Fusarium spp. in Loggerhead Sea Turtles (Caretta caretta): From Colonization to Infection.

With the aim of evaluating the presence of Fusarium spp. in sea turtles with and without lesions and assessing the risk factors favoring colonization and/or infection, 74 loggerhead sea turtles (Caretta caretta) admitted to rescue and rehabilitation clinics in Italy were analyzed. The study compared 31 individuals with no apparent macroscopic lesions and 43 individuals with macroscopic lesions. Shell and skin samples were analyzed using Calcofluor white with 10% potassium hydroxide, standard histopathological examination, and fungal cultures. Fusarium spp. were isolated more frequently from animals with superficial lesions (39%) than from those with no macroscopic lesions (16%). Isolates from animals with superficial lesions were Fusarium solani species complex (FSSC) lineages haplotypes 9, 12, and 27 (unnamed lineages), FSSC-2 (Fusarium keratoplasticum), Fusarium oxysporum (27%), and Fusarium brachygibbosum (3%). In contrast, only F. solani haplotypes 9 and 12 were isolated from animals with no macroscopic lesions. The presence of lesions was identified as a risk factor for the occurrence of Fusarium spp. Of the 74 animals, only 7 (9.5%) scored positive on microscopic examination with Calcofluor, and histological examination of those 7 animals revealed necrosis, inflammatory cells, and fungal hyphae in the carapace and skin. The results of this study suggest that fusariosis should be included in the differential diagnosis of shell and skin lesions in sea turtles. Direct examination using Calcofluor and potassium hydroxide was not useful to diagnose the infection. Histopathological examination and fungal culture should be performed to ensure correct treatment and infection control.

The role of Virus "X" (Tortoise Picornavirus) in kidney disease and shell weakness syndrome in European tortoise species determined by experimental infection.

Tortoise Picornavirus (ToPV) commonly known as Virus "X" was recently discovered in juvenile European tortoises suffering from soft carapace and plastron as well as kidney disease. Therefore, this virus was a potential candidate to be a causative agent for these disease patterns. Spur thighed tortoises (Testudo graeca) seemed to be more susceptible to establish clinical symptoms than other European species like T. hermanni. Thus this trial investigated the role of ToPV in the described syndrome. Two groups of juvenile European tortoises (T. graeca and T.hermanni) each of 10 animals, were cloacally, oronasally and intracoelomically inoculated with an infectious dose (~ 2000 TICD) of a ToPV strain isolated from a diseased T. graeca. A control group of two animals of each species received non-infected cell culture supernatant. The tortoises were examined daily and pharyngeal and cloacal swabs for detection of ToPV-RNA by RT-PCR were taken from each animal every six days for a period of 6 months. At the end of the study the remaining animals were euthanised and dissected. Bacteriological and parasitological tests were performed and organ samples of all tortoises were investigated by RT-PCR for the presence of ToPV and histopathology. Animals that were euthanised at the end of the experiment, were examined for presence of specific anti-ToPV antibodies. Several animals in both inoculated groups showed retarded growth and a light shell weakness, in comparison to the control animals. Three animals were euthanised during the trial, showing reduced weight gain, retarded growth, severe shell weakness and apathy, in parallel to clinical observations in naturally infected animals. In all inoculated animals of both species an intermittent virus shedding, starting from 18 days post inoculation (d.p.i.), till 164 d.p.i. was detected, while the control animals remained negative. The virus was successfully reisolated in terrapene heart cell culture in 16 of 20 inoculated animals of both species. Histopathology of most inoculated animals revealed a lack of bone remodeling and vacuolisation in kidney tubuli which supports the described pathogenesis of nephropathy and osteodystrophy. Anti- ToPV antibody titres ranged from 1:2 to >1:256 in 13 of 20 animals, whereas all control animals were seronegative. The study proofed the Henle Koch`s postulates of ToPV as causative agent for shell dystrophy and kidney disease in both testudo species. The proposed species specific sensitivity towards clinical disease was not observed.

Nutrition and husbandry conditions of Palearctic tortoises (Testudo spp.) in captivity.

Mediterranean and Russian tortoises (Testudo spp.) are popular companion animals (pets), despite ongoing controversy concerning privately keeping reptiles. The arguments used during these controversial discussions have often been based on outdated facts. Therefore, a survey was developed to evaluate the current population structure, husbandry conditions, diet regime, and health status of Testudo species in captivity. More than 75% of the 1075 respondents housed their tortoises in an outdoor enclosure containing a greenhouse or cold frame, which is considered the most species-appropriate way of husbandry. Of the respondents, 67.7% fed their tortoises with the optimum diet of more than 80% grasses and weeds during the summer vegetation period. Only 8.2% of respondents owned a tortoise with a diagnosed disease. According to the results, the likelihood of tortoises developing pyramidal growth syndrome, which can be used as an indicator of the quality of tortoise husbandry, was high in tortoises kept in a terrarium and/or fed a diet of less than 80% grasses and weeds in summer. This likelihood varied among species, with a higher incidence in Hermann's tortoises (Testudo hermanni).

Shell repair and the potential microbial causal in a shell disease of the pearl oyster Pinctada fucata.

The pearl oyster Pinctada fucata is famous for producing luxurious pearls. As filter feeders, they are confronted with various infectious microorganisms. Despite a long history of aquaculture, diseases in P. fucata are not well studied, which limits the development of the pearl industry. We report here a shell disease in P. fucata and a study of the shell repair processes. Scanning electron microscopy (SEM) revealed that the nacreous layer gradually recovered from disordered CaCO3 deposition, accompanied by a polymorphic transition from a calcite-aragonite mixture to an aragonite-dominant composition, as revealed by X-ray diffraction analysis. SEM also showed that numerous microbes were embedded in the abnormal shell layers. Similar indications were induced by a high concentration of microbes injected into the extrapallial space, suggesting the potential pathogenic effect of uncontrolled microbes. Furthermore, hemocytes were found to participate in pathogens resistance and might promote shell repair. These results further our understanding of pathogen-host interactions in pearl oysters and have implications for biotic control in pearl aquaculture.

Rising Temperatures, Molting Phenology, and Epizootic Shell Disease in the American Lobster.

Phenological mismatch-maladaptive changes in phenology resulting from altered timing of environmental cues-is an increasing concern in many ecological systems, yet its effects on disease are poorly characterized. American lobster (Homarus americanus) is declining at its southern geographic limit. Rising seawater temperatures are associated with seasonal outbreaks of epizootic shell disease (ESD), which peaks in prevalence in the fall. We used a 34-year mark-recapture data set to investigate relationships between temperature, molting phenology, and ESD in Long Island Sound, where temperatures are increasing at 0.4°C per decade. Our analyses support the hypothesis that phenological mismatch is linked to the epidemiology of ESD. Warming spring temperatures are correlated with earlier spring molting. Lobsters lose diseased cuticle by molting, and early molting increases the intermolt period in the summer, when disease prevalence is increasing to a fall peak. In juvenile and adult male lobsters, September ESD prevalence was correlated with early molting, while October ESD prevalence was correlated with summer seawater temperature. This suggests that temperature-induced molting phenology affects the timing of the onset of ESD, but later in the summer this signal is swamped by the stronger signal of summer temperatures, which we hypothesize are associated with an increased rate of new infections. October ESD prevalence was ∼80% in years with hot summers and ∼30% in years with cooler summers. Yearly survival of diseased lobsters is <50% that of healthy lobsters. Thus, population impacts of ESD are expected to increase with increasing seawater temperatures.

Immune status of the spiny lobster Jasus edwardsii with tail fan necrosis.

Tail fan necrosis (TFN), a disorder commonly found in some populations of commercially fished and cultured lobsters, is thought to be initiated by injuries caused by handling and containment. The unsightly appearance of affected lobster tails significantly lowers their commercial value. Knowledge about TFN is limited. In this study we describe the morphological features of TFN and apply 6 common methods for evaluating the immune status of wild-caught Australasian red spiny lobsters Jasus edwardsii with and without TFN. The disease was more frequent in uropods than in telsons of the tail fan, and more extensive on the ventral versus the dorsal surfaces of the tail fan. Missing appendages (i.e. antenna, pereiopod or pleopod) were significantly more common and greater in number for individual lobsters affected with TFN versus those without, possibly as a result of handling in the fishery or as an indirect effect of the disease. Two immune parameters, total haemocyte count and phenoloxidase activity in the haemocyte lysate supernatant (HLS), were significantly compromised in lobsters with TFN. No differences were found in the other immune parameters, i.e. haemocyte viability, haemolymph bacterial count and the protein content of haemolymph plasma and HLS. The results are consistent with injury sustained during prior capture and handling that initiates TFN in these natural caught lobsters. These results raise some potential concerns about the fitness of lobsters in natural populations that are affected by TFN, and some potential solutions are proposed.

The use of silver amalgam for treatment of shell ulcerations in turtles: 60 cases.

Shell ulcers were diagnosed based on clinical appearance in 60 turtles. Under general anaesthesia, surgical curettage was performed using a burr mounted on a micro-grinder. A calcium hydroxide paste was applied to the debrided lesions before the application of restorative material. Lesions were completely filled with rapidly hardening silver amalgam. Treated terrapins were hospitalised in dry containers and later were housed in tanks with controlled temperature 24 hours. Fillings were found spontaneously expulsed at various periods after placement. No signs of mercury poisoning or tissue reaction to the prosthesis were apparent.

Real-time PCR assay for Aquimarina macrocephali subsp. homaria and its distribution in shell disease lesions of Homarus americanus, Milne-Edwards, 1837, and environmental samples.

Epizootic shell disease (ESD) is causing major losses to the lobster fishery in southern New England. Potential pathogens have been identified in lesion communities, but there are currently no efficient means of detecting and quantifying their presence. A qPCR assay was developed for a key potential pathogen, Aquimarina macrocephali subsp. homaria found to be ubiquitous in ESD lesions but not the unaffected integument. Application of the assay to various samples demonstrated that A. macrocephali subsp. homaria is ubiquitous and abundant in lobster lesions, commonly associated with healthy surfaces of crabs and is scarce in water and sediment samples from southern New England suggesting the affinity of this microorganism to the Arthropod integument. The qPCR assay developed here can be applied in future in vivo and in vitro studies to better understand the ecology and role of A. macrocephali subsp.homaria. in shell disease.

Basal cell carcinoma in two Hermann's tortoises (Testudo hermanni).

Neoplastic disorders are frequently encountered in the practice of reptile medicine. Herein we report the clinical behavior, antemortem diagnosis, and histopathologic characteristics of a recurrent intraoral keratinizing basal cell carcinoma (BCC) and a metastatic BCC of the carapace in 2 Hermann's tortoises (Testudo hermanni). Although squamous cell carcinomas (SCCs) in tortoises show similar predilection sites and gross pathologic features, the BCCs described in our report were characterized by a remarkably fast and highly infiltrative growth in comparison to SCCs. Accordingly, early diagnosis including reliable discrimination from SCC is essential toward the management of this neoplastic entity in tortoises.

Investigating the tool marks on oracle bones inscriptions from the Yinxu site (ca., 1319-1046 BC), Henan province, China.

Oracle Bone Inscriptions in the Shang dynasty (1600-1046 BC) are the earliest well-developed writing forms of the Chinese character system, and their carving techniques have not been studied by tool marks analysis with microscopy. In this study, a digital microscope with three-dimensional surface reconstruction based on extended depth of focus technology was used to investigate tool marks on the surface of four pieces of oracle bones excavated at the eastern area of Huayuanzhuang, Yinxu site(ca., 1319-1046 BC), the last capital of the Shang dynasty, Henan province, China. The results show that there were two procedures to carve the characters on the analyzed tortoise shells. The first procedure was direct carving. The second was "outlining design," which means to engrave a formal character after engraving a draft with a pointed tool. Most of the strokes developed by an engraver do not overlap the smaller draft, which implies that the outlining design would be a sound way to avoid errors such as wrong and missing characters. The strokes of these characters have different shape at two ends and variations on width and depth of the grooves. Moreover, the bottom of the grooves is always rugged. Thus, the use of rotary wheel-cutting tools could be ruled out. In most cases, the starting points of the strokes are round or flat while the finishing points are always pointed. Moreover, the strokes should be engraved from top to bottom. When vertical or horizontal strokes had been engraved, the shell would be turned about 90 degrees to engrave the crossed strokes from top to bottom. There was no preferred order to engrave vertical or horizontal strokes. Since both sides of the grooves of the characters are neat and there exists no unorganized tool marks, then it is suggested that some sharp tools had been used for engraving characters on the shells. Microsc. Res. Tech. 79:827-832, 2016. © 2016 Wiley Periodicals, Inc.

Possible shell disease in 100 million-year-old crabs.

Modern organisms exhibit evidence of many diseases, but recognizing such evidence in fossils remains difficult, thus hampering the study of the evolution of disease. We report on 2 molts of the goniodromitid crabs Distefania incerta and Goniodromites laevis from the mid-Cretaceous (late Albian) of Spain, with both species exhibiting damage to the dorsal carapace in otherwise well-preserved specimens. The subcircular to quadratical holes, found in <0.2% of the specimens, resemble damage caused by bacterial infections on the cuticle of modern decapods in terms of size and shape. Abiotic damage, predation, and encrustation followed by damage to the shell provide less satisfactory explanations, although these agents cannot be completely excluded from a role in shell disease etiology. We hypothesize that the observed fossil lesions are caused primarily by bacterial disease that started prior to molting, with or without other agents of initiation. If correct, this is the only known example of such bacterial infections in decapod crustaceans from the fossil record thus far, pushing back the evolutionary history of this type of shell disease by ~100 million years.

Disease, parasite, and commensal prevalences for blue crab Callinectes sapidus at shedding facilities in Louisiana, USA.

Blue crab diseases, parasites, and commensals are not well studied in the Gulf of Mexico, and their prevalence rates have only been sporadically determined. Commercial soft shell shedding facilities in Louisiana experience high mortality rates of pre-molt crabs, and some of these deaths may be attributable to diseases or parasites. During the active shedding season in 2013, we determined the prevalence of shell disease, Vibrio spp., Lagenophrys callinectes, and Hematodinium perezi at 4 commercial shedding facilities along the Louisiana coast. We also detected Ameson michaelis and reo-like virus infections. Shell disease was moderately prevalent at rates above 50% and varied by shedding facility, collection month, and crab size. Vibrio spp. bacteria were prevalent in the hemolymph of 37% of the pre-molt crabs. Lagenophrys callinectes was highly prevalent in the pre-molt crabs, but because it is a commensal species, it may not cause high mortality rates. Hematodinium perezi was absent in all pre-molt crabs.

Soft plastron, soft carapace with skeletal abnormality in juvenile tortoises. Histopathology and isolation of a novel picornavirus from Testudo graeca and Geochelone elegans.

OBJECTIVE: A disease is described in juvenile tortoises (Testudo graeca and Geochelone elegans) consisting mainly of a soft carapace, soft plastron and deformed skeleton. The aim of this study was to determine histopathological lesions and the biological properties of the isolated viruses. MATERIALS AND METHODS: Clinical signs and gross pathology were determined on diseased and healthy appearing tortoises. Paraffin sections were stained with HE, PAS and Prussian Blue and histologically examined. Terrapene heart (TH-1) cell cultures served for virus isolations from 64 tissues and 104 swabs. One isolate (isolate 1243/37 tongue) was used in neutralization tests on 19 sera. RESULTS: Retarded growth and increasingly soft plastron and carapace were the prominent signs in diseased tortoises. Pathological lesions consisted of dilated urinary sac, enlarged kidneys and livers. Histopathologically, hepatic hemosiderosis, hypoplastic anaemia, congestive glomerulonephrosis and osteodystrophy were seen. A novel vi- rus ("virus X") was isolated from 64 organs and 79 of 104 swabs. The isolated viruses were identified as a novel chelonid picornavirus based on cytopathic effect, resistance to chloroform and stability at low pH. Co-cultivation with 5-iodo-2'-deoxyuridine and actinomycin D did not reduce virus titres. Electron microscopically, round, non-enveloped particles (25-30 nm) were detected. Neutralizing antibodies to the isolate 1243/37tongue were present in 17 of 19 sera from seven species of tortoises. CONCLUSION AND CLINICAL RELEVANCE: Nephropathy, osteodystrophy and virus isolations suggest a viral aetiology. Metabolic bone disease is the major differential diagnosis. Further investigations in vivo are needed to evaluate the likely effects of the picornavirus on tortoises.

Fusarium solani species complex associated with carapace lesions and branchitis in captive American horseshoe crabs Limulus polyphemus.

Captive American horseshoe crabs Limulus polyphemus housed at the National Aquarium presented with a variety of shell and gill lesions over a 3 yr period. Carapace lesions were located on both the dorsal and ventral prosoma and opisthosoma and included multifocal circular areas of tan discoloration, ulcerations, and/or pitting lesions, extending from superficial to full thickness. Gill lesions involved both the book gill cover (operculum) and individual book gill leaflets and included multifocal circular areas of tan discoloration, tan to off-white opaque proliferative lesions, and/or areas of black discoloration. Histopathology revealed fungal hyphae, with variable morphology throughout the thickened and irregular cuticle of the carapace and occasionally penetrating into subcuticular tissues, with associated amebocytic inflammation. Book gill leaflets were infiltrated by fungal hyphae and contained necrotic debris and amebocytes. Thirty-eight of 39 animals (97%) evaluated via histopathological examination had intralesional fungal hyphae. Fungal cultures of carapace and gill lesions were attempted in 26 tissue samples from 15 individuals and were positive in 13 samples (50%), with 10 cultures (77%) yielding identification to genus. Fusarium sp. was identified in 8 of the 10 cultures (80%) via culture morphology. The Fusarium solani species complex was confirmed in 6 of these 8 (75%) via polymerase chain reaction amplification of 2 different ribosomal-specific sequences of isolated fungal DNA. Ante-mortem systemic and topical treatments were performed on some affected individuals, but no appreciable change in lesions was observed. Mycotic dermatitis and branchitis are serious health issues for captive American horseshoe crabs.

Early Pleistocene aquatic resource use in the Turkana Basin.

Evidence for the acquisition of nutritionally dense food resources by early Pleistocene hominins has implications for both hominin biology and behavior. Aquatic fauna may have comprised a source of highly nutritious resources to hominins in the Turkana Basin at ∼1.95 Ma. Here we employ multiple datasets to examine the issue of aquatic resource use in the early Pleistocene. This study focuses on four components of aquatic faunal assemblages (1) taxonomic diversity, (2) skeletal element proportion, (3) bone fragmentation and (4) bone surface modification. These components are used to identify associations between early Pleistocene aquatic remains and hominin behavior at the site of FwJj20 in the Koobi Fora Fm. (Kenya). We focus on two dominant aquatic species: catfish and turtles. Further we suggest that data on aquatic resource availability as well as ethnographic examples of aquatic resource use complement our observations on the archaeological remains from FwJj20. Aquatic food items provided hominins with a valuable nutritional alternative to an exclusively terrestrial resource base. We argue that specific advantages afforded by an aquatic alternative to terrestrial resources include (1) a probable reduction in required investment of energy relative to economic return in the form of nutritionally dense food items, (2) a decrease in the technological costs of resource acquisition, and (3) a reduced level of inter-specific competition associated with carcass access and an associated reduction of predation risk relative to terrestrial sources of food. The combined evidence from FwJj20 suggests that aquatic resources may have played a substantial role in early Pleistocene diets and these resources may have been overlooked in previous interpretations of hominin behavior.

A comparison of the structure of American (Homarus americanus) and European (Homarus gammarus) lobster cuticle with particular reference to shell disease susceptibility.

The integument of arthropods is an important first-line defence against the invasion of parasites and pathogens. Once damaged, this can be subject to colonisation by microbial agents from the surrounding environment, which in crustaceans can lead to a condition termed shell disease syndrome. This condition has been reported in several crustacean species, including crabs and lobsters. The syndrome is a progressive condition where the outer cuticle becomes pitted and eroded, and in extreme cases is compromised, leaving animals susceptible to septicaemia. This study examined the susceptibility of juvenile American (Homarus americanus) and European (Homarus gammarus) lobsters to shell disease, as a result of mechanical damage. Scanning electron microscopy was used as a method to identify differences in the cuticle structure and consequences of mechanical damage. Claw regions were aseptically punctured, whilst carapaces were abraded using sterile sandpaper, to mimic natural damage. After a period of between 10 and 12 weeks, lobsters were sacrificed, fixed and stored for later examination. The carapace and claws of juvenile American lobsters were shown to be thinner and more vulnerable to abrasion damage than their European counterparts. In addition, the number and distribution of setal pits and pore canal openings also differed between the two species of lobster. Mechanical damage resulted in the formation of shell disease lesions on the claw and carapace of both lobster species. However, American lobsters, unlike their European counterparts, had extensive bacterial colonisation on the margins of these lesions. Overall, it is concluded that the cuticle of the American lobster is more susceptible to damage and resulting microbial colonisation. This may have implications for susceptibility of both species of lobster to shell disease syndrome.

Shell damage in the Tehuelche scallop Aequipecten tehuelchus caused by Polydora rickettsi (Polychaeta: Spionidae) infestation.

The different types of shell damage caused to the commercially valuable Tehuelche scallop (Aequipecten tehuelchus) by the polychaete Polydora rickettsi are described. X-rays, computerized tomography, shell sections, scanning electron microscopy, Energy Dispersive X-ray analysis (EDAX), mineralogical analyses and geometric morphometrics were applied to that end. Scallop shells presented three types of damage: (1) spots, (2) calcareous alterations, and (3) mud blisters. Microstructural alterations consisted of a simple conchiolin membranous layer in the case of spots, a series of interleaved layers of different degree of calcifications in calcareous alterations, and two different surface morphologies (muddy and mucous layers) in mud blisters. Damage was localized mainly along concentric growth rings, coincidentally with the location of most burrows, as shown by X-ray. Mineralogical analysis showed that in all cases (including non-infested shells) calcite was the calcium carbonate polymorph present. Geometric morphometrics showed that only 5% of shape variation was explained by infestation with P. rickettsi, irrespective of the type of damage. Number of worms per infested shell varied significantly among four beds. Left shells (upward-oriented) were significantly more affected than right shells, which are in closer contact with the bottom.