Mitochondrial Protein Synthesis Adapts to Influx of Nuclear-Encoded Protein.

Mitochondrial ribosomes translate membrane integral core subunits of the oxidative phosphorylation system encoded by mtDNA. These translation products associate with nuclear-encoded, imported proteins to form enzyme complexes that produce ATP. Here, we show that human mitochondrial ribosomes display translational plasticity to cope with the supply of imported nuclear-encoded subunits. Ribosomes expressing mitochondrial-encoded COX1 mRNA selectively engage with cytochrome c oxidase assembly factors in the inner membrane. Assembly defects of the cytochrome c oxidase arrest mitochondrial translation in a ribosome nascent chain complex with a partially membrane-inserted COX1 translation product. This complex represents a primed state of the translation product that can be retrieved for assembly. These findings establish a mammalian translational plasticity pathway in mitochondria that enables adaptation of mitochondrial protein synthesis to the influx of nuclear-encoded subunits.

MITRAC links mitochondrial protein translocation to respiratory-chain assembly and translational regulation.

Mitochondrial respiratory-chain complexes assemble from subunits of dual genetic origin assisted by specialized assembly factors. Whereas core subunits are translated on mitochondrial ribosomes, others are imported after cytosolic translation. How imported subunits are ushered to assembly intermediates containing mitochondria-encoded subunits is unresolved. Here, we report a comprehensive dissection of early cytochrome c oxidase assembly intermediates containing proteins required for normal mitochondrial translation and reveal assembly factors promoting biogenesis of human respiratory-chain complexes. We find that TIM21, a subunit of the inner-membrane presequence translocase, is also present in the major assembly intermediates containing newly mitochondria-synthesized and imported respiratory-chain subunits, which we term MITRAC complexes. Human TIM21 is dispensable for protein import but required for integration of early-assembling, presequence-containing subunits into respiratory-chain intermediates. We establish an unexpected molecular link between the TIM23 transport machinery and assembly of respiratory-chain complexes that regulate mitochondrial protein synthesis in response to their assembly state.

Differential Impact In Vivo of Pf4-ΔCre-Mediated and Gp1ba-ΔCre-Mediated Depletion of Cyclooxygenase-1 in Platelets in Mice.

BACKGROUND: Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1-derived thromboxane A2. Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. METHODS: To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1flox/flox mice to generate platelet Cox-1-/- mice on normolipidemic and hyperlipidemic (Ldlr-/-; low-density lipoprotein receptor) backgrounds. RESULTS: Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1-/-/Ldlr-/- and Gp1ba-ΔCre Cox-1-/-/Ldlr-/- mice. In a mouse model of tail injury, Pf4-ΔCre-mediated and Gp1ba-ΔCre-mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE2 (prostaglandin E) and PGD2 (prostanglandin D), activation of the inflammasome, elevated plasma levels of IL-1ß (interleukin), reduced plasma levels of HDL-C (high-density lipoprotein receptor-cholesterol), and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. CONCLUSIONS: Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1-dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.

Phylogenetic inferences based on distinct molecular markers reveals a novel Babesia (Babesia pantanalensis nov. sp.) and a Hepatozoon americanum-related genotype in crab-eating foxes (Cerdocyon thous).

Piroplasmids and Hepatozoon spp. Are apicomplexan protozoa that may cause disease in several canid species. The present study aimed to expand the knowledge on the diversity of piroplasmids and Hepatozoon in crab-eating foxes (Cerdocyon thous; n = 12) sampled in the Pantanal of Mato Grosso do Sul State, central-western Brazil. PCR assays based on the 18S rRNA were used as screening. Three (25%) and 11 (91.7%) were positive for piroplasmids and Hepatozoon spp., respectively. Co-infection was found in three C. thous. Phylogenetic analyses based on the near-complete 18S rRNA, cox-1 and hsp70 genes evidenced the occurrence of a novel of Babesia spp. (namely Babesia pantanalensis nov. sp.) closely related to Rangelia vitalii and Babesia sp. 'Coco'. This finding was supported by the genetic divergence analysis which showed (i) high divergence, ranging from 4.17 to 5.62% for 18 S rRNA, 6.16% for hps70 and 4.91-9.25% for cox-1 and (ii) the genotype network (which displayed sequences separated from the previously described Piroplasmida species by median vectors and several mutational events). Also, phylogenetic analysis based on the 18S rRNA gene of Hepatozoon spp. positioned the sequences obtained herein in a clade phylogenetically related to Hepatozoon sp. 'Curupira 2', Hepatozoon sp. detected in domestic and wild canids from Uruguay and Hepatozoon americanum. The present study described Babesia pantanalensis nov sp. and Hepatozoon closely related to H. americanum in crab-eating foxes from Brazil. Moreover, the coinfection by piroplasmids and Hepatozoon sp. for the first time in crab-eating foxes strongly suggesting that this wild canid species potentially acts as a bio-accumulate of hemoprotozoan in wild environment.

Integrating network pharmacology and renal metabonomics to reveal the protective mechanism of resveratrol on gouty nephropathy.

Resveratrol (Res) has been demonstrated to have beneficial effects on gouty nephropathy (GN). However, the mechanisms of Res on GN remain unclear. This study aimed to investigate the mechanisms of Res on GN. In this study, network pharmacology technology was used to predict the Res targets in the prevention and treatment of GN. Renal metabonomics was used to identify differential metabolites in kidney tissue of GN model rats. Finally, molecular docking technology was used to verify the binding ability of Res to key targets. Metabonomics analysis showed that 24 potentially important metabolites were involved in the prevention and treatment of GN with Res. After exposure to Res, metabolite levels normalized. The network pharmacology analysis showed that 24 key targets were involved in the prevention and treatment of GN disease. According to the metabolite-gene network diagram, we identified two core genes, PTGS1 and PTGS2, and found that both were involved in the arachidonic acid metabolism pathway. Molecular docking further verified the affinity of Res binding to PTGS1 and PTGS2. In conclusion, the mechanism of Res against GN may be the regulation of arachidonic acid metabolism through the regulation of PTGS 1 and PTGS 2.

Exposure to nicotine regulates prostaglandin E2 secretion and autophagy of granulosa cells to retard follicular maturation in mammals.

Exposure to nicotine by cigarette smoking have shown strongly defectives on the physiological function of ovaries, which in turn leads to disorders of fertility in women. However, the potential molecular mechanisms remain to be elucidated. In this study, we notably found that nicotine was likely to specifically raise the expression of histone deacetylase 3 (HDAC3) to promote the apoptosis and autophagy of granulosa cells (GCs) and block follicular maturation. Moreover, prostaglandin E2 (PGE2) inhibited the apoptosis of GCs and facilitated follicular maturation, and nicotine appeared to inhibit PGE2 secretion by freezing the expression of cyclooxygenase 1 (COX1), which was the rate-limiting and essential enzyme for PGE2 synthesis. Epigenetically, the nicotine was observed to diminish the histone H3 lysine 9 acetylation (H3K9ac) level and compact the chromatin accessibility in -1776/-1499 bp region of COX1 by evoking the expression of HDAC3, with the deactivated Cas9-HDAC3/sgRNA system. Mechanistically, the COX1 protein was found to pick up and degrade the autophagy related protein beclin 1 (BECN1) to control the autophagy of GCs. These results provided a potential new molecular therapy to recover the damage of female fertility induced by nicotine from cigarette smoking.

The specific deletion of cyclooxygenase-1 in megakaryocytes/platelets reduces intestinal polyposis in ApcMin/+ mice.

Clinical and experimental evidence sustain the role of cyclooxygenase (COX)-1 in intestinal tumorigenesis. However, the cell type expressing the enzyme involved and molecular mechanism(s) have not been clarified yet. We aimed to elucidate the role of platelet COX-1 (the target of low-dose aspirin in humans) in intestinal tumorigenesis of ApcMin/+ mice, considered a clinically relevant model. To realize this objective, we generated an ApcMin/+ mouse with a specific deletion of Ptgs1(COX-1 gene name) in megakaryocytes/platelets (ApcMin/+;pPtgs1-/-mice) characterized by profound inhibition of thromboxane(TX)A2 biosynthesis ex vivo (serum TXB2; by 99%) and in vivo [urinary 2,3-dinor-TXB2(TXM), by 79%]. ApcMin/+ mice with the deletion of platelet COX-1 showed a significantly reduced number (67%) and size (32%) of tumors in the small intestine. The intestinal adenomas of these mice had decreased proliferative index associated with reduced COX-2 expression and systemic prostaglandin(PG)E2 biosynthesis (urinary PGEM) vs. ApcMin/+mice. Extravasated platelets were detected in the intestine of ApcMin/+mice. Thus, we explored their contribution to COX-2 induction in fibroblasts, considered the primary polyp cell type expressing the protein. In the coculture of human platelets and myofibroblasts, platelet-derived TXA2 was involved in the induction of COX-2-dependent PGE2 in myofibroblasts since it was prevented by the selective inhibition of platelet COX-1 by aspirin or by a specific antagonist of TXA2 receptors. In conclusion, our results support the platelet hypothesis of intestinal tumorigenesis and provide experimental evidence that selective inhibition of platelet COX-1 can mitigate early events of intestinal tumorigenesis by restraining COX-2 induction.

Genetic diversity and structure of Oncomelania hupensis hupensis in two eco-epidemiological settings as revealed by the mitochondrial COX1 gene sequences.

BACKGROUND: Oncomelania hupensis hupensis is the only intermediate host of Schistosoma japonicum, the causative agent of schistosomiasis in China and is therefore of significant medical and veterinary health importance. Although tremendous progress has been achieved, there remains an understudied area of approximately 2.06 billion m2 of potential snail habitats. This area could be further increased by annual flooding. Therefore, an understanding of population genetics of snails in these areas may be useful for future monitoring and control activities. METHODS AND RESULTS: We sampled snails from Hexian (HX), Zongyang (ZY) and Shitai (ST) in Anhui (schistosomiasis transmission control), and from Hengtang (HT), Taicang (TC), Dongsan (DS) and Xisan (XS) in Jiangsu (schistosomiasis transmission interrupted), downstream of Anhui. ST, DS and XS are classified as hilly and mountainous areas, and HX, ZY, TC and HT as lake and marshland areas. The mitochondrial cytochrome c oxidase subunit I gene were sequenced. Out of 115 snails analyzed, 29 haplotypes were identified. We observed 56 (8.72%) polymorphic sites consisting of 51 transitions, four transversions and one multiple mutational change. The overall haplotype and nucleotide diversity were 0.899 and 0.01569, respectively. Snail populations in Anhui had higher genetic diversity than in Jiangsu. 73.32% of total variation was distributed among sites and 26.68% within sites. Snails were significantly separated according to eco-epidemiological settings in both network and phylogenetic analyses. CONCLUSION: Our results could provide important guidance towards assessing coevolutionary interactions of snails with S. japonicum, as well as for future molluscan host monitoring and control activities.

COX-1, COX-2 and CYP2C19 variations may be related to cardiovascular events due to acetylsalicylic acid resistance.

BACKGROUND: In some stent implanted patients, cardiovascular events (CE) may occur. Acetylsalicylic acid (ASA) is routinely administered to these patients in order to prevent the occurrence of CE. CE may be related to gene variations which cause ASA resistance (AR). Therefore, it was aimed to investigate the relationship between COX-1, COX-2, CYP2C9 and CYP2C19 variations with CE due to AR. MATERIALS AND RESULTS: Seventy-four stent implanted patients, using 100 mg of ASA per day during five years were enrolled into the study. Following stent implantation, thirty-eight patients who had a CE within five years due to AR and 36 patients without CE were enrolled in patient and control group, respectively. AR was confirmed by platelet aggregation testing. After DNA isolation from blood; COX-1, COX-2, CYP2C19 and CYP2C9 variations were investigated with real-time polymerase chain reaction. At the end of this study, heterozygous genotype of COX-1 was found statistically high in patients whereas heterozygous genotype of CYP2C19*17 was found statistically high in controls. The presence of C and G allele in COX-1 and COX-2 were found statistically high in patients, respectively. The presence of T allele in CYP2C19*17 was found statistically high in controls. Heterozygous genotype of COX-1 variation was found statistically high in patients who have AR. Additionally heterozygous genotype of CYP2C19*17 was found statistically high in patients who have low thrombosis risk. CONCLUSIONS: COX-1 and COX-2 gene mutations may increase the risk of CE due to AR whereas CYP2C19*17 may have a protective effect in this process.

Musa sp. Leaves Extract Ameliorates the Hepato-Renal Toxicities Induced by Cadmium in Mice.

Heavy metals intoxication causes several health problems that necessitate finding new protective and therapeutic approaches. This study aimed to evaluate the impact of Musa sp. leaves extract (MLE) on hepato-renal toxicities induced by cadmium (Cd) in male mice. The phytochemical screening, metal chelating activity (MCA), and the median lethal dose (LD50) of MLE were determined. Fifty CD-1 male mice were used and intraperitoneally (i.p.) injected with MLE (1000 to 5000 mg/kg b.wt) for MLE LD50 determination. Another 50 mice were used for evaluating the effect of MLE on Cd toxicity. Blood samples were collected for hematological, liver, and kidney functions assessments. Liver tissue homogenates were used for determination of oxidant/antioxidant parameters. Liver and kidney tissues were harvested for histopathological and molecular investigations. MLE showed potent in vitro antioxidant activities. The MCA and LD50 of the MLE were 75 µg/mL and 3000 mg/kg b.wt, respectively. MLE showed beneficial therapeutic activity against hepato-renal toxicities in Cd-intoxicated mice, evidenced by improving the hematological, biochemical, histopathological, and molecular alterations.

Morphological Identification and Phylogenetic Analysis of Laelapin Mite Species (Acari: Mesostigmata: Laelapidae) from China.

Laelapinae mites are involved in transmission of microbial diseases between wildlife and humans, with an impact on public health. In this study, 5 mite members in the subfamily Laelapinae (laelapin mites; LM) were morphologically identified by light microscopy, and the phylogenetic relationship of LM was analyzed in combination with the sequence information of part of the LM cytochrome oxidase subunit I (cox1) gene. The morphological identification revealed that 5 mites belonged to the genera Laelaps and Haemolaelaps, respectively. Sequence analysis showed that the ratio of non-synonymous mutation rate to synonymous mutation rate of LM was less than 1, indicating that the LM cox1 gene had undergone purifying selection. Phylogenetic analysis showed that the Laelapinae is a monophyletic group. The genera Haemolaelaps and Hyperlaelaps did not separated into distinct clades but clustered together with species of the genus Laelaps. Our morphological and molecular analyses to describe the phylogenetic relationships among different genera and species of Laelapinae provide a reference for the improvement and revision of the LM taxonomy system.

Identification of a homozygous recessive variant in PTGS1 resulting in a congenital aspirin-like defect in platelet function.

We have identified a rare missense variant on chromosome 9, position 125145990 (GRCh37), in exon 8 in PTGS1 (the gene encoding cyclo-oxygenase 1, COX-1, the target of anti-thrombotic aspirin therapy). We report that in the homozygous state within a large consanguineous family this variant is associated with a bleeding phenotype and alterations in platelet reactivity and eicosanoid production. Western blotting and confocal imaging demonstrated that COX-1 was absent in the platelets of three family members homozygous for the PTGS1 variant but present in their leukocytes. Platelet reactivity, as assessed by aggregometry, lumi-aggregometry and flow cytometry, was impaired in homozygous family members, as were platelet adhesion and spreading. The productions of COX-derived eicosanoids by stimulated platelets were greatly reduced but there were no changes in the levels of urinary metabolites of COX-derived eicosanoids. The proband exhibited additional defects in platelet aggregation and spreading which may explain why her bleeding phenotype was slightly more severe than those of other homozygous affected relatives. This is the first demonstration in humans of the specific loss of platelet COX-1 activity and provides insight into its consequences for platelet function and eicosanoid metabolism. Notably despite the absence of thromboxane A2 (TXA2) formation by platelets, urinary TXA2 metabolites were in the normal range indicating these cannot be assumed as markers of in vivo platelet function. Results from this study are important benchmarks for the effects of aspirin upon platelet COX-1, platelet function and eicosanoid production as they define selective platelet COX-1 ablation within humans.

Dectin-1/2-induced autocrine PGE2 signaling licenses dendritic cells to prime Th2 responses.

The molecular mechanisms through which dendritic cells (DCs) prime T helper 2 (Th2) responses, including those elicited by parasitic helminths, remain incompletely understood. Here, we report that soluble egg antigen (SEA) from Schistosoma mansoni, which is well known to drive potent Th2 responses, triggers DCs to produce prostaglandin E2 (PGE2), which subsequently-in an autocrine manner-induces OX40 ligand (OX40L) expression to license these DCs to drive Th2 responses. Mechanistically, SEA was found to promote PGE2 synthesis through Dectin-1 and Dectin-2, and via a downstream signaling cascade involving spleen tyrosine kinase (Syk), extracellular signal-regulated kinase (ERK), cytosolic phospholipase A2 (cPLA2), and cyclooxygenase 1 and 2 (COX-1 and COX-2). In addition, this pathway was activated independently of the actions of omega-1 (ω-1), a previously described Th2-priming glycoprotein present in SEA. These findings were supported by in vivo murine data showing that ω-1-independent Th2 priming by SEA was mediated by Dectin-2 and Syk signaling in DCs. Finally, we found that Dectin-2-/-, and to a lesser extent Dectin-1-/- mice, displayed impaired Th2 responses and reduced egg-driven granuloma formation following S. mansoni infection, highlighting the physiological importance of this pathway in Th2 polarization during a helminth infection. In summary, we identified a novel pathway in DCs involving Dectin-1/2-Syk-PGE2-OX40L through which Th2 immune responses are induced.

Cell-Specific Gene Deletion Reveals the Antithrombotic Function of COX1 and Explains the Vascular COX1/Prostacyclin Paradox.

RATIONALE: Endothelial cells (ECs) and platelets, which respectively produce antithrombotic prostacyclin and prothrombotic thromboxane A2, both express COX1 (cyclooxygenase1). Consequently, there has been no way to delineate any antithrombotic role for COX1-derived prostacyclin from the prothrombotic effects of platelet COX1. By contrast, an antithrombotic role for COX2, which is absent in platelets, is straightforward to demonstrate. This has resulted in an incomplete understanding of the relative importance of COX1 versus COX2 in prostacyclin production and antithrombotic protection in vivo. OBJECTIVE: We sought to identify the role, if any, of COX1-derived prostacyclin in antithrombotic protection in vivo and compare this to the established protective role of COX2. METHODS AND RESULTS: We developed vascular-specific COX1 knockout mice and studied them alongside endothelial-specific COX2 knockout mice. COX1 immunoreactivity and prostacyclin production were primarily associated with the endothelial layer of aortae; freshly isolated aortic ECs released >10-fold more prostacyclin than smooth muscle cells. Moreover, aortic prostacyclin production, the ability of aortic rings to inhibit platelet aggregation and plasma prostacyclin levels were reduced when COX1 was knocked out in ECs but not in smooth muscle cells. When thrombosis was measured in vivo after FeCl3 carotid artery injury, endothelial COX1 deletion accelerated thrombosis to a similar extent as prostacyclin receptor blockade. However, this effect was lost when COX1 was deleted from both ECs and platelets. Deletion of COX2 from ECs also resulted in a prothrombotic phenotype that was independent of local vascular prostacyclin production. CONCLUSIONS: These data demonstrate for the first time that, in healthy animals, endothelial COX1 provides an essential antithrombotic tone, which is masked when COX1 activity is lost in both ECs and platelets. These results help us define a new 2-component paradigm wherein thrombotic tone is regulated by both COX1 and COX2 through complementary but mechanistically distinct pathways.

Activated Platelets Induce Endothelial Cell Inflammatory Response in Psoriasis via COX-1.

OBJECTIVE: Patients with psoriasis have impaired vascular health and increased cardiovascular disease (CVD). Platelets are key players in the pathogenesis of vascular dysfunction in cardiovascular disease and represent therapeutic targets in cardiovascular prevention. The object of this study was to define the platelet phenotype and effector cell properties on vascular health in psoriasis and evaluate whether aspirin modulates the platelet-induced phenotype. Approach and Results: Platelets from psoriasis patients (n=45) exhibited increased platelet activation (relative to age- and gender-matched controls, n=18), which correlated with psoriasis skin severity. Isolated platelets from psoriasis patients demonstrated a 2- to 3-fold (P<0.01) increased adhesion to human aortic endothelial cells and induced proinflammatory transcriptional changes, including upregulation of IL 8 (interleukin 8), IL1ß, and Cox (cyclooxygenase)-2 Platelet RNA sequencing revealed an interferon signature and elevated expression of COX-1, which correlated with psoriasis disease severity (r=0.83, P=0.01). In a randomized trial of patients with psoriasis, 2 weeks of 81 mg low-dose aspirin, a COX-1 inhibitor, reduced serum thromboxane (Tx) B2 and reduced brachial vein endothelial proinflammatory transcript expression >70% compared with the no-treatment group (P<0.01). Improvement in brachial vein endothelial cell inflammation significantly correlated with change in serum TxB2 (r=0.48, P=0.02). CONCLUSIONS: In patients with psoriasis, platelets are activated and induce endothelial cell inflammation. Low-dose aspirin improved endothelial cell health in psoriasis via platelet COX-1 inhibition. These data demonstrate a previously unappreciated role of platelets in psoriasis and endothelial cell inflammation and suggests that aspirin may be effective in improving vascular health in patients with psoriasis. Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT03228017.

Molecular and morphological characterization of Bolbosoma balaenae (Acanthocephala: Polymorphidae), a neglected intestinal parasite of the fin whale Balaenoptera physalus.

Post-mortem examination of a fin whale Balaenoptera physalus stranded in the Mediterranean Sea led to the finding of Bolbosoma balaenae for the first time in this basin. In this work, we describe new structural characteristics of this parasite using light microscopy and scanning electron microscopy approaches. Moreover, the molecular and phylogenetic data as inferred from both ribosomal RNA 18S-28S and the mitochondrial DNA cytochrome oxidase c subunit 1 (cox1) for adult specimens of B. balaenae are also reported for the first time. Details of the surface topography such as proboscis's hooks, trunked trunk spines of the prebulbar foretrunk, ultrastructure of proboscis's hooks and micropores of the tegument are shown. The 18S + 28S rRNA Bayesian tree (BI) as inferred from the phylogenetic analysis showed poorly resolved relationships among the species of Bolbosoma. In contrast, the combined 18S + 28S + mtDNA cox1 BI tree topology showed that the present sequences clustered with the species of Bolbosoma in a well-supported clade. The comparison of cox1 and 18S sequences revealed that the present specimens are conspecific with the cystacanths of B. balaenae previously collected in the euphausiid Nyctiphanes couchii from the North Eastern Atlantic Ocean. This study provided taxonomic, molecular and phylogenetic data that allow for a better characterization of this poor known parasite.

The first case of periorbital human dirofilariasis in the Czech Republic.

Dirofilaria repens and Dirofilaria immitis are the most common filarial species affecting humans in Europe. Dirofilaria repens causes subcutaneous or ocular infection, whereas D. immitis is responsible mainly for the pulmonary form. In this report, we present the first human case of periorbital dirofilariasis in the Czech Republic. A 58-year-old woman suffered from an eyelid oedema, redness and pain in the left eye. After excising the parasite from her eyelid, all clinical symptoms disappeared. Based on the morphology and cytochrome oxidase I sequencing, the parasite was identified as D. repens. Histology revealed that the excised worm was female with absent microfilariae in uteri. With respect to the length of the incubation period and the sequence identity with a known Czech isolate, we concluded that D. repens was most likely of autochthonous origin.

Molecular identification of Sarcocystis species in diaphragm muscle tissue of European mouflon (Ovis gmelini musimon) from Austria.

Previous morphological studies suggested that mouflon may have sarcocysts similar to those of sheep. However, to date, no molecular-based studies of the species of Sarcocystis infecting mouflon have been done. The present study identified Sarcocystis species in diaphragm muscle samples from 20 European mouflon (Ovis gmelini musimon). Molecular identification using the cox1 sequence analysis was performed on sarcocysts excised from muscle tissue and on DNA from digested muscle samples. Both frequency and intensity of infection in mouflon were high with 19 of 20 animals testing Sarcocystis positive and > 50 cysts per gram of tissue recovered from 10 of the 19 Sarcocystis positive animals. Molecular analysis revealed dominant Sarcocystis tenella (18/19 animals) and Sarcocystis arieticanis (1/19 animals), whose known intermediate hosts are sheep. In addition, Sarcocystis capracanis, which is known to form sarcocysts in goats, was detected in two animals. The results of this study demonstrated the digestion method to be superior over the direct isolation of sarcocysts for the molecular identification of Sarcocystis species in a certain host. Future research of Sarcocystis diversity in wild ovine and caprine species is needed.

A discovery of two new Tetrahymena species parasitizing slugs and mussels: morphology and multi-gene phylogeny of T. foissneri sp. n. and T. unionis sp. n.

The presence of parasitic ciliates of the hymenostome genus Tetrahymena was examined in 150 mollusks belonging to six bivalve and 13 gastropod species in Slovakia, Central Europe. Tetrahymenids were detected only in two species, viz., in the invasive Lusitanian slug (Arion vulgaris) and in the native swollen river mussel (Unio tumidus). Although only 10.52% of the examined mollusk taxa were positive, their Tetrahymena infections were very intensive accounting for several hundreds of ciliates per host. Phylogenetic analyses of the 16S and 18S rRNA genes as well as of the barcoding region of the gene encoding for cytochrome c oxidase subunit I revealed that both isolates represent new taxa, T. foissneri sp. n. and T. unionis sp. n. The former species belongs to the 'borealis' clade and its nearest relative is T. limacis, a well-known parasite of slugs and snails. Besides molecular data, T. foissneri can be distinguished from T. limacis also morphologically by the body shape of the parasitic-phase form, dimensions of micronuclei, and the silverline system. On the other hand, T. unionis was classified within the 'paravorax' clade along with T. pennsylvaniensis, T. glochidiophila, and T. nigricans. Although these four species are genetically distinct, T. unionis could be morphologically separated only from T. nigricans by body shape and size. The present study suggests that both aquatic and terrestrial mollusks represent interesting hosts for the discovery of novel Tetrahymena lineages.

The first records of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidae), a causative agent of human sparganosis, in Latvian wildlife.

Diphyllobothriid tapeworms of the genus Spirometra are causative agents of sparganosis, food-borne zoonotic parasitic disease. They have been recorded in broad spectrum of hosts, including humans, in all continents except Antarctica. Spirometra tapeworms have been intensively studied in several Asian countries; however, they have been rather neglected in Europe. The aim of this study was to provide a pilot screening of Spirometra spp. in Latvia, where data on sparganosis are not available. Tapeworms morphologically identified as diphyllobothriid species were isolated from grey wolves Canis lupus and Eurasian lynxes Lynx lynx from Latvia during the hunting periods 2013-2019. The parasites were subjected to molecular genotyping using sequences of the partial large (LSU rDNA; 615 bp) and small (SSU rDNA; 720 bp) subunits of the nuclear ribosomal RNA gene and complete (1566 bp) cytochrome c oxidase subunit I gene of the mitochondrial DNA (cox1 mtDNA). Analyses of both ribosomal subunits of 13 tapeworms revealed no intraspecific variation within the respective rDNA subunits. On the other hand, sequence analysis of mitochondrial cox1 revealed intraspecific polymorphism displayed by 12 cox1 haplotypes. Comparison of the current data with sequences of the corresponding DNA regions deposited in the GenBank revealed 99.3-99.5% (LSU rDNA), 99.2% (SSU rDNA) and 99.6-100% (cox1 mtDNA) identity of studied tapeworms with Spirometra erinaceieuropaei, which provided the first confirmation of this diphyllobothriid tapeworm in Latvia. Since S. erinaceieuropaei is probably prevalent in Latvian wildlife and may also occur in other potential host species, further studies are needed in order to acquire complex data on its geographic distribution and transmission in the natural environment of Latvia, as well as on the spectrum of its intermediate, paratenic, and definitive hosts.