Estimated specific antibody-based true sero-prevalences of canine filariosis in dogs in Central Europe and the UK.

Dirofilariosis is a vector-borne disease mainly caused by Dirofilaria immitis and Dirofilaria repens. In contrast to the known endemicity of dirofilariosis in southern and south-eastern Europe, information on the distribution of D. repens in Central-Europe is fragmentary. We tested 8877 serum samples from dogs from Austria, Denmark, Germany, Italy, Lithuania, Poland, Switzerland and the UK using an ELISA detecting filarial-specific antibodies, hypothesising higher occurrence of D. repens. Based on two overlapping frequency distributions, presumed negative samples had a mean optical density (OD) value of 0.097, representing 97.45% of all samples. Presumed positive samples, representing 2.55% of all sera, had a mean OD value of 0.287. Test prevalence based on the calculated cut-off was 3.51% for all sera (4.36% for Austria, 1.94% for Denmark, 1.39% for Germany, 3.37% for Italy, 6.90% for Lithuania, 6.99% for Poland, 0.77% for Switzerland and 0.0% for the UK, respectively). The bimodal distribution, representing overlapping distributions of OD values from positive and negative dogs, enabled the assignment of a probability of true infection status to each dog. Mean probabilities of true infection status across groups, based on the postal codes of origin, allowed us to estimate and map true prevalences. For all countries, except the UK, the true prevalence was lower than the test prevalence. The large number of serum samples and the use of a non-gold standard analytical method allowed us to create a more realistic picture of the distribution of D. repens in Central Europe and the UK.

Agranulocytosis leads to intestinal Echinococcus multilocularis oncosphere invasion and hepatic metacestode development in naturally resistant Wistar rats.

Susceptibility to Echinococcus multilocularis infection considerably varies among intermediate (mostly rodents) and dead-end host species (e.g. humans and pig), in particular regarding intestinal oncosphere invasion and subsequent hepatic metacestode development. Wistar rats are highly resistant to infection and subsequent diseases upon oral inoculation with E. multilocularis eggs, however, after immunosuppressive treatment with dexamethasone, rats become susceptible. To address the role of the cellular innate immunity, Wistar rats were individually or combined depleted of natural killer (NK) cells, macrophages (MΦ) and granulocytes (polymorphonuclear cells, PMN) prior to E. multilocularis egg inoculation. Although NK cell and MΦ depletion did not alter the resistance status of rats, the majority of PMN-depleted animals developed liver metacestodes within 10 weeks, indicating that PMN are key players in preventing oncosphere migration and/or development in Wistar rats. In vitro studies indicated that resistance is not caused by neutrophil reactive oxygen species or NETosis. Also, light microscopical examinations of the small intestine showed that oral inoculation of E. multilocularis eggs does not elicit a mucosal neutrophil response, suggesting that the interaction of oncospheres and neutrophils may occur after the former have entered the peripheral blood. We suggest to consider granulocytes as mediators of resistance in more resistant species, such as humans.

An experimental genetically attenuated live vaccine to prevent transmission of Toxoplasma gondii by cats.

Almost any warm-blooded creature can be an intermediate host for Toxoplasma gondii. However, sexual reproduction of T. gondii occurs only in felids, wherein fertilisation of haploid macrogametes by haploid microgametes, results in diploid zygotes, around which a protective wall develops, forming unsporulated oocysts. Unsporulated oocysts are shed in the faeces of cats and meiosis gives rise to haploid sporozoites within the oocysts. These, now infectious, sporulated oocysts contaminate the environment as a source of infection for people and their livestock. RNA-Seq analysis of cat enteric stages of T. gondii uncovered genes expressed uniquely in microgametes and macrogametes. A CRISPR/Cas9 strategy was used to create a T. gondii strain that exhibits defective fertilisation, decreased fecundity and generates oocysts that fail to produce sporozoites. Inoculation of cats with this engineered parasite strain totally prevented oocyst excretion following infection with wild-type T. gondii, demonstrating that this mutant is an attenuated, live, transmission-blocking vaccine.

Activity of mefloquine and mefloquine derivatives against Echinococcus multilocularis.

The cestode E. multilocularis causes the disease alveolar echinococcosis (AE) in humans. The continuously proliferating metacestode (larval stage) of the parasite infects mostly the liver and exhibits tumor-like growth. Current chemotherapeutical treatment options rely on benzimidazoles, which are rarely curative and have to be applied daily and life-long. This can result in considerable hepatotoxicity and thus treatment discontinuation. Therefore, novel drugs against AE are urgently needed. The anti-malarial mefloquine was previously shown to be active against E. multilocularis metacestodes in vitro, and in mice infected by intraperitoneal inoculation of metacestodes when administered at 100 mg/kg by oral gavage twice a week for 12 weeks. In the present study, the same dosage regime was applied in mice infected via oral uptake of eggs representing the natural route of infection. After 12 weeks of treatment, the presence of parasite lesions was assessed in a liver squeeze chamber and by PCR, and a significantly reduced parasite load was found in mefloquine-treated animals. Assessment of mefloquine plasma concentrations by HPLC and modeling using a two-compartment pharmacokinetic model with first-order absorption showed that >90% of the expected steady-state levels (Cmin 1.15 mg/L, Cmax 2.63 mg/L) were reached. These levels are close to concentrations achieved in humans during long-term weekly dosage of 250 mg (dose applied for malaria prophylaxis). In vitro structure-activity relationship analysis of mefloquine and ten derivatives revealed that none of the derivatives exhibited stronger activities than mefloquine. Activity was only observed, when the 2-piperidylmethanol group of mefloquine was replaced by an amino group-containing residue and when the trifluoromethyl residue on position 8 of the quinoline structure was present. This is in line with the anti-malarial activity of mefloquine and it implies that the mode of action in E. multilocularis might be similar to the one against malaria.

Intense Focus of Alveolar Echinococcosis, South Kyrgyzstan.

Human alveolar echinococcosis (AE) is a highly pathogenic zoonotic parasitic disease caused by Echinococcus multilocularis. An ultrasound study in southern Kyrgyzstan during 2012 revealed a prevalence of 4.2% probable or confirmed AE and an additional 2.2% possible AE, representing an emerging situation. The risk for probable or confirmed AE was significantly higher in dog owners.

Evaluation of kinase-inhibitors nilotinib and everolimus against alveolar echinococcosis in vitro and in a mouse model.

Infection with the larval stage (metacestode) of the fox tapeworm Echinococcus multilocularis leads to a primary hepatic disease referred to as alveolar echinococcosis (AE). The progressive disease can be lethal if untreated. In cases where complete parasite resection by surgery is not feasible, the current treatment regimens of AE consist of chemotherapy with the parasitostatic benzimidazoles albendazole or mebendazole over decades. Kinase-inhibitors currently administered in various cancer treatments are of increasing interest also as anti-parasitic drugs due to previous promising in vitro results. In order to search for novel drug targets and treatment regimens, nilotinib (AMN107; Tasigna®), an Abl-tyrosine kinase inhibitor and everolimus (RAD001; Afinitor®), a serine/threonine-kinase inhibitor, were tested for their treatment efficacy against metacestode vesicles of E. multilocularis in vitro and in BALB/c mice. In vitro treatment with 200 µM nilotinib caused drug-induced alterations after 12 days, and everolimus exerted parasite damage at concentrations dosing from 40 to 100 µM after 5 and 12 days of in vitro exposure. Nilotinib (100 mg/kg) + erythromycin (to increase nilotinib plasma levels: 10 mg/kg intraperitoneal) or everolimus (5 mg/kg) were formulated in honey and administered daily for three weeks and subsequently twice a week for an additional three weeks in experimentally infected mice. Treatments did not result in any reduction of parasite growth compared to untreated control groups, whereas oral treatment with albendazole (200 mg/kg) was highly effective. Combined application of the kinase-inhibitors with albendazole did not lead to a synergistic or additive treatment efficacy compared to albendazole treatment alone. These results show that neither nilotinib nor everolimus represent valuable alternatives to the current treatment regimens against AE.

Total and Toxocara canis larval excretory/secretory antigen- and allergen-specific IgE in atopic and non-atopic dogs.

BACKGROUND: Total IgE concentrations are higher in dogs than in humans. Persistent Toxocara canis larval infection is prevalent in dogs and is associated with substantial specific antibody reactions. A correlation, however, between total IgE and T. canis-specific antibody levels in dogs has not been evaluated. OBJECTIVES: To determine the relationship between total IgE, T. canis-specific IgG and IgE, and allergen-specific IgE levels in atopic and non-atopic dogs, and to evaluate possible confounding factors. ANIMALS: Sera of 30 atopic and 30 non-atopic client-owned dogs. METHODS: Total IgE, T. canis-specific antibody and allergen-specific IgE levels were evaluated by ELISA. RESULTS: Total IgE, T. canis-specific antibody and allergen-specific IgE levels were significantly higher in non-atopic compared to atopic dogs. A positive correlation was demonstrated between T. canis-specific IgG and T. canis-specific IgE; T. canis-specific IgG and total IgE; T. canis-specific IgE and total IgE; and allergen-specific IgE and total IgE. No differences were detected on the basis of age, gender, vaccination status; deworming or season between atopic and non-atopic dogs. Previous immunomodulatory treatment and cause of atopy did not influence antibody levels of atopic dogs. CONCLUSIONS: Toxocara canis-specific IgE appears to be a major component of total IgE in dogs. Total and T. canis-specific IgE levels are higher in non-atopic compared to atopic dogs. It is speculated that T. canis infection may have a protective effect against the development of canine atopic dermatitis and/or that elevations in total serum IgE level are often not associated with atopic dermatitis.

[Postpartal clinically apparent alveolar echinococcosis in a female dog]. / Postpartal klinisch manifeste alveoläre Echinokokkose bei einer Hündin.

The lifecycle of the fox tapeworm Echinococcus multilocularis comprises primarily red foxes (definitive hosts) and small rodents (intermediate hosts). Dogs can additionally be definitive hosts and in rare cases they act as accidental or dead-end host species by developing alveolar echinococcosis (AE) primarily in the liver. This report describes a clinically apparent AE that appeared 5 weeks postpartum in a 3-year-old Labrador Retriever. The bitch was presented with loss of appe tite and apathy. Radiological examination revealed dense and partially calcified, space-occupying lesions in the abdomen that were sonographically further characterized as fluid-filled caverns. Intra operationem, a multifocal generalized cystic infiltration of the liver was observed with metastasis in the omentum. Because of the severe altera tions, the dog was euthanized. Histopathological examination revealed a laminated layer, which is typical for E. multilocularis and sporadic protoscoleces. E. multilocularis-specific PCR was positive. To the best of the authors' knowledge, this is the first case report of a postpartal clinically apparent AE described in a dog. Gestation may have led to progression of the disease.

Optimized dexamethasone immunosuppression enables Echinococcus multilocularis liver establishment after oral egg inoculation in a rat model.

Comparable with immunocompetent humans, rats are considered highly resistant to Echinococcus multilocularis oncosphere invasion, both in nature and after experimental oral inoculation with eggs. Pharmacological immunosuppression with dexamethasone (DMX) was shown to abrogate the resistance of RccHan™:WIST rats, but due to weight losses >20%, many animals had to be excluded from previous experiments. The optimized DXM (Dexafort, MSD Animal Health, Germany) dosage regime presented in this study (each animal: 750 µg DXM at day -13 and 600 µg DXM at day -9 before inoculation) applied subcutaneously to RccHan™:WIST rats, resulted in weight losses ≤20%, but led to liver alveolar echinococcosis (AE) in all eight inoculated animals. Untreated control groups (each n = 8) including RccHan™:WIST (Wistar) and F344/DuCrl (Fischer-344) rats showed no parasite establishment. Antibodies against E. multilocularis metacestode vesicle fluid were present in 7/8 of the infected RccHan™:WIST rats 70 days after inoculation but in none of the control animals. Serology can therefore be used to diagnose AE. This optimized animal model enables a high infection rate in rats and may be applied in future immunological and experimental studies.

Successful intestinal Echinococcus multilocularis oncosphere invasion and subsequent hepatic metacestode establishment in resistant RccHan™:WIST rats after pharmacological immunosuppression.

Susceptibility/resistance to larval Echinococcus multilocularis infection varies greatly depending on host species and strains. Whereas several mice strains and non-human primates are highly susceptible to alveolar echinococcosis, rats and most of humans are considered as more resistant. In this study, we aimed to elucidate factors responsible for host resistance in rats (Experiments A-D). (A) The parasite establishment was not observed in immunocompetent Wistar rats orally inoculated with sodium hypochlorite resistant eggs with/without pig bile, or activated/non-activated oncospheres (NAO). Peritoneal inoculation with NAO or metacestode tissue allowed the parasite establishment in rats. (B) T-cell-deficient athymic nude rats showed complete resistance against the metacestode establishment after oral inoculation with parasite eggs. This finding suggests that T-cell-independent parasite clearance occurred in the animals during early phase of the parasite invasion. Finally, Wistar rats that received pharmacological immunosuppression using either dexamethasone (DMS) alone or methotrexate (MTX) i.p. alone or a combination of these compounds were orally inoculated with the parasite's eggs. As a result (D), successful establishment of metacestode with protoscoleces was observed in all 3 rats treated with DMS (s.c.) alone or in all 6 rats treated with DMS (s.c.) plus MTX but not in 8 rats with MTX alone, suggesting that factors affected by DMS treatment are responsible to regulate the parasite invasion and establishment.

Vaccination with recombinant paramyosin against the bovine lungworm Dictyocaulus viviparus considerably reduces worm burden and larvae shedding.

BACKGROUND: The lungworm Dictyocaulus viviparus, causing parasitic bronchitis in cattle, induces a temporary protective immunity that prevents clinical disease. A radiation-attenuated larvae based vaccine is commercially available in a few European countries, but has the disadvantages of a live vaccine. As a recombinant subunit vaccine would overcome these disadvantages, the parasite's muscle protein paramyosin (PMY) was tested as a recombinant vaccine antigen. METHODS: D. viviparus-PMY was recombinantly expressed in Escherichia coli as a glutathione-S-transferase (GST)-fused protein. Emulsified in adjuvant Saponin Quil A, the protein was given intramuscularly into calves. Two independent recombinant PMY (rPMY) vaccination trials with negative control groups (first trial: adjuvant only; second trial: non-fused GST) as well as an additional positive control group in the second trial, using the Bovilis Dictol live vaccine to verify vaccination results, were performed. To determine the vaccination success, shedding of larvae as well as worm burden and worm sizes were analyzed. Additionally, ELISA-based determination of development of immunglobulins IgM, IgA, IgE, IgG as well as the subclasses IgG1 and IgG2 was performed. To analyze PMY localization in the bovine lungworm, immunohistochemical staining of adult worms was carried out. RESULTS: Immunohistochemical staining revealed that PMY is part of the bovine lungworm's pharyngeal and body wall muscles. Vaccination with rPMY resulted in 47% [geometric mean: 67%] and 57% (geometric mean: 71%) reduction of larvae shedding in the first and second vaccination trial, respectively. Worm burden was reduced by 54% (geometric mean: 86%) and 31% (geometric mean: 68%), respectively, and worms of rPMY-vaccinated cattle were significantly shorter in both trials. Furthermore, ELISAs showed a clear antibody response towards rPMY with exception of IgE for which titers could not be detected. After challenge infection, rPMY antibodies were only exceptionally elevated among study animals indicating PMY to be a hidden antigen. CONCLUSIONS: Even though vaccination with the attenuated live vaccine was with 94% (geometric mean: 95%) reduction in larvae shedding and 93% (geometric mean: 94%) reduction in worm burden superior to rPMY vaccination, results using the latter are promising and show the potential for further development of a recombinant PMY-based vaccine against the bovine lungworm.