Possible control of acute outbreaks of a marine fungal pathogen by nominally herbivorous tropical reef fish.

Primary producers in terrestrial and marine systems can be affected by fungal pathogens threatening the provision of critical ecosystem services. Crustose coralline algae (CCA) are ecologically important members of tropical reef systems and are impacted by coralline fungal disease (CFD) which manifests as overgrowth of the CCA crust by fungal lesions causing partial to complete mortality of the CCA host. No natural controls for CFD have been identified, but nominally herbivorous fish could play a role by consuming pathogenic fungi. We documented preferential grazing on fungal lesions by adults of six common reef-dwelling species of herbivorous Acanthuridae and Labridae, (surgeonfish and parrotfish) which collectively demonstrated an ~ 80-fold higher grazing rate on fungal lesions relative to their proportionate benthic coverage, and a preference for lesions over other palatable substrata (e.g. live scleractinian coral, CCA, or algae). Furthermore, we recorded a ~ 600% increase in live CFD lesion size over an approximately 2-week period when grazing by herbivorous fish was experimentally excluded suggesting that herbivorous reef fish could control CFD progression by directly reducing biomass of the fungal pathogen. Removal rates may be sufficient to allow CCA to recover from infection and explain historically observed natural waning behaviour after an outbreak. Thus, in addition to their well-known role as determinants of macroalgal overgrowth of reefs, herbivorous fish could thus also be important in control of diseases affecting crustose coralline algae that stabilize the foundation of coral reef substrata.

Quantifying Spirorchiid Eggs in Splenic Histological Samples from Green Turtles.

The present study proposes a new methodology for the quantification of parasite eggs in animal tissue. Quantification of parasites are important to understand epidemiology of spirorchiid infections in sea turtles, however different methodologies for quantifying Spirorchiidae eggs in turtle tissues have been used. The most representative way to quantify Spirorchiidae burdens in tissues is counting eggs / g of tissue, however, this method is very laborious. As an alternative, we propose quantifying number of Spirorchiidae eggs/ area of tissue on a microscope slide. We compared this method to number of eggs / slide, a common metric of egg burden in turtle tissues. Both methods correlated well with eggs / g with eggs/mm2 of tissue having better correlation.

Gross and Microscopic Lesions in Corals from Micronesia.

The authors documented gross and microscopic morphology of lesions in corals on 7 islands spanning western, southern, and eastern Micronesia, sampling 76 colonies comprising 30 species of corals among 18 genera, with Acropora, Porites, and Montipora dominating. Tissue loss comprised the majority of gross lesions sampled (41%), followed by discoloration (30%) and growth anomaly (29%). Of 31 cases of tissue loss, most lesions were subacute (48%), followed by acute and chronic (26% each). Of 23 samples with discoloration, most were dark discoloration (40%), with bleaching and other discoloration each constituting 30%. Of 22 growth anomalies, umbonate growth anomalies composed half, with exophytic, nodular, and rugose growth anomalies composing the remainder. On histopathology, for 9 cases of dark discoloration, fungal infections predominated (77%); for 7 bleached corals, depletion of zooxanthellae from the gastrodermis made up a majority of microscopic diagnoses (57%); and for growth anomalies other than umbonate, hyperplasia of the basal body wall was the most common microscopic finding (63%). For the remainder of the gross lesions, no single microscopic finding constituted >50% of the total. Host response varied with the agent present on histology. Fragmentation of tissues was most often associated with algae (60%), whereas necrosis dominated (53%) for fungi. Two newly documented potentially symbiotic tissue-associated metazoans were seen in Porites and Montipora. Findings of multiple potential etiologies for a given gross lesion highlight the importance of incorporating histopathology in coral disease surveys. This study also expands the range of corals infected with cell-associated microbial aggregates.

Effects of Coralliophila violacea on tissue loss in the scleractinian corals Porites spp. depend on host response.

We investigated interactions between the corallivorous gastropod Coralliophila violacea and its preferred hosts Porites spp. Our objectives were to experimentally determine whether tissue loss could progress in Porites during or after Coralliophila predation on corals with and without tissue loss and to histologically document snail predation. In 64% of feeding scars, tissue regenerated within 3 wk, leaving no trace of predation. However, in roughly 28% of scars, lesions progressed to subacute tissue loss resembling white syndrome. In feeding experiments, scars from snails previously fed diseased tissue developed progressive tissue loss twice as frequently as scars from snails previously fed healthy tissue. Scars from previously healthy-fed snails were 3 times as likely to heal as those from previously diseased-fed snails. Histology revealed marked differences in host responses to snails; P. cylindrica manifested a robust inflammatory response with fewer secondary colonizing organisms such as algae, sponges, and helminths, whereas P. rus showed no evident inflammation and more secondary colonization. We conclude that lesion progression associated with Coralliophila may be associated with secondary colonization of coral tissues damaged by predator-induced trauma and necrosis. Importantly, variation at the cellular level should be considered when explaining interspecific differences in host responses in corals impacted by phenomena such as predation.

Dynamics of Virus Shedding and In Situ Confirmation of Chelonid Herpesvirus 5 in Hawaiian Green Turtles With Fibropapillomatosis.

Cancers in humans and animals can be caused by viruses, but virus-induced tumors are considered to be poor sites for replication of intact virions (lytic replication). Fibropapillomatosis (FP) is a neoplastic disease associated with a herpesvirus, chelonid herpesvirus 5 (ChHV5), that affects green turtles globally. ChHV5 probably replicates in epidermal cells of tumors, because epidermal intranuclear inclusions (EIIs) contain herpesvirus-like particles. However, although EIIs are a sign of herpesvirus replication, they have not yet been firmly linked to ChHV5. Moreover, the dynamics of viral shedding in turtles are unknown, and there are no serological reagents to confirm actual presence of the specific ChHV5 virus in tissues. The investigators analyzed 381 FP tumors for the presence of EIIs and found that overall, about 35% of green turtles had lytic replication in skin tumors with 7% of tumors showing lytic replication. A few (11%) turtles accounted for more than 30% cases having lytic viral replication, and lytic replication was more likely in smaller tumors. To confirm that turtles were actively replicating ChHV5, a prerequisite for shedding, the investigators used antiserum raised against F-VP26, a predicted capsid protein of ChHV5 that localizes to the host cell nucleus during viral replication. This antiserum revealed F-VP26 in EIIs of tumors, thus confirming the presence of replicating ChHV5. In this light, it is proposed that unlike other virus-induced neoplastic diseases, FP is a disease that may depend on superspreaders, a few highly infectious individuals growing numerous small tumors permissive to viral production, for transmission of ChHV5.

Skin pathology in Hawaiian goldring surgeonfish, Ctenochaetus strigosus (Bennett).

Twenty-eight goldring surgeonfish, Ctenochaetus strigosus (Bennett), manifesting skin lesions and originating from the north-western and main Hawaiian Islands were examined. Skin lesions were amorphous and ranged from simple dark or light discolouration to multicoloured tan to white sessile masses with an undulant surface. Skin lesions covered 2-66% of the fish surface, and there was no predilection for lesions affecting a particular part of the fish. Males appeared over-represented. Microscopy revealed the skin lesions to be hyperplasia, melanophoromas or iridophoromas. The presence of skin tumours in a relatively unspoiled area of Hawaii is intriguing. Explaining their distribution, cause and impact on survivorship of fish all merit further study because C. strigosus is an economically important fish in the region.

Histological observations in the Hawaiian reef coral, Porites compressa, affected by Porites bleaching with tissue loss.

The scleractinian finger coral Porites compressa is affected by the coral disease Porites bleaching with tissue loss (PBTL). This disease initially manifests as bleaching of the coenenchyme (tissue between polyps) while the polyps remain brown with eventual tissue loss and subsequent algal overgrowth of the bare skeleton. Histopathological investigation showed a loss of symbiont and melanin-containing granular cells which was more pronounced in the coenenchyme than the polyps. Cell counts confirmed a 65% reduction in symbiont density. Tissue loss was due to tissue fragmentation and necrosis in affected areas. In addition, a reduction in putative bacterial aggregate densities was found in diseased samples but no potential pathogens were observed.

Identification of a small, naked virus in tumor-like aggregates in cell lines derived from a green turtle, Chelonia mydas, with fibropapillomas.

Serial cultivation of cell lines derived from lung, testis, periorbital and tumor tissues of a green turtle (Chelonia mydas) with fibropapillomas resulted in the in vitro formation of tumor-like cell aggregates, ranging in size from 0.5 to 2.0 mm in diameter. Successful induction of tumor-like aggregates was achieved in a cell line derived from lung tissue of healthy green turtles, following inoculation with cell-free media from these tumor-bearing cell lines, suggesting the presence of a transmissible agent. Thin-section electron microscopy of the cell aggregates revealed massive collagen deposits and intranuclear naked viral particles, measuring 50+/-5 nm in diameter. These findings, together with the morphological similarity between these tumor-like cell aggregates and the naturally occurring tumor, suggest a possible association between this novel virus and the disease. Further characterization of this small naked virus will clarify its role in etiology of green turtle fibropapilloma, a life-threatening disease of this endangered marine species.

Comparative susceptibility of Culex tarsalis, Anopheles franciscanus, and Culiseta inornata (Diptera: Culicidae) to Plasmodium relictum (Haemosporidia: Plasmodiiae).

Repeated laboratory attempts failed to infect Culiseta inornata (Williston) and Anopheles franciscanus McCracken with Plasmodium relictum as efficiently as Culex tarsalis Coquillett controls. Of 210 An. franciscanus that imbibed a replete meal from a parasitemic canary, two were found with oocysts and none with sporozoites. Of 112 Cs. inornata similarly fed, seven contained oocysts and one contained sporozoites. In contrast, of 94 Cx. tarsalis tested, 80 (85%) were found with oocysts, thus confirming that this mosquito is a suitable host of P. relictum. Lack of An. franciscanus and Cs. inornata with sporozoites precluded transmission studies and indicated that these species do not play a major role in the maintanance of P. relictum during fall and winter in the Sacramento Valley, Calif.

Effects of anesthetization and storage temperature on bluetongue virus recovery from Culicoides variipennis (Diptera: Ceratopogonidae) and sheep blood.

Monitoring infection rates of Culicoides variipennis (Coquillett) with bluetongue virus in rangeland deer and cattle often requires prolonged field excursions. Methodology that fits field processing and storage was evaluated under controlled laboratory conditions. Triethylamine was used to anesthetize Culicoides for sorting. A portable liquid nitrogen vapor shipping container offered a convenient means of insect storage at temperatures that ensured preservation of the virus and eliminated fungal and bacterial contamination of cell cultures. Triethylamine and liquid nitrogen storage had little effect on recovery of bluetongue virus from experimentally infected C. variipennis and sheep blood stored under similar conditions.

Assessing humoral and cell-mediated immune response in Hawaiian green turtles, Chelonia mydas.

Seven immature green turtles, Chelonia mydas, captured from Kaneohe Bay on the island of Oahu were used to evaluate methods for assessing their immune response. Two turtles each were immunized intramuscularly with egg white lysozyme (EWL) in Freund's complete adjuvant, Gerbu, or ISA-70; a seventh turtle was immunized with saline only and served as a control. Humoral immune response was measured with an indirect enzyme linked immunosorbent assay (ELISA). Cell-mediated immune response was measured using in vitro cell proliferation assays (CPA) using whole blood or peripheral blood mononuclear cells (PBM) cultured with concanavalin A (ConA), phytohaemagglutinin (PHA), or soluble egg EWL antigen. All turtles, except for one immunized with Gerbu and the control, produced a detectable humoral immune response by 6 weeks which persisted for at least 14 weeks after a single immunization. All turtles produced an anamnestic humoral immune response after secondary immunization. Antigen specific cell-mediated immune response in PBM was seen in all turtles either after primary or secondary immunization, but it was not as consistent as humoral immune response; antigen specific cell-mediated immune response in whole blood was rarely seen. Mononuclear cells had significantly higher stimulation indices than whole blood regardless of adjuvant, however, results with whole blood had lower variability. Both Gerbu and ISA-70 appeared to potentiate the cell-mediated immune response when PBM or whole blood were cultured with PHA. This is the first time cell proliferation assays have been compared between whole blood and PBM for reptiles. This is also the first demonstration of antigen specific cell-mediated response in reptiles. Cell proliferation assays allowed us to evaluate the cell-mediated immune response of green turtles. However, CPA may be less reliable than ELISA for detecting antigen specific immune response. Either of the three adjuvants appears suitable to safely elicit a detectable immune response in green turtles.

MPTP-induced Parkinson-like disease in sheep: clinical and pathologic findings.

Eight ewes, divided into two groups based on age, with group 1 7-8 and group 2 1-3 years old, respectively, were administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intravenously (IV) at cumulative doses of 2.0 to 34.6 mg/kg body weight. Two group 1 sheep, given cumulative doses of 2 and 8.5 mg/kg, developed persistent severe neurologic signs of body stiffness and rigidity, paucity of movement, intention body tremors, and abnormal body posture and stance similar to those signs in MPTP-induced disease in people and primates. After their acute onset, these persistent signs were nonprogressive up to the observation period of 32 days post infusion. None of the younger ewes had persistent neurologic symptoms at equivalent cumulative doses (9.0 mg/kg). The only pathologic changes were microscopic lesions in the central nervous system, consisting of bilaterally symmetrical neuronal chromatolysis and necrosis limited to the substantia nigra and locus ceruleus. These lesions were found in two persistently affected and two younger sheep, suggesting age-based differences in dose response and the threshold of clinical expression of disease. Serum MPTP half-life was 11 days. Thus sheep exposed to MPTP could be an alternative model to the primate for the comparative study of clinical, pathologic, and biochemical mechanisms in MPTP neurotoxicity and Parkinson's disease in people.

Establishment and characterization of 13 cell lines from a green turtle (Chelonia mydas) with fibropapillomas.

Thirteen cell lines were established and characterized from brain, kidney, lung, spleen, heart, liver, gall bladder, urinary bladder, pancreas, testis, skin, and periorbital and tumor tissues of an immature male green turtle (Chelonia mydas) with fibropapillomas. Cell lines were optimally maintained at 30 degrees C in RPMI 1640 medium supplemented with 10% fetal bovine serum. Propagation of the turtle cell lines was serum dependent, and plating efficiencies ranged from 13 to 37%. The cell lines, which have been subcultivated more than 20 times, had a doubling time of approximately 30 to 36 h. When tested for their sensitivity to several fish viruses, most of the cell lines were susceptible to a rhabdovirus, spring viremia carp virus, but refractory to channel catfish virus (a herpesvirus), infectious pancreatic necrosis virus (a birnavirus), and two other fish rhabdoviruses, infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus. During in vitro subcultivation, tumor-like cell aggregates appeared in cell lines derived from lungs, testis, and periorbital and tumor tissues, and small, naked intranuclear virus particles were detected by thin-section electron microscopy. These cell lines are currently being used in attempts to isolate the putative etiologic virus of green turtle fibropapilloma.

Erysipelas in a free-ranging Hawaiian crow (Corvus hawaiiensis).

We describe a case of erysipelas in a free-ranging endangered Hawaiian crow. The partially scavenged carcass exhibited gross emaciation and petechial hemorrhages in both lungs. Microscopy revealed multiple necrotic foci associated with gram-positive rods in the liver and adrenal, diffuse acute proximal tubular necrosis of kidney, diffuse necrosis and inflammation of proventricular mucosa associated with gram-positive rods, and multiple intravascular aggregates of gram-positive rods associated with thrombi. Culture of the kidney revealed the bacterium to be Erysipelothrix rhusiopathiae. The implications of this finding to free-ranging crows remain unclear.

Necrotizing enteritis as a cause of mortality in Laysan albatross, Diomedea immutabilis, chicks on Midway Atoll, Hawaii.

A necropsy survey of Laysan albatross, Diomedea immutabilis, chicks on Midway Atoll in June 1993, 1994, and 1995 revealed 54% (21/39), 67% (49/71), and 93% (15/16), respectively, to have enteritis as the most severe pathologic finding. The lesion was limited to the ileum, ceca, and large intestine. We were unable to attribute a single infectious etiology to this lesion. Many birds with enteritis also exhibited renal lesions similar to those encountered in chickens experimentally deprived of water. We propose that enteritis is a significant cause of mortality in Laysan albatross chicks on Midway and that it may be a sequela to dehydration. It is likely that the pathology of dehydration in Laysan albatross differs from that in chickens largely because of diet.

Description and epizootiology of Babesia poelea n. sp. in brown boobies (Sula leucogaster (Boddaert)) on Sand Island, Johnston Atoll, Central Pacific.

We describe a new species of piroplasm from brown boobies (Sula leucogaster) on Sand Island, Johnston Atoll National Wildlife Refuge, central Pacific. Mean parasitemia in adults and chicks was less than 1%, with the parasitemia in chicks significantly greater than in adults. There was no significant relation between the age of chicks and the degree of parasitemia. Parasitized red cells and red cell nuclei were significantly smaller than those of unparasitized cells, and infected birds appeared clinically normal. Prevalence of the parasite in chicks (54%) was significantly greater than in adults (13%), and the geographic distribution of parasitized chicks was skewed toward the eastern end of Sand Island. On the basis of morphologic characteristics, we named it Babesia poelea. The specific name is a concatenation of the Hawaiian names for dark (po'ele) and booby ('a). This is the second documentation of an endemic avian hemoparasite in seabirds from the central Pacific.

Haemoproteus iwa n. sp. in great frigatebirds (Fregata minor [Gmelin]) from Hawaii: parasite morphology and prevalence.

We describe a new species of Haemoproteus Kruse, 1890 from great frigatebirds (Fregata minor [Gmelin]) captured on Tern Island-French Frigate Shoals and Laysan Island in Hawaii. Parasite prevalence on Laysan Island (35%) was not significantly different than that of Tern Island (36%). On Laysan, prevalence was highest in juveniles (52%), followed by adult males (29%) and adult females (19%). Prevalence on Tern was 36% both for adult females and juveniles, and 28% for adult males. Parasitemia was low (mean < 2 parasites/10, 000 red blood cell). Parasitized red cells had significantly greater areas than unparasitized cells. We named this parasite Haemoproteus iwa after the Hawaiian name for frigatebirds (iwa). This is the first documentation of a hemoparasite from tropical pelagic seabirds in Hawaii and the first description of an endemic hemoparasite in the archipelago.

Morphologic and cytochemical characteristics of blood cells from Hawaiian green turtles.

OBJECTIVE: To identify and characterize blood cells from free-ranging Hawaiian green turtles, Chelonia mydas. SAMPLE POPULATION: 26 green turtles from Puako on the island of Hawaii and Kaneohe Bay on the island of Oahu. PROCEDURE: Blood was examined, using light and electron microscopy and cytochemical stains that included benzidine peroxidase, chloroacetate esterase, alpha naphthyl butyrate esterase, acid phosphatase, Sudan black B, periodic acid-Schiff, and toluidine blue. RESULTS: 6 types of WBC were identified: lymphocytes, monocytes, thrombocytes, heterophils, basophils, and eosinophils (small and large). Morphologic characteristics of mononuclear cells and most granulocytes were similar to those of cells from other reptiles except that green turtles have both large and small eosinophils. CONCLUSIONS: Our classification of green turtle blood cells clarifies improper nomenclature reported previously and provides a reference for future hematologic studies in this species.

Weights, hematology, and serum chemistry of seven species of free-ranging tropical pelagic seabirds.

I established reference values for weight, hematology, and serum chemistry for seven species of free-ranging Hawaiian tropical pelagic seabirds comprising three orders (Procellariiformes, Pelecaniformes, Charadriiformes) and six families (Procellariidae, Phaethontidae, Diomedeidae, Sulidae, Fregatidae, and Laridae). Species examined included 84 Hawaiian darkrumped petrels (Pterodoma phaeopygia), 90 wedge-tailed shearwaters (Puffinus pacificus), 151 Laysan albatrosses (Diomedea immutabilis), 69 red-footed boobies (Sula sula), 154 red-tailed tropicbirds (Phaeton rubricauda), 90 great frigatebirds (Fregata minor), and 72 sooty terns (Sterna fuscata). Hematocrit, total plasma solids, total and differential white cell counts, serum glucose, calcium, phosphorus, uric acid, total protein, albumin, globulin, aspartate aminotransferase and creatinine phosphokinase were analyzed. Among and within species, hematology and chemistry values varied with age, sex, season, and island of collection. Despite this variation, order-wide trends were observed.

Causes of owl mortality in Hawaii, 1992 to 1994.

Eighty-one barn owls (Tyto alba) and five Hawaiian owls or pueo (Asio flammeus sandwichensis) from Kauai, Oahu, Lanai, Molokai, Maui and Hawaii (USA) were evaluated for cause of death, November 1992 through August 1994. The most common cause of death in barn owls was trauma (50%) followed by infectious disease (28%) and emaciation (22%). Most traumas apparently resulted from vehicular collisions. Trichomoniasis was the predominant infectious disease and appeared to be a significant cause of death in barn owls in Hawaii. Pasteurellosis and aspergillosis were encountered less commonly. No predisposing cause of emaciation was detected. Stomach contents from 28 barn owls contained mainly insects (64%) of the family Tetigoniidae and Gryllidae, and rodents (18%); the remainder had mixtures of rodents and insects or grass. Three pueo died from trauma and one each died from emaciation and pasteurellosis. We found no evidence of organochlorine, organophosphorus, or carbamate pesticides as causes of death in pueo or barn owls.