The golden jackal (Canis aureus): A new host for Echinococcus multilocularis and Trichinella britovi in Switzerland.

INTRODUCTION: The golden jackal (Canis aureus) is a wild canid new to Switzerland. It is an officially monitored species and all deceased individuals are submitted for post-mortem examination to collect baseline health data. This includes parasitological examinations, with an emphasis on zoonotic, reportable infections, such as those caused by Trichinella spp. or Echinococcus spp. From 2016 to 2021, five golden jackals originating from four Swiss cantons were submitted for full post-mortem examination. In one case only organ samples were available, and therefore parasitological examination was not possible. Parasite stages recovered during necropsy, as well as by routine coproscopical techniques, were morphologically identified. Taeniid eggs and adult tapeworms were processed for molecular species identification. Additionally, tongue and diaphragm were analysed for Trichinella spp. by the artificial digestion technique followed by multiplex-PCR in positive cases. Of the four jackals investigated for parasites, hookworm eggs were detected in one animal, both adult worms and eggs of Echinococcus multilocularis were present in another case, and one animal was free of parasites. Eggs of E. multilocularis as well as eggs of Toxocara canis and sporocysts of Sarcocystis sp. were detected in the intestinal content, and Trichinella britovi larvae were found in the muscle samples of the last case. The health monitoring programme in place for protected carnivores in Switzerland allowed us to add the golden jackal to the list of hosts for the endemic zoonotic parasites E. multilocularis and T. britovi in this country. Hunters, farmers, and other persons who could come in contact with golden jackals should be aware of the associated health risk and handle faeces and carcasses with caution.

INTRODUCTION: Le chacal doré (Canis aureus) est un canidé sauvage nouvellement présent en Suisse. Il s'agit d'une espèce officiellement surveillée et tous les individus morts sont soumis à un examen post-mortem afin de recueillir des données sanitaires de base. Cela inclut un examen parasitologique mettant l'accent sur les infections zoonotiques à déclaration obligatoire, telles que celles causées par Trichinella spp. ou Echinococcus spp. De 2016 à 2021, cinq chacals dorés originaires de quatre cantons suisses ont été soumis à un examen post-mortem complet. Dans un cas, seuls des échantillons d'organes ont été envoyés, l'examen parasitologique n'a pas été possible pour cet animal. Les stades parasitaires trouvés lors de l'examen pathologique et de la coprologie de routine ont été identifiés morphologiquement. Les espèces de ténias (œufs et stades adultes) ont été déterminées par des techniques de biologie moléculaire. En outre, la recherche de Trichinella spp. a été effectuée sur du tissu musculaire lingual et diaphragmatique par la technique de digestion artificielle suivie d'une PCR multiplex dans les cas positifs. Sur les quatre chacals ayant fait l'objet d'une recherche de parasites, des œufs d'ankylostomes ont été détectés chez un animal, des vers adultes et des œufs d'Echinococcus multilocularis étaient présents chez un autre animal, et aucun parasite n'a été trouvé dans un autre cas. Chez le dernier cas, des œufs d'E. multilocularis ainsi que des œufs de Toxocara canis et des sporocystes de Sarcocystis sp. ont été détectés dans le contenu intestinal, et des larves de Trichinella britovi ont été trouvées dans les échantillons de muscle. Le programme de surveillance sanitaire mis en place pour les carnivores protégés en Suisse a donc permis d'ajouter le chacal doré à la liste des hôtes des parasites zoonotiques endémiques E. multilocularis et T. britovi. Les chasseurs, agriculteurs et autres personnes susceptibles d'entrer en contact avec le chacal doré doivent être conscients du risque sanitaire associé et manipuler les fèces et les carcasses avec précaution.

Efficacy of novel albendazole salt formulations against secondary cystic echinococcosis in experimentally infected mice.

In this study, we evaluated the efficacy, expressed as a mean weight decrease of the whole echinococcal cyst mass, of novel benzimidazole salt formulations in a murine Echinococcus granulosus infection model. BALB/c mice were intraperitoneally infected with protoscoleces of E. granulosus (genotype G1). At 9 months post-infection, treatment with albendazole (ABZ), ricobendazole (RBZ) salt formulations, and RBZ enantiomer salts (R)-(+)-RBZ-Na and (S)-(-)-RBZ-Na formulations were initiated. Drugs were orally applied by gavage at 10 mg kg-1 body weight per day during 30 days. Experimental treatments with benzimidazole sodium salts resulted in a significant reduction of the weight of cysts compared to conventional ABZ treatment, except for the (S)-(-)-RBZ-Na enantiomer formulation. Scanning electron microscopy and histological inspection revealed that treatments impacted not only the structural integrity of the parasite tissue in the germinal layer, but also induced alterations in the laminated layer. Overall, these results demonstrate the improved efficacy of benzimidazole salt formulations compared to conventional ABZ treatment in experimental murine cystic echinococcosis.

To see or not to see: non-invasive imaging for improved readout of drug treatment trials in the murine model of secondary alveolar echinococcosis.

Alveolar echinococcosis (AE) is an emerging zoonotic disease caused by the cestode Echinococcus multilocularis. The secondary infection model of AE is based on intraperitoneal injection of disease-causing metacestodes into the peritoneal cavity of mice, which allows investigations on novel drugs or immunotherapeutical treatment options in vivo. So far, such in vivo studies assessed exclusively the parasite weight at the endpoint of a given treatment period. We here developed an ultrasound (US)-based scoring system that allows to follow-up parasite development in the living animal, and provides insights into parasite growth during the treatment phase. By this method a statistically significant difference between untreated and medicated mice with E. multilocularis infection was observed at 2 months post-infection, and the growth curve of the parasite load was described by a linear mixed model. High correlation and similar levels of variation were observed for the standard method based on parasite weight measurement, the novel US-based scoring system, as well volume segmentation by post-mortem magnetic resonance imaging. Thus, US-based scoring in the live animal has the potential to assist the 3R concept by contributing to the refinement and reduction of animal use in experimental echinococcosis.

An anionic phosphenium complex as an ambident nucleophile.

A unique anionic phosphenium complex was prepared from reaction of an N-heterocyclic chlorophosphine with Collman's reagent or K[HFe(CO)4]/NaH and characterized by spectral and XRD data. The complex behaves as an ambident nucleophile. Reactions with acetic acid, ClSnPh3, and a further equivalent of an N-heterocyclic chlorophosphine proceed via electrophilic functionalization at the metal site to yield appropriate mono- or bis-phosphenium complexes. Reaction with MeI at -70 °C produces a P-alkylation product as the first spectroscopically detectable intermediate, which decays at a higher temperature to give a mixture of free P-methylated N-heterocyclic phosphine and its Fe(CO)4 complex. The different reaction products were characterized by spectral and XRD data. Computational studies indicate that the NHP units in all complexes display π-acceptor behaviour but show no disposition to adopt phosphide-like character or formally oxidize the metal centre.

Echinococcus metacestodes as laboratory models for the screening of drugs against cestodes and trematodes.

Among the cestodes, Echinococcus granulosus, Echinococcus multilocularis and Taenia solium represent the most dangerous parasites. Their larval stages cause the diseases cystic echinococcosis (CE), alveolar echinococcosis (AE) and cysticercosis, respectively, which exhibit considerable medical and veterinary health concerns with a profound economic impact. Others caused by other cestodes, such as species of the genera Mesocestoides and Hymenolepis, are relatively rare in humans. In this review, we will focus on E. granulosus and E. multilocularis metacestode laboratory models and will review the use of these models in the search for novel drugs that could be employed for chemotherapeutic treatment of echinococcosis. Clearly, improved therapeutic drugs are needed for the treatment of AE and CE, and this can only be achieved through the development of medium-to-high throughput screening approaches. The most recent achievements in the in vitro culture and genetic manipulation of E. multilocularis cells and metacestodes, and the accessability of the E. multilocularis genome and EST sequence information, have rendered the E. multilocularis model uniquely suited for studies on drug-efficacy and drug target identification. This could lead to the development of novel compounds for the use in chemotherapy against echinococcosis, and possibly against diseases caused by other cestodes, and potentially also trematodes.

Innovative chemotherapeutical treatment options for alveolar and cystic echinococcosis.

Echinococcus granulosus and Echinococcus multilocularis are cestode parasites, of which the metacestode (larval) stages cause the diseases cystic echinococcosis (CE) and alveolar echinococcosis (AE), respectively. Albendazole and mebendazole are presently used for chemotherapeutical treatment. However, these benzimidazoles do not appear to be parasiticidal in vivo against AE. In addition, failures in drug treatments as well as the occurrence of side-effects have been reported. New drugs are needed to cure AE and CE, which are considered to be neglected diseases. Strategies currently being implemented to identify novel chemotherapeutical treatment options include (i) conventional primary in vitro testing of broad-spectrum anti-infective drugs, either in parallel with, or followed by, animal experimentation; (ii) studies of drugs which interfere with the proliferation of cancer cells and of Echinococcus metacestodes; (iii) exploitation of the similarities between the parasite and mammalian signalling machineries, with a special focus on targeting specific signalling receptors; (iv) in silico approaches, employing the current Echinococcus genomic database information to search for suitable targets for compounds with known modes of action. In the present article, we review the efforts toward obtaining better anti-parasitic compounds which have been undertaken to improve chemotherapeutical treatment of echinococcosis, and summarize the achievements in the field of host-parasite interactions which may also lead to new immuno-therapeutical options.

Molecular phylogeny of New World Myotis (Chiroptera, Vespertilionidae) inferred from mitochondrial and nuclear DNA genes.

Recent studies have shown that species in the genus Myotis have evolved a number of convergent morphological traits, many of which are more related to their mode of food procurement than to their phylogeny. Surprisingly, the biogeographic origins of these species are a much better predictor of phylogenetic relationships, than their morphology. In particular, a monophyletic clade that includes all New World species was apparent, but only a third of the 38 species have been analysed. In order to better understand the evolution of this clade, we present phylogenetic reconstructions of 17 Nearctic and 13 Neotropical species of Myotis compared to a number of Old World congeners. These reconstructions are based on mitochondrial cytochrome b (1140 bp), and nuclear Rag 2 genes (1148 bp). Monophyly of the New World clade is strongly supported in all analyses. Two Palaearctic sister species, one from the west (M. brandtii) and one from the east (M. gracilis), are embedded within the New World clade, suggesting that they either moved across the Bering Strait, or that they descended from the same ancestor that reached the New World. An emerging feature of these phylogenetic reconstructions is that limited faunal exchanges have occurred, including between the North and South American continents, further emphasizing the importance of biogeography in the radiation of Myotis. A fossil-calibrated, relaxed molecular-clock model was used to estimate the divergence time of New World lineages to 12.2+/-2.0 MYA. Early diversification of New World Myotis coincides with the sharp global cooling of the Middle Miocene. Radiation of the temperate-adapted Myotis may have been triggered by these climatic changes. The relative paucity of species currently found in South America might result from a combination of factors including the early presence of competitors better adapted to tropical habitats.

In vitro phosphorylation of purified glycosylphosphatidylinositol-specific phospholipase D.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was phosphorylated in vitro by cAMP-dependent protein kinase (PKA) and by tyrosine kinase. Phosphorylation by PKA occurred in the 110 kDa native form of GPI-PLD as well as in multiple proteolytic degradation products and caused a significant decrease in enzyme activity. Dephosphorylation by treatment with alkaline phosphatase completely restored GPI-PLD activity. In addition, incubation of GPI-PLD with trypsin, which results in the generation of distinct peptide fragments, resulted in complete dephosphorylation of radiolabeled GPI-PLD. The site of phosphorylation by PKA was assigned to Thr-286. Tyrosine phosphorylation was only observed in a proteolytically processed fragment of GPI-PLD but not in the 110 kDa native form and had no effect on GPI-PLD activity.

Expression of intracellular and GPI-anchored forms of GPI-specific phospholipase D in COS-1 cells.

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is a secretory protein present in high amounts in mammalian body fluids. Its cDNA has been isolated and encodes a signal peptide of 23 amino acids and the mature protein of 816 amino acids. We generated cDNAs encoding a signal peptide-deficient and a GPI-anchored form of GPI-PLD and transiently transfected these constructs into COS-1 cells. The signal peptide-deficient form of GPI-PLD was expressed as a 90-kDa protein that was catalytically active and was localized intracellularly. Cells transfected with cDNA encoding the GPI-anchored form of GPI-PLD expressed a catalytically active enzyme of 100 kDa that could be labelled with [3H]ethanolamine demonstrating its modification by a GPI structure. Expression of the GPI-anchored form of GPI-PLD resulted in the release of endogenous GPI-anchored alkaline phosphatase from COS-1 cells, whereas expression of the intracellular form of GPI-PLD had no effect on membrane attachment of endogenous alkaline phosphatase. Similarly, in cells cotransfected with GPI-anchored placental alkaline phosphatase (PLAP) and the GPI-anchored form of GPI-PLD, PLAP was released into the cell culture supernatant while expression of the signal peptide-deficient form of GPI-PLD did not affect the amount of cell-associated PLAP.

Addition of G418 and other aminoglycoside antibiotics to mammalian cells results in the release of GPI-anchored proteins.

Resistance to the neomycin analogue G418 forms the basis of a dominant marker selection system for mammalian cells transfected with the bacterial neomycin gene. We found that COS-1 cells stably transfected with the neomycin resistance gene had a greater than 50% reduction in cell-associated glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (AP). A similarly reduced amount of AP was also observed in wild-type COS-1 cells incubated in the presence of G418 or other aminoglycoside antibiotics. The AP was released from cells into the culture supernatant in its GPI-anchored form. Our data suggest that the G418-induced reduction of AP involves a vesiculation process of COS-1 cells.

The C-terminus of glycosylphosphatidylinositol-specific phospholipase D is essential for biological activity.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) (EC 3.1.4.50) from mammalian serum is a 115 kDa glycoprotein consisting of 816 amino acids. We found that C-terminal deletions of only two to five amino acids reduced GPI-PLD enzymatic activity by roughly 70% as compared to wild-type protein. C-terminal deletions of more than five amino acids resulted in a complete loss of GPI-PLD enzymatic activity. Point mutations at position 811 indicate that Tyr-811 may play a major role in maintaining the biological activity of GPI-PLD.

Distribution of glycosylphosphatidylinositol-specific phospholipase D mRNA in bovine tissue sections.

It has been reported that mammalian serum, and to a lower extent mammalian liver, brain, pancreas, udder, and milk, contain glycosylphosphatidylinositol-specific phospholipase D activity. However, the sites of synthesis have not been determined. In order to study in which cell(s) of the organism synthesis of glycosylphosphatidylinositol-specific phospholipase D takes place, we undertook a systematic screening of 12 different bovine tissues. In situ hybridization experiments with a specific anti-sense RNA probe, derived from a bovine liver cDNA, revealed that glycosylphosphatidylinositol-specific phospholipase D mRNA is present in mast cells of the adrenal gland, lung, and liver. On the other hand, our specific probe detected no mRNA in bovine pancreas, brain, and udder, although enzyme activity has been reported in these tissues. Northern blot analysis of total bovine liver RNA demonstrated two distinct glycosylphosphatidylinositol-specific phospholipase D mRNAs of approximately 3.3 kb and 4 kb length suggesting that two forms of the enzyme may exist.

Immunogenotyping with antigen receptor gene probes as a diagnostic tool in childhood acute lymphoblastic leukaemia.

13 cases of childhood acute lymphoblastic leukaemia (ALL) were studied combining cell surface marker analysis with immunogenotyping by Southern blot hybridisation with a panel of antigen receptor gene probes. The immunophenotypes were unequivocal: 7 patients had B-phenotype and 6 patients T-phenotype ALL. In several patients immunogenotypes were not fully consistent with the respective phenotypes. For example, 2 B-cell precursor ALL had rearranged TCR beta chain genes and 2 T-ALL rearrangement of Ig heavy-chain genes. All cases showed clonal rearrangement or deletions within the TCR delta gene locus. TCR delta gene rearrangements might, therefore, serve as markers of clonality but not of B- or T-lineage in immature lymphoid neoplasms. We conclude that in current diagnostic practice immunogenotyping is a supplement rather than an alternative to immunophenotyping by surface marker analysis.

Repression of nuclear lamin A and C gene expression in human acute lymphoblastic leukemia and non-Hodgkin's lymphoma cells.

The lamins A, B and C which are differentially expressed during ontogenesis and differentiation are karyoskeletal proteins forming a polymeric meshwork at the inner nuclear membrane. Using Northern blot analyses we investigated the steady state levels of the three lamin specific RNA transcripts in neoplastic cells derived from 16 untreated patients with acute lymphoblastic leukemia (ALL) or non-Hodgkin's lymphoma (NHL) and in ALL and NHL established cell lines. Whereas lamin B mRNA was present in all, lamin A and C transcripts were observed in none of the malignant cell samples except one of a common-ALL patient (precursor B-ALL, cytoplasmic mu chain negative). All three lamin mRNAs were detected in normal peripheral blood lymphocytes, however, only after mitogenic stimulation with concanavalin A. Our results provide evidence that expression of lamin A and C is repressed in neoplastic blast cells derived from patients with ALL or NHL and suggest that lamin A and C gene repression is not related to cell proliferation but might be relevant to the differentiated stages of the lymphoid cells in vivo.

Antisuppressor mutations and sulfur-carrying nucleosides in transfer RNAs of Schizosaccharomyces pombe.

Antisuppressor mutations reduce the efficiency of nonsense suppressors. A mutation in the gene sin4 of Schizosaccharomyces pombe leads to loss of 5-(methoxycarbonylmethyl) thiouridine (mcm5s2U) from the first anticodon position of tRNAs. This resembles the phenotype of sin3 (Heyer, W. D., Thuriaux, P., Kohli, J., Ebert, P., Kersten, H., Gehrke, C., Kuo, K. C., and Agris, P. F. (1984) J. Biol. Chem. 259, 2856-2862), but the mutations reside in different genes. In vivo 35S-labeled tRNA from the parental suppressor strain sup3, the antisuppressor strains sin3 and sin4, and the double mutant sin3 sin4 has been digested to nucleosides and analyzed with high performance liquid chromatography methods. The major sulfur-carrying nucleoside in wild-type S. pombe tRNA is mcm5s2U. It is reduced in the mutant strains. Two other thiolated nucleosides are also present: 2-thiouridine and a nucleoside of unknown structure. Neither was affected by the antisuppressor mutations. Thiocytidine has not been found. Independent from their effect on suppressors, the two mutations sin3 and sin4 reduce the growth rate of cells, and sin3 also increases cell length. In vivo decoding of the serine codon UCG by the UCA reading serine tRNA is not promoted by the two antisuppressor mutations.

The Schizosaccharomyces pombe sup3-i suppressor recognizes ochre, but not amber codons in vitro and in vivo.

The inefficient suppressor sup3-i of the fission yeast Schizosaccharomyces pombe is an ochre suppressor. Sup3-i was derived from the efficient serine inserting UGA suppressor sup3-e. The cloning and sequencing of the sup3-i gene indicate that the suppressor is different from the parent sup3-e by a C----T substitution in the sequence coding for the middle position of the anticodon. In vitro translation assays supplemented with purified sup3-i tRNA and programmed with Xenopus globin mRNAs lead to the accumulation of a readthrough product in response to UAA termination signals, but not in response to UGA termination codons. Transformation of Saccharomyces cerevisiae nonsense mutant strains with plasmid DNA carrying the S. pombe sup3-i gene, led to ochre, but not amber or UGA suppression in vivo.