Gastrointestinal Parasites and Bacteria in Free-Living South American Sea Lions (Otaria flavescens) in Chilean Comau Fjord and New Host Record of a Diphyllobothrium scoticum-Like Cestode

Present study aimed to characterize gastrointestinal parasites and culturable bacteria from free-living South American sea lions (Otaria flavescens) inhabiting waters of Comau Fjord, Patagonia, Chile. Therefore, a total of 28 individual fecal samples were collected from sea lions within their natural marine habitat during several diving expeditions. Using classical parasitological techniques, study revealed infections with five different gastrointestinal parasite genera. In addition, bacterial cultures showed presence of at least 28 different bacterial genera. Referring to parasites, protozoan, and metazoan species were found with some of them bearing anthropozoonotic potential and/or pathogenic impact for these marine mammals. As such, four of identified parasite genera harbored zoonotic potential (i.e., Entamoeba, Balantidium, Diphyllobothrium, Anisakis) and one genus (Parafilaroides) represented a specific lungworm of marine pinnipeds. Proglottids from fecal samples showed high morphological homology to “Diphyllobothrium” scoticum (Rennie and Reid, 1912; Meggitt, 1924), which was found in Antarctic sea leopards (Hydrurga leptonyx; Phocidae), but contained eggs of smaller size. Molecular characterization revealed 97–100% identity to a new “Diphyllobothrium” species which was recently isolated from a Californian sea lion (Zalophus californianus; Otariidae) in San Francisco. As such, O. flavescens represents a new host record for this parasite species. Furthermore, potential zoonotic bacteria (i.e., Clostridium, Escherichia, Vibrio, Yersinia, Salmonella) were identified amongst others in O. flavescens indicating a reservoir role for these pinnipeds in marine ecosystem. Current data should be considered as a baseline study for future monitoring surveys on anthropozoonotic pathogens circulating in wild free-living sea lions and their possible impact on public health issues and marine wildlife

The Syk Kinase SmTK4 of Schistosoma mansoni Is Involved in the Regulation of Spermatogenesis and Oogenesis

The signal transduction protein SmTK4 from Schistosoma mansoni belongs to the family of Syk kinases. In vertebrates, Syk kinases are known to play specialized roles in signaling pathways in cells of the hematopoietic system. Although Syk kinases were identified in some invertebrates, their role in this group of animals has not yet been elucidated. Since SmTK4 is the first Syk kinase from a parasitic helminth, shown to be predominantly expressed in the testes and ovary of adult worms, we investigated its function. To unravel signaling cascades in which SmTK4 is involved, yeast two-/three-hybrid library screenings were performed with either the tandem SH2-domain, or with the linker region including the tyrosine kinase domain of SmTK4. Besides the Src kinase SmTK3 we identified a new Src kinase (SmTK6) acting upstream of SmTK4 and a MAPK-activating protein, as well as mapmodulin acting downstream. Their identities and colocalization studies pointed to a role of SmTK4 in a signaling cascade regulating the proliferation and/or differentiation of cells in the gonads of schistosomes. To confirm this decisive role we performed biochemical and molecular approaches to knock down SmTK4 combined with a novel protocol for confocal laser scanning microscopy for morphological analyses. Using the Syk kinase-specific inhibitor Piceatannol or by RNAi treatment of adult schistosomes in vitro, corresponding phenotypes were detected in the testes and ovary. In the Xenopus oocyte system it was finally confirmed that Piceatannol suppressed the activity of the catalytic kinase domain of SmTK4. Our findings demonstrate a pivotal role of SmTK4 in gametogenesis, a new function for Syk kinases in eukaryotes

Prevalence survey on lungworm (Angiostrongylus vasorum, Crenosoma vulpis, Eucoleus aerophilus) infections of wild red foxes (Vulpes vulpes) in central Germany

Abstract Background Angiostrongylus vasorum, Crenosoma vulpis and Eucoleus aerophilus are a source of increasing concern, potentially causing significant pulmonary and severe cardiac/systemic diseases in domestic dogs and wild canids, especially red foxes (Vulpes vulpes). To investigate the prevalence and geographical distribution of these parasites in central Germany, a total of 569 foxes were examined by dissection. Methods Pluck (heart and lung) and faecal samples of red foxes were collected from three regions of Germany. Lungs, hearts and adjacent vessels were processed for adult nematode detection. Parasitological diagnoses of faecal samples were performed by SAF technique, Giardia- and Cryptosporidium-Coproantigen-ELISAs and by a duplex copro-PCR for the detection of A. vasorum and C. vulpis DNA. Results Foxes originated from three Federal States of central Germany: Thuringia (n = 359); Rhineland-Palatinate (n = 121) and Hesse (n = 89). High prevalences for all three nematodes were detected, with E. aerophilus (69.4%; 395/569), followed by C. vulpis (32.3%; 184/569) and A. vasorum (14.1%; 80/569). In case of A. vasorum, prevalences varied significantly between Federal States, with the highest prevalence of 27.3% in Rhineland-Palatinate, followed by 19.1% and 8.4% in Hesse and Thuringia, respectively. The presence of A. vasorum in fox populations showed a rather patchy distribution, increasing from north-eastern to south-western regions. Analyses on C. vulpis revealed prevalences of 35.1%, 30.3% and 25.6% (Thuringia, Hesse and Rhineland-Palatinate, respectively). The most prevalent lungworm nematode was E. aerophilus, with a prevalence of 75.2%, 71.9% and 66.9% (Rhineland-Palatinate, Hesse and Thuringia, respectively) and an almost area-wide equal distribution. Significant differences for single parasite prevalences within geographical regions of the Federal States could be detected whilst no correlation between age or gender and parasite occurrence was estimated. Weak seasonality for the winter months for A. vasorum, stronger correlation to spring and late summer for C. vulpis and no correlation to any season for E. aerophilus were detected. The method of dissection revealed a significantly higher sensitivity for C. vulpis when compared with the results of the duplex copro-PCR. Conclusions A sylvatic cycle was confirmed for all three lungworm nematodes in the examined area. The prevalences for all three lungworm nematodes are some of the highest recorded so far in German foxes. The data suggest that A. vasorum might be spreading from south-western to north-eastern parts of Germany

Subcutaneous merocercoids of Clistobothrium sp. in two Cape fur seals (Arctocephalus pusillus pusillus)

Fur seals represent intermediate hosts of the cestode Clistobothrium. Large sharks are definitive hosts for these parasites. Two female, 25– and 27-year-old fur seals, caught in the 1980s at the South African coast, were examined pathomorphologically. Both animals showed multifocal, up to 1 cm in diameter large cavities of the thoracic and abdominal subcutaneous adipose tissue containing intraluminal metacestodes of tapeworms, which were surrounded by a locally extensive, pyogranulomatous panniculitis. The metacestodes (merocercoids) of one fur seal were isolated from the subcutaneous adipose tissue and characterized morphologically and for the first time from this host by molecular techniques. The morphometric data corresponded with ‘delphini'-morphotype merocercoids, but the sequence of the partial 28S ribosomal RNA gene identified them as conspecific with merocercoids of the morphotype ‘grimaldii’. These merocercoid types are morphologically Type XV metacestodes of marine tapeworms and represent different species of Clistobothrium. Sequence data were generated for 18S, ITS1, 5.8S, ITS2, partial 28S ribosomal DNA and partial mitochondrial cox1 gene and phylogenetic analysis of 18S rRNA and partial 28S rRNA genes identified the fur seal merocercoids as Clistobothrium species. However, it cannot yet be assigned to species level because of limited molecular data from adult stages. Most likely, both fur seals were infected as juveniles in their original habitat, the coastal regions of South Africa. The metacestode infection is probably an incidental finding, however, there is a chronic inflammatory reaction next to the subcutaneous merocercoids. It is noteworthy, that the merocercoids remain in a potentially infective stage even after more than 20 years. Keywords: Arctocephalus pusillus pusillus, Clistobothrium sp., Fur seal, Cestode, Subcutaneous parasites, Monorygma grimaldi

The seal louse (Echinophthirius horridus) in the Dutch Wadden Sea: investigation of vector-borne pathogens

Background!#!Belonging to the anopluran family Echinophthiriidae, Echinophthirius horridus, the seal louse, has been reported to parasitise a broad range of representatives of phocid seals. So far, only a few studies have focused on the vector function of echinophthiriid lice, and knowledge about their role in pathogen transmission is still scarce. The current study aims to investigate the possible vector role of E. horridus parasitising seals in the Dutch Wadden Sea.!##!Methods!#!E. horridus seal lice were collected from 54 harbour seals (Phoca vitulina) and one grey seal (Halichoerus grypus) during their rehabilitation period at the Sealcentre Pieterburen, The Netherlands. DNA was extracted from pooled seal lice of individual seals for molecular detection of the seal heartworm Acanthocheilonema spirocauda, the rickettsial intracellular bacterium Anaplasma phagocytophilum, and the cell wall-less bacteria Mycoplasma spp. using PCR assays.!##!Results!#!Seal lice from 35% of the harbour seals (19/54) and from the grey seal proved positive for A. spirocauda. The seal heartworm was molecularly characterised and phylogenetically analysed (rDNA, cox1). A nested PCR was developed for the cox1 gene to detect A. spirocauda stages in seal lice. A. phagocytophilum and a Mycoplasma species previously identified from a patient with disseminated 'seal finger' mycoplasmosis were detected for the first time, to our knowledge, in seal lice.!##!Conclusions!#!Our findings support the potential vector role of seal lice in the transmission of A. spirocauda and reveal new insights into the spectrum of pathogens occurring in seal lice. Studies on vector competence of E. horridus, especially for bacterial pathogens, are essentially needed in the future as these pathogens might have detrimental effects on the health of seal populations. Furthermore, studies on the vector role of different echinophthiriid species infecting a wide range of pinniped hosts should be conducted to extend the knowledge of vector-borne pathogens

The invasive giant African snail Lissachatina fulica as natural intermediate host of Aelurostrongylus abstrusus, Angiostrongylus vasorum, Troglostrongylus brevior, and Crenosoma vulpis in Colombia.

BackgroundSeveral metastrongyloid lungworms are unreported pathogens in Colombia. Angiostrongylus vasorum and Crenosoma vulpis target the cardiopulmonary system of domestic and wild canids. Aelurostrongylus abstrusus and Troglostrongylus brevior infect felids and considering that six wild felid species exist in Colombia, knowledge of feline lungworm infections is important for their conservation. The zoonotic metastrongyloids Angiostrongylus costaricensis and Angiostrongylus cantonensis can cause severe gastrointestinal and neurological diseases. Angiostrongylus costaricensis has been reported in Colombia, while Ang. cantonensis is present in neighbouring countries. Research on the epidemiology of metastrongyloids in Colombia and South America more broadly requires evaluating the role that gastropods play as intermediate hosts in their life cycles. This study assessed the prevalence of metastrongyloid larvae in populations of the invasive giant African snail, Lissachatina fulica, in Colombia.Methodology/principal findingsA total of 609 Lissachantina fulica were collected from 6 Colombian municipalities. The snails were then cryo-euthanized, artificially digested and the sediments examined microscopically for the presence of metastrongyloid larvae. Based on morphological characteristics 53.3% (56/107) of the snails from Puerto Leguízamo (Department of Putumayo) were infected with Ael. abstrusus larvae, 8.4% (9/107) with Ang. vasorum larvae, 6.5% (7/107) with T. brevior larvae and 5.6% (6/107) with C. vulpis larvae, being the region with highest prevalences of the four species. Snails from Andes (Department of Antioquia) and Tulúa (Department of Valle del Cauca) were positive for Ang. vasorum larvae with a prevalence of 4.6 (11/238) and 6.3% (4/64), respectively. Species identifications were confirmed by PCR and sequencing.Conclusions/significanceThis epidemiological survey reports for first time the presence of Ael. abstrusus, T. brevior, C. vulpis and Ang. vasorum in L. fulica in a number of regions of Colombia

Molecular Detection of Gurltia paralysans by Semi-Nested PCR in Cerebrospinal Fluid and Serum Samples from Domestic Cats (Felis catus)

Gurltia paralysans is an angio-neurotropic metastrongyloid nematode that infects domestic and wild cats, invading the veins of the subarachnoid space of the spinal cord and mainly causing progressive paralysis of the pelvic limbs. The definitive diagnosis of feline gurltiosis can only be achieved by post-mortem examination that reveals the presence of the nematode in the spinal cord vein vasculature. An early diagnosis with conclusive results is required since laboratory and imaging findings are not sufficient. Therefore, the purpose of this study was to detect the presence of G. paralysans, via semi-nested PCR, in samples of cerebrospinal fluid (CSF) and the sera of domestic cats naturally infected with the parasite. A total of 12 cats with a diagnosis suggestive of feline gurltiosis were selected, and they underwent a complete neurological and imaging examination. DNA samples were analysed by semi-nested PCR, with universal (AaGp28Sa1/AaGp28Ss1) and specific (Gp28Sa3/Aa28Ss2) primers, for G. paralysans (G. paralysans 18S rRNA gene, partial sequence; ITS 1, 5.8S rRNA gene, and ITS 2, complete sequence; and 28S rRNA gene, partial sequence) and Aelurostrongylus abstrusus, obtaining amplifications of 356 and 300 bp, which indicated the presence or absence of nematode DNA, respectively. The presence of G. paralysans was detected in the CSF of four out of nine cats, and the sera of seven out of seven cats. In the sera analysis of five out of seven cats, a mixed infection with A. abstrusus was found, despite no alterations of the respiratory tract being observed during the necropsies. It is proposed that serum samples could be more effective than CSF in detecting the parasite by PCR analysis. Sequencing analysis showed high percentages of identity with G. paralysans, which indicated the feasibility of detection and the sensitivity/specificity of the method used, suggesting the implementation of semi-nested PCR as a routine diagnostic test for early and timely detection of feline gurltiosis