Infected Turtles Research Articles

Genomic features and comparative analysis of a multidrug-resistant Acinetobacter bereziniae strain infecting an animal: a novel emerging one health pathogen?

Acinetobacter bereziniae has recently gained medical notoriety due to its emergence as a multidrug resistance and healthcare-associated pathogen. In this study, we report the whole-genome characterization of an A. bereziniae strain (A321) recovered from an infected semiaquatic turtle, as well as a comparative analysis of A. bereziniae strains circulating at the human-animal-environment interface. Strain A321 displayed a multidrug resistance profile to medically important antimicrobials, which was supported by a wide resistome. The novel Tn5393m transposon and a qnrB19-bearing ColE1-like plasmid were identified in A321 strain. Novel OXA-229-like β-lactamases were detected and expression of OXA-931 demonstrated a 2-64-fold increase in the minimum inhibitory concentration for β-lactam agents. Comparative genomic analysis revealed that most A. bereziniae strains did not carry any antimicrobial resistance genes (ARGs); however, some strains from China, Brazil, and India harbored six or more ARGs. Furthermore, A. bereziniae strains harbored conserved virulence genes. These results add valuable information regarding the spread of ARGs and mobile genetic elements that could be shared not only between A. bereziniae but also by other bacteria of clinical interest. This study also demonstrates that A. bereziniae can spill over from anthropogenic sources into natural environments and subsequently be transmitted to non-human hosts, making this a potential One Health bacteria that require close surveillance.

Hemoparasite occurrence and hematological/serum chemistry variations in Podocnemis vogli turtles: A comparative analysis between wild-residing infected specimens and captive non-infected counterparts.

Reptiles show a high occurrence of hemoparasites in the wild; however, little is known about the impact of such infections on their hosts' physiology and health status. Podocnemis vogli is an ancient turtle distributed in South America, frequently infected by blood parasites. Specifically, we analyzed the hematological and serum chemistry parameters of 78 wild turtles. We compared these values with those obtained from non-infected turtles of the same species in ex-situ conditions, considering factors such as sex and coinfections. Two orders of hemoparasites were detected under microscopic analyses: Adelorina, represented by Haemogregarina sp. (98.72±0.28%), and Haemosporida, represented by Haemocystidium sp. (30.77±1.16%), the latter genus being always in coinfection with Haemogregarina. Significant differences were observed in 20 parameters between infected (free living) and uninfected (living in captivity) turtles. The ALP, PCV, Hb, and MCV were significantly different by sex; albumin, cholesterol, creatinine, percentage of heterophils, lymphocytes, and basophils differed in coinfection. This is the first report of reference intervals of P. vogli in the wild and the first study comparing hematological and blood biochemistry values between hemoparasite-infected and uninfected P. vogli turtles. However, the limited knowledge of these parameters in wild reptiles and the wide range of interval values makes it difficult to determine any significant impact on turtle health. Nevertheless, this study highlights the need for more in-depth research to explore the potential effects of blood parasite infections on turtles, including immune response and coevolution studies.

Eight-Year Study of Haemogregarina stepanowi Infection in Poached European Pond Turtles (Emys orbicularis) Held in Belgrade Zoo Quarantine.

The eight-year study (2015-2023) was performed on a large sample of poached European pond turtles infected with Haemogregarina stepanowi and held in a pond that belongs to a quarantine section of Belgrade Zoo. The protected species of European pond turtles have been found in poor health, with general weakness, anorexia, and low motility. Comprehensive cytological, hematological, molecular, and postmortem evaluations have been performed. Initially, Diff Quick staining of the blood smears revealed rounded or elongated erythrocytes, often bearing premeront or U-shaped gamont of the hemogregarines inside. The reduced erythrocyte numbers, hemoglobin, and hematocrit values found in the examined population of infected turtles indicated anemia. Macroscopically, shell necrosis and massive skin hemorrhages were the most prominent findings observed in diseased turtles. Microscopically, the lungs, liver, kidneys, and spleen revealed hyperemia, hemorrhages, and the presence of parasitic stages in tissue samples in 31 of 40 necropsied turtles. Cytological and microscopic examination of the samples proved to be sufficient for establishing the infection, but molecular analyses of the 18S sequence were used for phylogenetic studies. Over the years, the number of diseased and dead turtles has decreased, which could be hypothetically attributed to the elimination of leeches as the definitive host.

Evidence of chelonid herpesvirus 5 infection in green turtle (Chelonia mydas) indicated a possible tumorigenesis activation by transcriptome analysis

Chelonia mydas (green turtles) are being threatened worldwide by fibropapillomatosis (FP), which has seriously affected their survival. The presence of FP on the body surface and visceral organs of green turtles found dead was confirmed, causing obstruction of the gastrointestinal tract, changes in foraging behavior, and reduction of visceral functions. The etiology of FP has not yet been elucidated, and previous research generally considers that the occurrence of FP is related to the chelonid alphaherpesvirus 5 (ChHV5), associated with low animal immunity, and also with marine environmental factors, such as poor water quality and eutrophication. However, there is no evaluation on the induction of FP pathogenesis associated with the green turtle. In this study, we evaluated blood samples from green turtles with and without FP using de novo transcriptome assembly. Results indicated that 3,090 differentially expressed genes (DEGs) (p < 0.05) were identified, including 1,357 upregulated genes and 1,733 downregulated genes in turtles with or without FP. We observed that DEGs, which are significantly upregulated, are found in cancer development, namely, MAPK1IP1L and APAF1. Furthermore, the infected green turtle indicated that the greater number of DEGs was contributed by the NOD-like receptor signaling pathway, which can be activated through an endocytosis of the viral particle by the immune system cells, and the Wnt signaling pathway, which is believed to have played a role in FP tumorigenesis. We validated the more upregulated/downregulated DEGs in cancer development and immunization, and DEGs such as LEF1, BTRC, and FOSL1 participating in the NOD-like receptor signaling pathway, as well as ERBIN, TRAF6, and NFKB1 in the Wnt signaling pathway, using real-time quantitative polymerase chain reaction (RT-qPCR). Altogether, this study provided some genes as potential markers during FP infection and a further evidence of FP in endangered green turtles in Taiwan.

Single-cell transcriptomes from turtle livers reveal sensitivity of hepatic immune cells to bacteria-infection

The liver is important in the synthesis, metabolism and storage of nutrients, detoxification and immune response of the body, and the liver immune response against exogenous pathogens from the intestinal tract plays a key role in the immune activities. However, the cellular composition of the liver immune atlas remains sparsely studied in reptiles. We used single-cell RNA sequencing to identify the cellular profile of the liver of the Chinese soft-shelled turtle (Pelodiscus sinensis). We obtained the transcriptional landscape based on 9938 cells from the fractionation of fresh hepatic tissues from two individuals, uninfected and infected with bacteria (Aeromonas hydrophila). We identified seven hepatic immune cell subsets, including plasma, erythroid, T/NK, B, endothelial, dendritic and Kupffer cells. Bacteria-infection altered the number of liver immune cells, as revealed by the fact that the infected turtle had more plasma, endothelial and Kupffer cells and fewer T/NK, dendritic and erythroid cells than did the uninfected turtle. Our study is the first to provide a comprehensive view of the hepatic immune landscape of P. sinensis at the single-cell resolution that outlines the characteristics of immune cells in the turtle liver and provides a liver transcriptome baseline for turtle immunology.

Pathologic, transcriptomic and microbiomic insight into the pathogenesis of intestinal parasitic tapeworm in cultured Chinese soft-shelled turtle (Pelodiscus sinensis)

Tapeworms are infamous and highly successful invasive parasites in aquatic animals, especially in fish. However, tapeworm-caused diseases in soft-shelled turtles are rarely discovered. In this study, a case of cultured Chinese soft-shelled turtle (Pelodiscus sinensis) infected with tapeworm in Changde city of China was reported, and the taxonomic identification of the tapeworm and the effects of tapeworm on the Chinese soft-shelled turtle were revealed. The 28S rRNA sequences of the tapeworm from P. sinensis had the highest similarity (99.82%) with Testudotaenia testudo of Proteocephalidea (Eucestoda), suggesting they are identical species. Infected turtles showed typical clinical symptoms of cestodiasis, which included nutrition loss, and histopathological lesions in the liver and intestine. The serum of infected turtles had significantly lower (P < 0.05) concentrations of glucose, creatinine, total protein, and low-density lipoprotein, and significantly higher (P < 0.05) alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), superoxide dismutase (SOD) activities. Trancriptional analyses showed that 158 differentially expressed genes (DEGs) were found in liver of infected turtles compared with uninfected groups. These DEGs mainly involved in metabolism, including “Arachidonic acid metabolism”, “Linoleic acid metabolism”, “Metabolism of xenobiotics by cytochrome P450”, “alpha-Linolenic acid metabolism”, “Ether lipid metabolism”, “Retinol metabolism” and “Glycerophospholipid metabolism”, which suggested that the tapeworm infection seriously affected the nutrient metabolism of P. sinensis. Besides, 12 immune-related DEGs were discovered and 7 of them were down-regulated, which inferred that tapeworm may regulate immune responses of host. Gut microflora analysis showed that there were no significant differences in richness and diversity between the infected and uninfected groups. Nevertheless, some opportunistic pathogens such as Citrobacter, Edwardsiella, and Lawsonia were significant higher (P < 0.05) in infected turtles, and the intestinal microflora in infected turtles were associated with “Bacterial invasion of epithelial cells” based on functional prediction. Overall, our results suggested that the tapeworm infection caused mechanical damage in the intestine, metabolism disorder in liver, and increased abundance of pathogenic bacteria in gut microflora. Our results provided a first insight into the pathogenesis of tapeworm infection in the Chinese soft-shelled turtle.

Texture quality, histological and extracellular matrix‐related molecular changes of the calipash of Chinese soft‐shelled turtle Pelodiscus sinensis in response to Aeromonas hydrophila challenge

Chinese soft-shelled turtle Pelodiscus sinensis possesses a distinctively soft and pliable apron around its dorsal carapace termed calipash, which is of great importance to evaluate the nutritional quality in turtle industry. As bacterial infection such as Aeromonas hydrophila often brings severe economic losses to P. sinensis culturing in China, it is necessary to study the response of this most distinctive calipash tissue to bacterial infection, which has not been reported. In this study, the texture quality, histology and extracellular matrix-related molecules of calipash in P. sinensis after A. hydrophila challenge were evaluated. After A. hydrophila infection, calipash tissue samples were subjected to biological evaluation, histological analyses using hematoxylin and eosin staining, Masson trichrome staining, scanning electron microscopy and real-time polymerase chain reaction for measuring mRNA expression of collagen biosynthesis as well as collagen degradation-related genes. Picrosirius red staining was used to reveal the type I collagen distribution. The results showed histologically observable connective tissue disorganization with a notable increase in collagen-characteristic amino acid hydroxyproline level, greater collagen I density and more collagen content in infected turtles when compared to the control. For the infected, significantly decreased mRNA levels of type I collagen and proline-4-hydroxylase coding genes, increased matrix metalloproteinases and lowered tissue inhibitor of metalloproteinase expression were observed. These results suggest that collagen metabolism or mobilization characterized using collagen degradation may play an important role in the maintenance of calipash in response to the A. hydrophila challenge.

Bacillus cereus causes fatal disease in soft-shelled turtle (Trionyx sinensis)

Soft-shelled turtle (Trionyx sinensis) is one of the most important aquacultural species in China. In recent years, an infectious disease frequently occurred in soft-shelled turtle, which exhibited similar symptoms, including craned necks, ulcerated pores on shells, hemorrhagic intestinal mucosa and heart, intumescent liver and spleen. In this study, a Gram-positive rod bacterial strain BC12 with vigorous hemolytic and lipolytic activity isolated from the sick soft-shelled turtles. Strain BC12 was identified as Bacillus cereus via 16S rRNA phylogenetic analysis and species-specific gene amplification. The juvenile soft-shelled turtles challenged with B. cereus BC12 exhibited similar symptoms as those natural infections, and the 50% lethal dose (LD50) is 3.89 × 105 colony-forming unit (CFU). Pure B. cereus re-isolated from the heart and liver of turtles infected with BC12. Noticeable pathological changes, including necrosis in the heart and liver and damage in the germinal center of the spleen, were observed in the B. cereus BC12 infected turtles for the first time. Antibiotics susceptibility assay showed that B. cereus BC12 is sensitive to the aminoglycosides, polypeptides, most of the cephalosporins and quinolones, which might be chosen as candidates to prevent and treat B. cereus infection.

Application of a recombinant replicase to localize the Trionyx sinensis hemorrhagic syndrome virus and evaluate its effects on antiviral genes of T. sinensis

Trionyx sinensis Hemorrhagic Syndrome Virus (TSHSV) is an arterivirus newly discovered in Chinese softshell turtles. Little is known about the effect of antibodies against the virus or the distribution of the virus in different organs of infected turtles. In this study, a partial protein of TSHSV-HP4 was produced using a prokaryotic expression system, and its polyclonal antibody was generated. The polyclonal antibody was confirmed by western blot and dot enzyme-linked immunosorbent assay (dot-ELISA). The distribution of TSHSV in different organs of T. sinensis was examined by immunohistochemistry (IHC) and the expression of immune-related genes was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). The results indicated that the recombinant TSHSV-HP4 protein was successfully expressed, and the generated polyclonal antibody showed specific binding to viral particles in the lung tissues of infected turtles. The IHC assay indicated that the virus was highly localized in various cells, including intestinal lymphocytes, enterocytes, kidney epithelial cells, spleen cells, lung macrophages, and cardiomyocytes. The qRT-PCR analysis revealed that TSHSV was detected in all organs tested, including the lungs, liver, kidneys, spleen, and heart. The numbers of viral mRNA copies in lung and heart tissues were significantly higher in the virus-antibody group than in the virus group. The interferon-stimulated genes (ISGs), myxovirus resistance protein 2 (MX2) and radical S-adenosyl methionine domain containing 2 (RSAD2) were highly upregulated in all groups of infected turtles. Antibody-dependent enhancement (ADE) seemed to occur after stimulation by the polyclonal antibody, because significantly greater expression of the two genes was detected in the virus-antibody group than in the virus group. Overall, these results are important in understanding the cell localization of TSHSV and the immune response of infected turtles.

Long-term survey of sea turtles (Caretta caretta) reveals correlations between parasite infection, feeding ecology, reproductive success and population dynamics

Long-term monitoring of host-parasite interactions is important for understanding the consequences of infection on host fitness and population dynamics. In an eight-year survey of the loggerhead sea turtle (Caretta caretta) population nesting in Cabo Verde, we determined the spatiotemporal variation of Ozobranchus margoi, a sanguivorous leech best known as a vector for sea turtle fibropapilloma virus. We quantified O. margoi association with turtles’ δ15N and δ13C stable isotopes to identify where infection occurs. We then measured the influence of infection on reproduction and offspring fitness. We found that parasite prevalence has increased from 10% of the population in 2010, to 33% in 2017. Stable isotope analysis of host skin samples suggests transmission occurs within the host’s feeding grounds. Interestingly, we found a significant interaction between individual size and infection on the reproductive success of turtles. Specifically, small, infected females produced fewer offspring of poorer condition, while in contrast, large, infected turtles produced greater clutch sizes and larger offspring. We interpret this interaction as evidence, upon infection, for a size-dependent shift in reproductive strategy from bet hedging to terminal investment, altering population dynamics. This link between infection and reproduction underscores the importance of using long-term monitoring to quantify the impact of disease dynamics over time.

Epidemiology of blood flukes (Digenea: Spirorchiidae) in sea turtles from Tyrrhenian and Adriatic Seas, off Italy

BackgroundThe Spirorchiidae is a family of blood flukes parasitizing turtles. Spirorchiids may cause a wide range of inflammatory reactions in the vascular system of their host being frequently implicated with stranding and death of sea turtles worldwide. Recent studies revealed the presence of two spirorchiid species in the Mediterranean basin. Our study presents comparative epidemiological data of spirorchiid infections in loggerhead turtles (Caretta caretta) stranded during an eight-year period from Adriatic and Tyrrhenian Seas, and the first report of Neospirorchis Neogen-11 in a green turtle (Chelonia mydas).MethodsWe screened a total of 319 carcasses of loggerhead turtles stranded from January 2011 to December 2018 along the Tyrrhenian coast (n = 111) and the north-western Adriatic coast (n = 208) of Italy using traditional (copromicroscopy and histopathology) and molecular assays. Three green turtles from the Tyrrhenian coast were also included in the study.ResultsA total of 56 (17.5%) loggerhead turtles and one green turtle (33.3%) were found to be infected with spirorchiid flukes. Amplification, sequencing of the ITS2 region of the ribosomal RNA gene cluster and BLAST analysis confirmed the presence of Hapalotrema mistroides and Neospirorchis Neogen-11 in 51 (16.0%) and 24 (7.5%) loggerhead turtles, respectively, and Neospirorchis Neogen-11 in an infected green turtle. Differences in prevalence of infection between the two sampling areas were found.ConclusionsThe risk of spirorchiid infection in the Tyrrhenian Sea is lower than in the Adriatic Sea and in general the risk of infection in the Mediterranean is lower than in other geographical locations. Differences in the prevalence of infection between the two sampling areas were related to the differences of regional habitats supporting different abundance of spirorchiid intermediate hosts. A systematic monitoring to evaluate the progress of the infection is recommended, as well as studies on the occurrence and distribution of spirorchiid species from other Mediterranean areas.

Presence of chelonid herpesvirus 5 (ChHV5) in sea turtles in northern Sinaloa, Mexico.

The presence of fibropapilloma and its associated chelonid herpesvirus 5 (ChHV5) was assessed in 82 wild sea turtles. Olive ridley turtles Lepidochelys olivacea (n = 58) were caught in the pelagic Area of Marine Influence (AMI) (off the coast of Guasave, Sinaloa), and black turtles Chelonia mydas agassizii (n = 24) were captured in the Navachiste Lagoon System. The apparent physical condition was evaluated as 'good' or 'poor' by physical examination. The population structure and general health status was determined by condition index, hematocrit and total plasma protein. Detection of ChHV5 from skin samples was done by PCR. The overall physical condition of black turtles was good and all the individuals were tumor-free. Likewise, the physical condition of most olive ridley turtles was good, except for 10 individuals with poor condition. Of these, 4 had fibropapilloma-like tumors. PCR analyses showed that 3 tumors were ChHV5-positive. The DNA sequence showed 96% identity with ChHV5. All other skin samples from black or olive ridley turtles were ChHV5-negative. This is the first report of fibropapillomatosis-ChHV5 in foraging grounds off northern Sinaloa. The virus was present in a small proportion of L. olivacea individuals, a free-ranging species. It is suggested that infected turtles acquired the virus at a different location somewhere during their development before arriving in the AMI zone. This finding makes the case for setting up a health monitoring program for turtle populations in the area, enforcing sanitary measures to reduce the spread of the pathogen.

Roles of polymorphic cathelicidins in innate immunity of soft-shell turtle, Pelodiscus sinensis

Cathelicidins are a class of gene-encoded immune effectors in vertebrate innate immune system. Though being extensively studied in mammals, little is known about the roles of cathelicidins in turtles, the water-dwelling vertebrates in the order Testudines. In the present study, six novel cathelicidins (Ps-CATH1-6) with different tissue and inducible expression patterns were characterized from the turtle of economic importance, Chinese soft-shell turtle (Pelodiscus sinensis). Although the structures of Ps-CATH1-6 precursors were identical with most of the other known cathelicidins, the mature peptides of Ps-CATH1-6 showed low sequence similarity with the other cathelicidins. Functional studies indicated that some of them either directly kill pathogens via inducing the permeabilization in bacterial membrane (Ps-CATH4, 6), or boost infection-resolving immunity by selectively inhibiting pro-inflammatory responses (Ps-CATH2, 4, 6) through MAPKs and NF-κB pathways. Ps-CATH2, 4, and 6, which assume the hallmark amphipathic α-helical conformations as most of the other host defense peptides, exhibit evident in vivo protection by significantly reducing the bacterial loads in bacterial infected turtles. Collectively, the discovery of novel Ps-CATHs with pleiotropic structures and functions helps elucidating the roles of cathelicidins in the Chinese softshell turtle innate immunity, and better understanding the survival strategy of Chinese softshell turtle in harsh habitat.

EXPERIMENTAL TRANSMISSION OF FROG VIRUS 3-LIKE RANAVIRUS IN JUVENILE CHELONIANS AT TWO TEMPERATURES.

The pathogenicity of frog virus 3 (FV3)-like ranavirus varies in adult chelonian species at different environmental temperatures, but differences in pathogenicity at different temperatures has yet to be determined in juveniles. Our objective was to determine the susceptibility to FV3-like ranavirus in four species of juvenile chelonians: red-eared sliders (RES; Trachemys scripta elegans), Mississippi map turtles ( Graptemys pseudogeographica kohnii), false map turtles (FMT; Graptemys pseudogeographica), and eastern river cooters ( Pseudemys concinna concinna) at two environmental temperatures. Two simultaneous trials ( n=8 treatment and n=4 controls of each species) were conducted in separate temperature-controlled rooms with animals maintained at 22 C or 27 C. All of the inoculated animals of each species at each temperature died, but no mortality was observed in control animals. Median survival times varied between 8 d and 11 d, based on species and temperature, with RES in the 27 C trial surviving the shortest time and the FMT in the 22 C trial surviving the longest. Combining all species, turtles in the 27 C trial survived for fewer days than those housed at 22 C, despite all turtles in both trials having similar viral copies detected in postmortem tissues. Lesions in inoculated turtles resembled those noted in natural and experimental FV3-like ranavirus infections and included vasculitis, thrombosis, hemorrhage in multiple organs, renal tubular necrosis, and hepatic necrosis. Myositis was not present in any juvenile, infected turtles in this study. This study confirmed that juvenile chelonians have a high susceptibility to ranaviral disease.

Cardiovascular flukes (Trematoda: Spirorchiidae) in Caretta caretta Linnaeus, 1758 from the Mediterranean Sea

BackgroundThe northern Adriatic Sea represents one of the most important neritic foraging grounds for the loggerhead sea turtle Caretta caretta L. in the Mediterranean Sea. Four genera of blood flukes with variable prevalence and pathogenic impact have been reported worldwide in this species. Hapalotrema Looss, 1899 and Amphiorchis Price, 1934 are the only two genera reported in Mediterranean waters; however, updated data describing spirorchiidiasis in the central and eastern Mediterranean and infection prevalence are still lacking. This work aimed to investigate the presence and pathology of spirorchiidiasis in C. caretta in the Mediterranean Sea.MethodsOne hundred sixty-eight animals stranded along the northwestern Adriatic coast between 2009 and 2015 were submitted to necropsy and subsequent analyses for the detection of adult flukes, detection of eggs in the faeces and spleen and histopathology. Molecular analyses were carried out on hosts (mitochondrial D-loop) and parasites (28S gene and ITS2 spacer) to trace the turtle origins and identify the fluke phylogenetic relationships.ResultsSpirorchiidiasis was detected in 16.7% of the animals. Hapalotrema mistroides (Monticelli, 1899) and Neospirorchis sp. were found in twenty-six and ten cases, respectively. Adult flukes were found in six cases, while eggs were detectable through copromicroscopic examination for all infected turtles, and the results for the detection of eggs in the spleen agreed with the copromicroscopic analysis. Only mild lesions were observed. Eggs of types 1 and 3 were grossly visible in the gastrointestinal mucosa, vasculitis was rarely observed in the heart and great vessels, and multifocal granulomas were widespread in the tissues. Molecular identification unambiguously assigned the spirorchiid samples to H. mistroides and Neospirorchis sp. Genetic characterization of loggerhead mtDNA pointed to a Mediterranean origin of the turtle hosts.ConclusionThis survey provides new data on the spread of spirorchiidiasis in the Mediterranean loggerhead sea turtle population and reports for the first time the presence of Neospirorchis spp. in this basin. The infections did not have a causal effect on the death nor a strong impact on the general health status of the animals.

Emendation and new species of Hapalorhynchus Stunkard, 1922 (Digenea: Schistosomatoidea) from musk turtles (Kinosternidae: Sternotherus) in Alabama and Florida rivers

Hapalorhynchus Stunkard, 1922 is emended based on morphological study of existing museum specimens (type and voucher specimens) and newly-collected specimens infecting musk turtles (Testudines: Kinosternidae: Sternotherus spp.) from rivers in Alabama and Florida (USA). Hapalorhynchus conecuhensis n. sp. is described from an innominate musk turtle, Sternotherus cf. minor, (type host) from Blue Spring (31°5′27.64″N, 86°30′53.21″W; Pensacola Bay Basin, Alabama) and the loggerhead musk turtle, Sternotherus minor (Agassiz, 1857) from the Wacissa River (30°20′24.73″N, 83°59′27.56″W; Apalachee Bay Basin, Florida). It differs from congeners by lacking a body constriction at level of the ventral sucker, paired anterior caeca, and a transverse ovary as well as by having a small ventral sucker, proportionally short posterior caeca, nearly equally-sized anterior and posterior testes, a small cirrus sac, and a uterus extending dorsal to the ovary and the anterior testis. Specimens of Hapalorhynchus reelfooti Byrd, 1939 infected loggerhead musk turtles, stripe-necked musk turtles (Sternotherus peltifer Smith and Glass, 1947), Eastern musk turtles (Sternotherus odoratus [Latreille in Sonnini and Latreille, 1801]), and S. cf. minor. Those of Hapalorhynchus cf. stunkardi infected S. minor and S. odoratus. Sternothorus minor, S. peltifer, and S. cf. minor plus S. minor and S. odoratus are new host records for H. reelfooti and H. cf. stunkardi, respectively. This is the first report of an infected musk turtle from the Coosa and Tallapoosa Rivers (Mobile-Tensaw River Basin), Pensacola Bay Basin, or Apalachee Bay Basin. Sequence analysis of the large subunit rDNA (28S) showed a strongly-supported clade for Hapalorhynchus.

Potential Noncutaneous Sites of Chelonid Herpesvirus 5 Persistence and Shedding in Green Sea Turtles Chelonia mydas.

Chelonid herpesvirus 5 (ChHV5), the likely etiologic agent of sea turtle fibropapillomatosis (FP), is predicted to be unevenly distributed within an infected turtle, in which productive virus replication and virion shedding occurs in cutaneous tumor keratinocytes. In this study, we measured and compared ChHV5 DNA quantities in tumors, skin, urine, major organs, and nervous tissue samples from green turtles Chelonia mydas. These samples were taken from the carcasses of 10 juvenile green turtles with and without clinical signs of FP that stranded in Florida during 2014. Quantitative PCR for ChHV5 UL30 was used to identify ChHV5 DNA in tumors, skin, heart, kidney, nerves, and urine sampled from five out of five FP-positive and three out of five FP-free turtles. The most frequently co-occurring sites were cutaneous tumor and kidney (n = 4). Novel data presented here include the identification of ChHV5 DNA in kidney, heart, and nerve samples from three FP-free turtles. These data support candidate nontumored anatomic sites of ChHV5 DNA localization and mobilization during two different disease states that may be involved in the ChHV5 infection cycle. Received September 8, 2016; accepted April 17, 2017.

Report of the presence of 'Hapalotrema synorchis' and 'H. postorchis' (Digenea: Spirorchiidae) in marine turtles (Reptilia: Cheloniidae) in Northern Territory waters

The spirorchiid digenean 'Hapalotrema synorchis' was recovered from the heart of a juvenile Hawksbill Turtle ('Eretmochelys imbricata') found deceased on a local Darwin beach. The turtle was in poor condition, showing many characteristics associated with spirorchiid infection. A second necropsied turtle showed signs of infection with spirorchiids but adult specimens were not recovered. Examination of specimens held at the Berrimah Veterinary Laboratory found another 'E. imbricata' infected with 'H. synorchis' and a Green Turtle ('Chelonia mydas') infected with the related 'H. postorchis'. Despite previous reports of infected turtles, this is the first confirmed identification of 'H. synorchis' and 'H. postorchis' from Northern Territory waters.

Non-cultured faecal and gastrointestinal seed samples fail to detect Trichomonad infection in clinically and sub-clinically infected columbid birds

Trichomonosis, caused by the protozoan Trichomonas gallinae, is an emerging infectious disease in finches, and is more commonly found in columbids and raptors. Infections can be sub-clinical or cause morbidity and mortality, but the parasite is currently only detectable by incubation of an oral swab. Here, we test whether T. gallinae parasites can be detected by PCR from faecal or non-cultured samples from the oral cavity and gastrointestinal tract of infected Turtle Doves (Streptopelia turtur). PCR did not detect T. gallinae parasites in any faecal samples screened, and in only 1 of 11 oral/gastrointestinal samples (from the mouth of a nestling suspected to have died from trichomonosis). We conclude that both oral swabs and parasite culture are still necessary to detect the sub-clinical presence of T. gallinae infection in birds.

Cytological And Molecular Identification Of Haemogregarina Stepanowi In Blood Samples Of The European Pond Turtle (Emys Orbicularis) From Quarantine At Belgrade Zoo

Abstract Blood smears stained with Diff Quick are the initial tool for cytological diagnosis of Haemogregarina spp. However, the development of sensitive and specific molecular methods enabled the detection and identification of parasites in the sample and to clarify the evolutionary relationships of adeleorinid parasites within the Apicomplexa. The current study was attempted in order to perform cytological investigation and molecular identification of the hemoparasites in thirty European pond turtles (Emys orbicularis) from the quarantine section at Belgrade Zoo, which have been found in poor health condition with massive skin hemorrhages, based on intraerythrocytic parasitic forms on hematological smears and 18S rDNA sequence, respectively. Different life cycle stages of the Haemogregarina sp. were noticed within the erythrocytes in the peripheral blood. Biochemical analysis indicated lower values of AST and iron in most of the infected turtles while hematological analysis showed a changed hematocrit value, a decrease in the number of red blood cells and low hemoglobin levels. Amplifications of the 18S rDNA sequence of Haemogregarina were detected in 30/30 (100%) turtles with clinical symptoms. The identity of PCR products was confirmed by direct DNA sequencing. Future research concerning H. stepanowi in Serbia should be applied to its definitive host-the leech.