Seroepidemiology of TmPV1 infection in captive and wild Florida manatees (Trichechus manatus latirostris)

In 1997, cutaneous papillomatosis caused by Florida manatee (Trichechus manatus latirostris [Tm]) papillomavirus 1 (TmPV1) was detected in seven captive manatees at the Homosassa Springs Wildlife State Park, Florida, USA, and, subsequently, in two wild manatees from the adjacent Homosassa River. Since then, papillomatosis has been reported in captive manatees housed in other locations, but not in wild animals. To determine TmPV1 antibody prevalence in captive and wild manatees sampled at various locations throughout Florida coastal regions, virus-like particles, composed of the L1 capsid protein of TmPV1, were generated with a baculovirus expression system and used to measure anti-TmPV1 antibodies in an enzyme-linked immunosorbent assay. Serologic analysis of 156 manatees revealed a TmPV1 antibody prevalence of 26.3%, with no significant difference between captive (n=39) and wild (n=117) manatees (28.2% and 25.6%, respectively). No antibody-positive wild animal showed PV-induced cutaneous lesions, whereas papillomatosis was observed in 72.7% of antibody-positive captive manatees. Our data indicate that Florida manatees living in the wild are naturally infected by TmPV1 but rarely show TmPV1-induced papillomatosis. Hence, it appears that the wild population would not be harmed in a case of contact with captive animals without visible lesions and productive infections, which could be thus released into the wild.     

Identification of Hendra virus G glycoprotein residues that are critical for receptor binding

Hendra virus (HeV) is an emerging paramyxovirus capable of infecting and causing disease in a variety of mammalian species, including humans. The virus infects its host cells through the coordinated functions of its fusion (F) and attachment (G) glycoproteins, the latter of which is responsible for binding the virus receptors ephrinB2 and ephrinB3. In order to identify the receptor binding site, a panel of G glycoprotein constructs containing mutations was generated using an alanine-scanning mutagenesis strategy. Based on a predicted G structure, charged amino acids residing in regions that could be homologous to those in the measles virus H attachment glycoprotein known to be involved in its protein receptor interaction were targeted. Using a coprecipitation-based assay, seven single-amino-acid substitutions in HeV G were identified as having significantly impaired binding to both the ephrinB2 and ephrinB3 viral receptors: D257A, D260A, G439A, K443A, G449A, K465A, and D468A. The impairment of receptor interaction conferred a concomitant diminution in their abilities to promote membrane fusion when coexpressed with F. The G glycoprotein mutants were also recognized by three or more conformation-dependent monoclonal antibodies of a panel of five, were expressed on the cell surface, and retained their abilities to bind and coprecipitate F. Interestingly, some of these mutant G glycoproteins coprecipitated with F more efficiently than wild-type G. Taken together, these data provide strong biochemical and functional evidence that some of these residues could be part of a conformation-dependent, discontinuous, and overlapping ephrinB2 and -B3 binding domain within the HeV G glycoprotein.     

The effect of a preparation made from Solco lactobacteria on the survival rate and intestinal microflora of irradiated animals

Lactobacillus acidophilus strain Lat 11/83 Solco has been used for the normalization of intestinal microflora in experimental post-irradiation intestinal dysbacteriosis in mice. The results of these experiments indicate that live Solco lactobacteria, introduced intragastrically, facilitate the enhancement of the survival rate of irradiated animals and the normalization of microflora in their gastrointestinal tract. The strain can be used as bacterial preparation for the regulation of intestinal microbiocenosis.     

Enhancement in leghemoglobin content of root nodules by exclusion of solar UV-A and UV-B radiation in soybean

The impact of exclusion of solar UV-B (280–320 nm) and UV-A+B (280–400 nm) radiation on the root nodules was studied in soybean(Glycine max var. MACS 330). Soybean plants were grown in the tropical region of Indore (Latitude-22.4°N), India under field conditions in metal cages covered with polyester exclusion filters that specifically cut off UV-B (<320 nm) and UV-A+B (<400 nm) radiation; control plants were grown under ambient solar radiation. Leghemoglobin content was analyzed in the root nodules on the 50^th day after emergence of seedlings. Exclusion of UV radiations significantly enhanced the leghemoglobin content in the nodules on fresh weight basis; 25% and 45% higher amount of leghemoglobin were present in the nodules after the exclusion of UV-B and UV-A+B radiation respectively. Analysis by native and SDS-PAGE showed high intense bands of leghemoglobin after the exclusion of UV-A+B as compared to control. Exclusion of UV radiation also enhanced the growth of roots as well as aerial parts of the plants. UV Exclusion increased nodulation by increase in the number and size of nodules. The results are discussed in the light of advantage of exclusion for enhancing protein/nitrogen content in the plants.     

Examination of naturally exposed bottlenose dolphins (Tursiops truncatus) for Microsporidia, Cryptosporidium, and Giardia

Bottlenose dolphins (Tursiops truncatus) captured in the estuarine waters off the coasts of South Carolina and Florida were examined for the presence of Microsporidia, Cryptosporidium sp., and Giardia sp. DNA extracted from feces or rectal swabs was amplified by polymerase chain reaction using parasite-specific small subunit ribosomal RNA gene primers. All positive specimens were subjected to gene sequence analysis. Of 83 dolphins, 17 were positive for Microsporidia. None was positive for Cryptosporidium or Giardia. Gene sequence data for each of the positive specimens were compared with data in GenBank. Fourteen specimens were found similar to, but not identical to, the microsporidian species Kabatana takedai, Tetramicra brevifilum, and Microgemma tinca, reported from fish, and possibly represent parasites of fish eaten by dolphins. Gene sequence data from 3 other specimens had approximately 87% similarity to Enterocytozoon bieneusi, a species known primarily to infect humans and a variety of terrestrial mammals, including livestock, companion animals, and wildlife. It is not clear if these specimens represent a species from a terrestrial source or a closely related species unique to dolphins. There were neither clinical signs nor age- or gender-related patterns apparent with the presence of these organisms.     

Residues in the stalk domain of the hendra virus g glycoprotein modulate conformational changes associated with receptor binding

Hendra virus (HeV) is a member of the broadly tropic and highly pathogenic paramyxovirus genus Henipavirus. HeV is enveloped and infects cells by using membrane-anchored attachment (G) and fusion (F) glycoproteins. G possesses an N-terminal cytoplasmic tail, an external membrane-proximal stalk domain, and a C-terminal globular head that binds the recently identified receptors ephrinB2 and ephrinB3. Receptor binding is presumed to induce conformational changes in G that subsequently trigger F-mediated fusion. The stalk domains of other attachment glycoproteins appear important for oligomerization and F interaction and specificity. However, this region of G has not been functionally characterized. Here we performed a mutagenesis analysis of the HeV G stalk, targeting a series of isoleucine residues within a hydrophobic α-helical domain that is well conserved across several attachment glycoproteins. Nine of 12 individual HeV G alanine substitution mutants possessed a complete defect in fusion-promotion activity yet were cell surface expressed and recognized by a panel of conformation-dependent monoclonal antibodies (MAbs) and maintained their oligomeric structure. Interestingly, these G mutations also resulted in the appearance of an additional electrophoretic species corresponding to a slightly altered glycosylated form. Analysis revealed that these G mutants appeared to adopt a receptor-bound conformation in the absence of receptor, as measured with a panel of MAbs that preferentially recognize G in a receptor-bound state. Further, this phenotype also correlated with an inability to associate with F and in triggering fusion even after receptor engagement. Together, these data suggest the stalk domain of G plays an important role in the conformational stability and receptor binding-triggered changes leading to productive fusion, such as the dissociation of G and F.     

Recreational exposure to aerosolized brevetoxins during Florida red tide events

During two separate Karenia brevis red tide events, we measured the levels of brevetoxins in air and water samples, conducted personal interviews, and performed pulmonary function tests on people before and after they visited one of two Florida beaches. One hundred and twenty-nine people participated in the study, which we conducted during red tide events in Sarasota and Jacksonville, FL, USA. Exposure was categorized into three levels: low/no exposure, moderate exposure, and high exposure. Lower respiratory symptoms (e.g. wheezing) were reported by 8% of unexposed people, 11% of the moderately exposed people, and 28% of the highly exposed people. We performed nasal–pharyngeal swabs on people who experienced moderate or high exposure, and we found an inflammatory response in over 33% of these participants. We did not find any clinically significant changes in pulmonary function test results; however, the study population was small. In future epidemiologic studies, we plan to further investigate the human health impact of inhaled brevetoxins.     

Prevalence of Sarcocystis sp. in stranded Atlantic white-sided dolphins (Lagenorhynchus acutus)

In January 1998 and 1999, two mass strandings of dolphins occurred in Wellfleet, Massachusetts. The strandings were composed of 97 and 53 animals, respectively. Tissues from 35 Atlantic white-sided dolphins (Lagenorhynchus acutus) from the 1998 stranding and 52 from the 1999 stranding were examined histologically. In the 1998 stranding, unidentified protozoal tissue cysts were seen in skeletal muscle from 11 of 28 (39%) dolphins. In addition, two dolphins had a protozoal tissue cyst in cardiac muscle. In the 1999 stranding, nine of 23 (39%) dolphins had the same protozoal tissue cysts in skeletal muscle. The identification of these protozoal tissue cysts as Sarcocystis sp. was confirmed by light and transmission electron microscopy. The high prevalence of sarcocysts in these dolphins suggests that they are likely intermediate hosts for previously undescribed Sarcocystis spp. The ultrastructure of the sarcocyst walls suggests that more than one species of Sarcocystis are present in dolphins.     

Iron Indices in Bottlenose Dolphins (Tursiops truncatus)

Bottlenose dolphins can have iron overload (that is, hemochromatosis), and managed populations of dolphins may be more susceptible to this disease than are wild dolphins. Serum iron, total iron-binding capacity (TIBC), transferrin saturation, and ferritin were measured in 181 samples from 141 dolphins in 2 managed collections and 2 free-ranging populations. Although no iron indices increased with age among free-ranging dolphins, ferritin increased with age in managed collections. Dolphins from managed collections had higher iron, ferritin, and transferrin saturation values than did free-ranging dolphins. Dolphins with high serum iron (exceeding 300 μg/dL) were more likely to have elevated ferritin but not ceruloplasmin or haptoglobin, demonstrating that high serum levels of iron are due to a true increase in total body iron. A time-series study of 4 dolphins with hemochromatosis that were treated with phlebotomy demonstrated significant decreases in serum ferritin, iron, and TIBC between pre- and posttreatment samples; transferrin saturation initially fell but returned to prephlebotomy levels by 6 mo after treatment. Compared with those in managed collections, wild dolphins were 15 times more likely to have low serum iron (100 μg/dL or less), and this measure was associated with lower haptoglobin. In conclusion, bottlenose dolphins in managed collections are more likely to have greater iron stores than are free-ranging dolphins. Determining why this situation occurs among some dolphin populations and not others may improve the treatment of hemochromatosis in dolphins and provide clues to causes of nonhereditary hemochromatosis in humans.Abbreviations: TIBC, total iron-binding capacityBottlenose dolphins (Tursiops truncatus) are susceptible to iron overload (that is, hemochromatosis).^19,32,33,35 Dolphins with hemochromatosis have high serum iron levels that progress over years, have high transferrin saturation (85% and greater), and are responsive to phlebotomy therapy.¹⁹ Left untreated, dolphins with hemochromatosis are more likely to have increased liver aminotransferases, chronic inflammation, and hyperlipidemia than are healthy controls.³⁵ A 25-y retrospective study of one population demonstrated that 67% of dolphins had excessive hepatic hemosiderin deposition at time of death, 92% of which had hemosiderin deposition in Kupffer cells; hemolytic anemia, anemia of chronic disease, and viral infections were not associated with hemosiderin deposition, and the primary hypothesis is that dolphins in managed collections may be susceptible to iron storage disease.³²The etiology of dolphin iron overload is unknown. The most common cause in humans is hereditary hemochromatosis, which is most often attributed to a point mutation of the HFE gene.¹² Other causes of iron overload include primary liver disease, excessive iron intake, and insulin resistance.^3,36 Comparisons of the HFE gene among dolphins with and without hemochromatosis showed no significant differences (unpublished data), and iron deposition in Kupffer cells (compared with hepatocytes) is not supportive of a hereditary cause.^3,32 Liver enzymes decrease in response to the removal of body iron through phlebotomy; this effect indicates that iron overload leads to liver disease rather than that primary liver disease causes iron overload.¹⁹ At least one dolphin population from which cases of hemochromatosis have been reported have not received iron supplementation for more than 10 y, although the iron content in their frozen–thawed fish diet has not been assessed fully.¹⁹ Dolphins with hemochromatosis have significantly higher 2-h postprandial insulin levels than do controls, suggesting that insulin resistance plays a role in this disease, as occurs in humans.³³In addition to dolphins and humans, iron overload occurs in diverse captive birds, callitrichids, black rhinos, Egyptian fruit bats, lemurs, northern fur seals, California sea lions, tapirs, and Saler cattle.^{5,11,15,18,20,21,23,29,31} Similar to that in other exotic species, iron overload in dolphins is thought to be a disease that is associated with managed care, but comparisons of iron levels among managed collections and free-ranging dolphin populations have been lacking.¹⁹ Further, measurements of serum ferritin, ceruloplasmin, and haptoglobin have not typically been included in iron overload evaluations in dolphins. Although serum ferritin levels can be an indicator of total iron body stores, ferritin also increases in response to inflammation or hemolytic anemia.⁴ In humans, ceruloplasmin is a plasma protein that increases with acute and chronic inflammation and plays a role in iron metabolism. Haptoglobin is a protein that, in humans, decreases with intravascular hemolytic anemia, among other conditions.² As such, concurrent measurements of iron, ferritin, ceruloplasmin, and haptoglobin provide insight into the true cause of changing iron and ferritin levels.Low serum iron is used as a general indicator of inflammation in marine mammals, and return of serum iron to normal levels during illness is seen as a positive indicator for recovery.^13,24 The dynamics of iron, erythrocyte sedimentation rate, and fibrinogen in inflammation have remained important as indicators of dolphin health for decades, yet little has been published about dolphin iron stores and associated acute-phase proteins in the past 30 y.²⁴ One barrier to the use of acute-phase proteins, including C-reactive protein, has been the need for species-specific assays; development of tests that better detect early phases of inflammation and infection could lead to earlier treatment and better outcomes. Other barriers include the need for consecutive samples from dolphins with known health status to validate indicators for inflammation, including ceruloplasmin.To better understand the prevalence of high serum iron and potential risks for hemochromatosis among various dolphin populations, indices of serum iron, ferritin, total iron-binding capacity (TIBC), percentage transferrin saturation, ceruloplasmin, and haptoglobin concentrations were measured in 2 populations of free-ranging dolphins and 2 managed collections, including the cohort of Navy dolphins. These variables were compared by age, sex, and population. Serum ferritin was measured by using a newly developed dolphin-specific assay.     

Yin Yang 1 Orchestrates a Metabolic Program Required for Both Neural Crest Development and Melanoma Formation

1. Download high-res image (168KB)
2. Download full-size image