus infection, ruptured aorta, cerebral hemorrhage and a perforated post-pyloric ulcer. Captive females appear to have a higher rate of mortality than males. Growth rates for whales that died were greater than for those that survived.

Abscess↗

Genital papillomatosis in sperm whale bulls.

Examination of 31 male sperm whales (Physeter catodon) caught off the western coast of Iceland revealed three cases of genital papillomatosis involving the unsheathed penis. One subadult and two sexually mature bulls were affected. Gross lesions resembled papillomas common in terrestrial mammalian species. Transmission electron microscopy of these lesions revealed nonenveloped intranuclear virus particles 28-40 nm in diameter and round to hexagonal in shape. In two cases immunoperoxidase staining was negative for group-specific papillomavirus antigen. These findings indicate that the spectrum of animal species affected with virus-associated genital papillomatosis includes at least one globally distributed species of the order Cetacea (whales, dolpins, and porpoises).

Animals↗

Unusual multisystemic pathology in a sperm whale bull.

This report describes an unusual combination of lesions and apparent behavioral abnormalities in a sexually mature sperm whale bull taken in whaling operations off Iceland. Lesions included heavy combative scarring of the head, grossly roughened and thickened skin on the lower left flank, cutaneous maculae, genital papillomatosis, partial duodenal obstruction by plastic debris, colo-rectal obstruction by ambergris, cystic degeneration of the right kidney, and a deeply ulcerative gastric nematodiasis. Sealskin was found in the stomach. Gross and histopathologic observations suggested that the disease complex in this animal may have been related both to habitat degradation and health risks naturally associated with its ecology and age.

Animals↗

Concentrations and interactions of selected essential and non-essential elements in bowhead and beluga whales of arctic Alaska.

In this study, we evaluated concentrations of twelve essential and non-essential elements (As, Cd, Co, Cu, Pb, Mg, Mn, Hg, Mo, Se, Ag, and Zn) in tissues of bowhead (Balaena mysticetus) and beluga (Delphinapterus leucas) whales from arctic Alaska (USA) and northwestern Canada. Tissue samples were collected between 1983 and 1997, mostly in 1995-97. The essential elements are reported to develop reference ranges for health status determination, and to help assess known or suspected interactions affecting toxicoses of cadmium (Cd) and mercury (Hg). In some tissues, Cd, Hg, and selenium (Se) were present at concentrations that have been associated with toxicoses in some domestic animals. Nevertheless, tissue levels of all elements were within ranges that have been reported previously in marine mammals. While mean Ag concentrations in beluga whale liver were relatively high (15.91 micrograms/g ww), Ag was not associated with hepatic Se levels or age, contrary to previous findings. Significant associations included: Cd with age, Zn, or Cu; Cu with age, Zn or Ag; and Hg with age, Se, Zn, or Cu. This study found hepatic Hg:Se molar ratios to be consistently lower than unity and different between species. Possible explanations for observed elemental correlations (i.e., interactions) and ancillary mechanisms of Cd and Hg detoxification are discussed.

Age Factors↗

Struvite penile urethrolithiasis in a pygmy sperm whale (Kogia breviceps).

Massive urolithiasis of the penile urethra was observed in an adult pygmy sperm whale (Kogia breviceps) stranded on Topsail Island, North Carolina, USA. Calculi occupied the urethra from just distal to the sigmoid flexure to the tip of the penis for a length of 43 cm. A urethral diverticulum was present proximal to the calculi. The major portion of the multinodular urolith weighed 208 g and was 16 cm long x 3.7 cm diameter at the widest point. The urolith was composed of 100% struvite (magnesium ammonium phosphate) and on culture yielded Klebsiella oxytoca, a urease-positive bacterium occasionally associated with struvite urolith formation in domestic animals. Reaction to the calculi was characterized histologically by moderate multifocal to coalescing plasmacytic balanitis and penile urethritis. Role of the urethrolithiasis in the whale's stranding is speculative but could have involved pain or metabolic perturbations such as uremia or hyperammonemia.

Animals↗

A novel gammaherpesvirus associated with genital lesions in a Blainville's beaked whale (Mesoplodon densirostris).

An adult male Blainville's beaked whale (Mesoplodon densirostris) was found stranded on the Atlantic coast of the USA on 28 January 2004. Necropsy revealed a focal papilloma-like penile lesion, the cells from which revealed single 4-6 microm basophilic intranuclear inclusions. Total DNA extracted from lesion material was tested using a pan-herpes-virus PCR assay that targets the DNA polymerase gene and found to be positive. When the amplified DNA fragment was cloned, sequenced, and compared to GenBank-deposited herpesvirus DNA polymerase sequences, the detected virus was determined to be a distinct member of the Gammaherpesvirinae subfamily of herpesviruses. This new virus, tentatively named Ziphiid herpesvirus type 1, was associated with but not determined to be the cause of genital disease in the Blainville's beaked whale.

Amino Acid Sequence↗

Intramolecular translocation of the protein radical formed in the reaction of recombinant sperm whale myoglobin with H2O2.

A sperm whale myoglobin gene containing multiple unique restriction sites has been constructed in pUC 18 by sequential assembly of chemically synthesized oligonucleotide fragments. Expression of the gene in Escherichia coli DH5 alpha cells yields protein that is identical to native sperm whale myoglobin except that it retains the terminal methionine. Site-specific mutagenesis has been used to prepare all the possible tyrosine----phenylalanine mutants of the recombinant myoglobin, including the three single mutants at Tyr-103, -146, and -151, the three double mutants, and the triple mutant. All of the mutant proteins are stable except the Tyr-103 mutant. Introduction of a second mutation (Lys-102----Gln) stabilizes the Tyr-103 mutant. Absorption spectroscopy suggests that the active sites of the mutant proteins are intact. EPR and absorption spectroscopy show that all the proteins, including the triple mutant devoid of tyrosine residues, react with H2O2 to give a ferryl species and a protein radical. The presence of a protein radical in all the mutants suggests that the radical center is readily transferred from one amino acid to another. Cross-linking studies show, however, that protein dimers are only formed when Tyr-151 is present. Tyr-103, shown earlier to be the residue that primarily cross-links to Tyr-151 (Tew, D., and Ortiz de Montellano, P. R. (1988) J. Biol. Chem. 263, 17880-17886), is not essential for cross-linking. Electron transfer from Tyr-151 to the heme, which are 12 A apart, occurs in the absence of the intervening tyrosines at positions 103 and 146. The present studies show that the peroxide-generated myoglobin radical readily exchanges between remote loci, including non-tyrosine residues, but protein cross-linking only occurs when radical density is located on Tyr-151.

Animals↗

[The oxidation of sperm whale, horse, and pig oxymyoglobins, catalyzed by ferrocyanide ions: kinetics and mechanism].

The influence of pH, ionic strength of the solution, and [Fe(CN)6]4- concentration on the rate of oxidation of sperm whale, horse, and pig oxymyoglobins, which is catalyzed by ferrocyanide ions, was studied. These myoglobins have homologous spatial structures and identical redox potentials but differ by the amount of His residues located on the protein surface. The effect of the MbO2 complexing with redox-inactive Zn2+ ion on the reaction rate was also examined. At the equimolar Zn2+ concentration, zinc ions form a stable complex with His119(GH1). It was found that the kinetic behavior of horse MbO2, which lacks His12(A10) substituted for by Gln, is fully analogous to one of sperm whale MbO2, while the oxidation of pig MbO2, three histidines of which, His12, His113(G14), and His116(G17), are replaced by Gln, is strongly inhibited. The mechanism of the catalysis was shown to involve specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) site, which is in accord with the large positive electrostatic potential of this site and the presence here of a cavity large enough to accommodate [Fe(CN)6]4-. The nearby His113 and His116 residiues, which are absent in pig Mb, also play a very important role in the catalysis, because their protonation (especially of the last residue) is most likely responsible for the week oxidation of bound [Fe(CN)6]4- by dissolved oxygen.

Animals↗

[The mechanism of oxymyoglobin oxidation catalyzed by ferrocyanide ions: chemically modified and mutant sperm whale myoglobins].

A comparative study of the rate of ferrocyanide-catalyzed oxidation of native sperm whale MbO2, its chemically modified derivative in which all accessible His residues are alkylated by sodium bromoacetate, (CM-MbO2), and mutant sperm whale MbO2 with His119 replaced by Asp residiue, [MbO2(His119-->Asp)] was carried out. The influence of pH, ionic strength, and [Fe(CN)6]4- concentration on the oxidation rate was investigated, as well as the effect of complexing MbO2 with redox-inactive Zn2+ ion, which, at the equimolar Zn2+ concentration, forms a stable complex with His119(GH1) on the protein surface. It was shown that the mechanism of the catalysis involves specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) region, which is in agreement with a large positive electrostatic potential and the presence at this site of Mb of a cavity large enough to accommodate [Fe(CN)6]4- anion. The protonation of nearby His113 and His116 residiues (especially of the latter) plays a very important role in the catalysis, promoting the fast oxidation of bound [Fe(CN)6]4- by dissolved oxygen. Only the presence of these both necessary conditions in MbO2 structure provides its effective oxydation catalyzed by ferrocyanide.

Amino Acid Substitution↗

Evidence for cooperative hydrogen ligand binding to sperm whale metmyoglobin.

Potentiometric titrations of sperm whale metmyoglobin from pH 10 to 3 shows the well-known exposure of groups between pH 4.5 and 4. However the reverse titration, at low protein concentration, results in the regeneration of its ionic property in the form of a reversible hysteresis, which was obtained by titrating the same solution twice. The Soret intensity band indicates reversibility for about 94%. Apomyoglobin shows an acid-base reversible titration from pH 9.5 to 3. Since the analysis by electrostatic interaction models do not adequately describe the overall ionic equilibria of metmyoglobin, a cooperative model was introduced. The model exactly reproduces the titration curve, indicating that 6 groups ionize with a cooperative coefficient of 9. The cooperative model explains the anomalous behaviour toward hydrogen ions and the incomplete spectral reversibility of sperm whale metmyoglobin as a molecular mechanism with physiological significance.

Animals↗

Nuclear magnetic resonance titration curves of histidine ring protons. Human metmyoglobin and the effects of azide on human, horse, and sperm whale metmyoglobins.

Four titrating histidine ring C2 and C4 proton resonances are observed in 220 MHz proton NMR spectra of human metmyoglobin as a function of pH. Values of ionization constants determined from the NMR titration data using an equation describing a simple proton association-dissociation equilibrium are curves (1) 6.6, (2) 7.0, (3) 5.8, and (4) 7.4. Four histidine residues have also been found to be solvent-accessible in human metmyoglobin by carboxymethylation studies (Harris, C.M., and Hill, R.L. (1969) J. Biol. Chem. 244, 2195-2203). Two of the titration curves (3 and 4) deviate significantly from the chemical shift values normally observed for histidine C2 proton resonances. Curve 3, with a low pKa, is shifted downfield at high values of pH and also exhibits a second minor inflection with a pKa value of 8.8. On the other hand, the high pKa curve, 4, is shifted upfield at all values of pH. The characteristics of the NMR titration curves with the lowest and highest pKa values (3 and4) are very similar to curves observed previously with sperm whale and horse metmyoglobins (Cohen, J.S., Hagenmaier, H., Pollard, H., and Schechter, A.N. (1972) J. Mol. Biol. 71, 513-519). These results indicate that the histidine residues from which these curves are derived have unusual and characteristic environments in this series of homologous proteins. The NMR spectra of all three metmyoglobins are changed extensively as a result of azide ion binding, indicating conformational changes affecting the environments of several imidazole side chains. The presence of azide ion causes a selective downfield chemical shift for the low pKa curve and a selective upfield chemical shift for the high pKa curve in all three proteins. Azide also abolishes the second inflection seen in the low pKa curve at high pH. In addition to these effects, the presence of azide ion permits the observation of two additional titrating proton resonances for all three metmyoglobins. Increasing the azide to protein ratio at several fixed values of pH yields results which show that a slow exchange process is occurring with each of the metmyoglobins. In the azide titration studies the maximum changes in the NMR spectra occurred at approximately equimolar concentrations. The NMR results for these proteins in the absence and presence of azide ion are related to x-ray crystallographic studies of sperm whale metmyoglobin and the known alkylation properties of the histidine residues. Tentative assignments of the titrating resonances observed are suggested.

Animals↗

Taxonomy and distribution of a Crassicauda species (Nematoda: Spirurida) infecting the kidney of the common fin whale (Balaenoptera physalus Linné, 1758).

Nematode parasites were found in the kidney of common fin whales (Balaenoptera physalus Linné, 1758) captured in the North Atlantic Ocean (Denmark Strait area). These were identified as Crassicauda boopis Baylis, 1920, on the basis of morphologic and morphometric descriptions of this species' posterior end. The cephalic end of this parasite was described for the first time, however, and was indistinguishable from that of Crassicauda pacifica Margolis and Pike, 1955, a species whose posterior portion is unknown. It was also found that the eggs, size, host, and site of infection of both C. boopis and C. pacifica are indistinguishable. This established the synonymy of C. boopis with the more recently named taxon, C. pacifica. In light of previous descriptions of Crassicauda infections in balaenopterids, this implied that C. boopis should at present be considered a renal parasite of fin whales, and perhaps other rorquals, throughout the world's oceans.

Animals↗

[Primary structure of whale prolactin].

Two CNBr fragments of sea whale prolactin containing 69 and 41 amino acid residues, respectively, were hydrolyzed by trypsin, and the hydrolytic products were separated by the paper peptide mapping technique. The amino acid sequence of 17 homogeneous peptides was studied by the Edman method as well as by hydrolysis with carboxypeptidases A and B. Based on the experimental data and the previously published results the primary structure of sea whale prolactin made up of 199 amino acid residues was proposed.

Amino Acid Sequence↗

Binding of alkylisocyanides with soybean leghemoglobin. Comparisons with sperm whale myoglobin.

The binding of various linear and branched chain alkylisocyanides to soybean leghemoglobin has been studied with respect to association and dissociation kinetics and the results compared with those obtained in parallel on sperm whale and horse heart myoglobins; the linear ligands used (methyl to n-heptyl) cover a greater distribution of chain lengths than hitherto used. The association rate constants are much higher for leghemoglobin than for myoglobin, while the dissociation rates are slower. For a given protein, the dissociation rate constants are not much different when different isocyanides are used (except for methyl), whereas the association rates show complex behavior in relation with the alkyl chain length; singular differences are observed between leghemoglobin and sperm whale myoglobin in this regard. For myoglobin, the binding rate constants decrease from methyl to n-propyl, but remain approximately the same when the ligand carries a still longer alkyl chain. In contrast, for leghemoglobin, although the rate constants decrease from methyl to n-propyl, they show a progressive and important rise with longer alkyl substituents: n-butyl and n-pentyl.

Animals↗

Topographic antigenic determinants recognized by monoclonal antibodies to sperm whale myoglobin.

Monoclonal antibodies of high affinity (approximately 10(9) M-1) for sperm whale myoglobin were studied to pinpoint the antigenic determinants with which they interact. None of 6 different monoclonal antibodies tested reacted with any of the 3 CNBr cleavage fragments which encompass the whole sequence of myoglobin, an indication that they react with determinants present only on the native structure. To identify these sites, we compared the affinities of each antibody for a series of 14 mammalian myoglobins of known sequence and similar tertiary structure. Correlation of sequence differences with relative affinities allowed us, thus far, to identify critical antigenic residues recognized by 3 of the antibodies. Two of these antibodies recognize groups of residues which are far apart in primary structure but close together in the 3-dimensional structure of the native myoglobin molecule, i.e. topographic determinants. The third antibody distinguishes 140 Lys leads to Asn plus, probably, surface residues nearby. These determinants differ from previously reported antigenic sites on sperm whale myoglobin both in that they are topographic, rather than sequential, and in that almost all the critical residues recognized by these antibodies are outside the previously reported sites. Monoclonal antibodies are sensitive to subtle changes, e.g. Glu leads to Asp, in the antigenic site.

Amino Acid Sequence↗

[Isolation and partial characterization of the amino acid sequence of sea whale prolactin].

The protein hormone prolactin was isolated from sea whale hypophysis and its amino acid sequence was partially characterized. The hormone was hydrolyzed by BrCN and trypsin, the products of hydrolysis were separated and purified by gel filtration on Sephadex G-50, G-100 and G-25, by ion-exchange chromatography on CM-cellulose and on Dowex 50 x 8 and by partition paper chromatography. The structure of the obtained peptides was determined by the Edman method and by carboxypeptidase A hydrolysis. The partial amino acid sequence of sea whale prolactin making up to 75% of the hormone molecule was established.

Amino Acid Sequence↗