3D holotomographic monitoring of Ca++ dynamics during ionophore-induced Neospora caninum tachyzoite egress from primary bovine host endothelial cells.

Neospora caninum represents an obligate intracellular parasite that belongs to the phylum Apicomplexa and is a major abortive agent in bovines. During merogony, N. caninum tachyzoites invade and proliferate in host cells in vivo, including endothelial cells of lymphatic and blood vessels. The egress at the end of the lytic cycle is tightly regulated in apicomplexans. Evidence in Toxoplasma gondii shows that Ca++ signalling governs tachyzoite egress. Much less is known on egress mechanisms of N. caninum. Here, we show, using 3D live cell holotomographic microscopy in fluo-4 AM-loaded N. caninum-infected BUVEC, that treatments with the calcium ionophore A23187 at 24- and 42-h post-infection (h p. i.) induced a fast and sustained increase in Ca++ signals in parallel to tachyzoite egress. A23187 treatments exclusively triggered tachyzoite release at 42-h p. i. but failed to do so at 24-h p. i. indicating a role for meront maturation in calcium-induced tachyzoite egress. Overall, we show that live cell 3D holotomographic analysis in combination with epifluorescence is a suitable tool to study calcium dynamics related to coccidian egress or other important cell functions.

Impacto de las complicaciones postoperatorias en el pronóstico del cáncer gástrico / Impact of postoperative complications on the prognosis of gastric cancer

Introducción. La cirugía sigue siendo el pilar fundamental en el tratamiento del cáncer gástrico, con una tasa de morbilidad quevaría entre 18-55%. El objetivo del estudio fue analizar el impacto de las complicaciones postoperatorias en la supervivencia global(SG) del cáncer gástrico. Material y métodos. Estudio retrospectivo y unicéntrico sobre 200 pacientes intervenidos con intencióncurativa por neoplasia gástrica entre el 1 de enero del 2010 y el 31 de diciembre del 2014. Para analizar la gravedad de las complicaciones se empleó la clasificación de Clavien-Dindo (CD). Se realizó un análisis univariante y multivariante de los factores queinfluyeron en la SG, la cual fue estimada mediante el método de Kaplan-Meier. Resultados. La morbilidad postoperatoria fue del47,5%, con tasas de incidencia de complicaciones leves (CD II) del 24,5%, y de complicaciones moderadas-graves (CD III-IV) del22,5%. En el análisis multivariante de la SG se observó que las complicaciones postoperatorias afectaron de forma significativa enla mortalidad (HR 1,61 [1,09;2,42], p=0,018). Las complicaciones médicas tuvieron un impacto significativo en la supervivenciaglobal (HR 1,89 [1,20;2,96], p=0,006), pero las complicaciones quirúrgicas no pudieron ser relacionadas estadísticamente (HR1,49 [0,92;2,43], p=0,108). Conclusión. El presente estudio muestra el impacto negativo de las complicaciones médicas postquirúrgicas sobre la supervivencia global del cáncer gástrico tratado con intención curativa. (AU)

Introduction. Surgery is the cornerstone in curative treatment of gastric cancer with complication rates ranging from 18% to 55%.The aim of the study was to analyze the impact of postoperative complications on overall survival (OS) of gastric cancer. Methods.This is a retrospective study of a single-center series. Patients who underwent curative gastrectomy between January 2010 and December 2014 were included. The severity of postoperative complications was studied according to the Clavien-Dindo classification.Patients OS was estimated using the method of Kaplan-Meier. Besides, univariate and multivariate analysis of the factors influencing on OS was performed. Results. 200 patients were included. Postoperative morbidity was 47.5%, with incidence rates of mildcomplications (CD II) of 24.5% and moderate-serious complications (CD III-IV) of 22.5%. The multivariate analysis of OS showedthat postoperative complications significantly affected mortality (HR 1.61 [1.09, 2.42], p = 0.018). Medical compli-cations had animpact on OS (HR 1.89 [1.20; 2.96], p = 0.006), but surgical complications could not be statistically related (HR 1.49 [0.92; 2.43],p = 0.108). Conclusion. The present study shows the negative impact of postoperative medical complications on the overall survivalof curative gastrectomy in patients with gastric cancer. (AU)

Pneumothorax in a persistent canine Angiostrongylus vasorum infection.

Canine angiostrongylosis is a gastropod-borne parasitosis caused by the cosmopolitan nematode Angiostrongylus vasorum. It is associated with cardiorespiratory disorders and eventually fatal coagulopathies. A 6-months-old female dog previously diagnosed with bronchopneumonia and pneumothorax, treated accordingly without success, was hospitalized for further treatment. Complementary diagnostics confirmed previous findings plus pneumomediastinum, thoracic exudate, leucocytosis, thrombocytosis, hypercoagulability, and hyperglobulinemia. Coprological analysis showed a severe patent A. vasorum infection. After the third daily fenbendazole administration, all A. vasorum larvae were found dead in follow-up coprological analyses while in intensive care. Upon release, the dog was monitored until full clinical recovery 3 months later. Up to date, A. vasorum infection-related pneumothorax was rarely described, particularly with full recovery of such a young patient. Thus, a parasitological aetiology should be considered for dogs with severe bronchopneumonia or pneumothorax, especially in Europe and South America where increasing numbers of canine angiostrongylosis cases call for enhanced awareness of veterinary practitioners.

Primer consenso cubano sobre el uso de terapia convencional y biológica en pacientes con artritis reumatoide / First Cuban consensus on the use of conventional and biological therapy in patients with rheumatoid arthritis

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Introduction: The development of recommendations for the treatment of rheumatoid arthritis (RA) in the Cuban context may be one of the ways to achieve better control of this disease. Objective: To reach a consensus and update relevant aspects of conventional and biological RA modifier therapy in Cuba. Methods: 18 specialists from 8 Cuban provinces, experts in RA care, were summoned, according to the years of dedication to the specialty, the conferences on this topic and their publications. The first meeting took place in March 2016 in the provincial hospital of Villa Clara, Cuba, with the participation of all the experts. A review of the literature on conventional and biological therapy previously collected by the participants was developed, and two teams were formed: the first would address everything related to conventional therapy in RA (HRCT) and the other, biological therapy in RA (TBAR). Three questionnaires related to the use of corticosteroids, HRCT and TBAR, were prepared, answered by the participants via email. In a second meeting, held in October 2016 in Havana, the analysis of all the responses provided was carried out. Questions with a response of 90% or more votes were considered as recommendations. Results: The questionnaires were answered by 95% of the participants. 9 recommendations and 1 algorithm were established. The recommendations are as follows: methotrexate is the drug of choice in the treatment of RA after diagnosis; The administration of another conventional drug (DMARDc) (azathioprine, salazosulfapyridine, antimalarials and leflunomide) is recommended in patients with a diagnosis of active RA in whom methotrexate is contraindicated or there is a failure in response - consider the administration of low doses of prednisone or equivalent (<7.5 mg/d) associated with DMARDc in patients with active moderate to severe RA, for the shortest possible time; perform serological control including tests for hepatitis B and C viruses and screening for HIV in all patients diagnosed with RA before starting treatment with DMARDc and biologics; in patients in remission or, at least, with a DAS-28 below 3.2, consideration should be given to withdrawing one of the DMARDs or reducing, to the minimum possible expression, the dose of both disease modifiers; if methotrexate fails, tocilizumab in combination with methotrexate or as monotherapy will be indicated. Conclusions: Aspects related to conventional therapy with methotrexate, azathioprine, salazosulfapyridine, antimalarials and leflunomide were agreed. The value of early diagnosis and immediate initiation of DMARDc therapy and the use of glucocorticoids was analyzed. Treatment with tocilizumab, the only biological available in Cuba against RA, will be administered when there is a failure in the response to conventional therapy and combinations between these drugs. It is recommended to hold educational conferences through the mass media aimed at patientshttp(AU)

Swine spermatozoa trigger aggregated neutrophil extracellular traps leading to adverse effects on sperm function.

In pigs, the number of PMN in uterus lumen increases within few hours after natural or artificial AI resulting in early PMN-derived innate immune reactions. Sperm-NETs formation was recently reported to occur in various mammalian species. Aim of this study was to investigate direct interactions of boar spermatozoa with swine PMN, the release of sperm-mediated NETs, and to assess NET-derived effects on sperm functionality. Sperm-triggered NETs were visualized by SEM- and immunofluorescence analyses. Sperm-mediated NETosis was confirmed by presence of extruded DNA with global histones and NE. Largest sizes of sperm-mediated aggNETs were detected after 5 h thereby resulting in effective massive sperm entrapment. The number of aggNETs increased from 3 h onwards. Kinetic studies of swine sperm-mediated NETosis showed to be a time-dependent cellular process. In addition, number of NETs-entrapped spermatozoa increased at 3 h of exposure whilst few free spermatozoa were detected after 3 h. Anchored NETs also increased from 3 h onwards. The cytotoxicity of NETs was confirmed by diminution of the total motility and the progressive motility. Spermatozoa membrane integrity and function loss exposed to NETs was confirmed from 3 h. Experiments revealed NETs-derived damaging effects on swine spermatozoa in membrane integrity, motility and functionality. We hypothesize that swine sperm-triggered aggNETs might play a critical role in reduced fertility potential in swine reproductive technique. Thus, aggNETs formation needs to be considered in future studies about uterine environment as well as advance of sperm in the porcine female reproductive tract.

Endoparasitic Insights of Free-Living Fin (Balaenoptera physalus), Humpback (Megaptera novaeangliae) and North Atlantic Right Whales (Eubalaena glacialis) from Eastern Canadian Waters.

PURPOSE: To date, little is still known on parasite infections affecting free-living large whale populations worldwide. Data presented should be considered as a baseline study for future monitoring surveys on endoparasites affecting whales, thereby enhancing investigations on impacts of zoonotic parasitoses not only on vulnerable or endangered baleen whale population health but also on public health. METHODS: The presented study is a first report on gastrointestinal parasites infecting different free-living baleen whales inhabiting East Canadian waters using non-invasive methods. Individual faecal samples from fin (n = 3; Balaenoptera physalus), humpback (n = 4; Megaptera novaeangliae) and North Atlantic right whales (n = 1; Eubalaena glacialis) were collected without animal disturbance, within their natural habitats on an ecological expedition during annual surveys in summer 2017. Faecal samples were assessed by standardized diagnostic methods, such as sodium acetate acetic formalin (SAF) technique, carbol fuchsin-stained faecal smears, Giardia/Cryptosporidium coproantigen ELISAs and were applied for further identification. RESULTS: Parasitological infections included three different potentially zoonotic parasite species, one protozoa (Entamoeba spp.) and two metazoans (Diphyllobothriidae gen. sp., Ascaridida indet.). No positive Giardia/Cryptosporidium coproantigen ELISA could be found in the studied whales. CONCLUSION: This study adds to the current knowledge of intestinal and zoonotic parasite infections of vulnerable to partly endangered free-ranging baleen whales. Only few or no parasitological studies exist for these whale species, usually dealing with only one dead specimen. We call for more research in this field especially for the importance of conservation of free-living marine mammals using non-invasive methods.

Eimeria ninakohlyakimovae casts NOX-independent NETosis and induces enhanced IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in caprine PMN.

Eimeria ninakohlyakimovae represents a highly pathogenic coccidian parasite causing severe haemorrhagic typhlocolitis in goat kids worldwide. NETosis was recently described as an efficient defense mechanism of polymorphonuclear neutrophils (PMN) acting against different parasites in vitro and in vivo. In vitro interactions of caprine PMN with parasitic stages of E. ninakohlyakimovae (i. e. oocysts and sporozoites) as well as soluble oocyst antigens (SOA) were analyzed at different ratios, concentrations and time spans. Extracellular DNA staining was used to illustrate classical molecules induced during caprine NETosis [i. e. histones (H3) and neutrophil elastase (NE)] via antibody-based immunofluorescence analyses. Functional inhibitor treatments with DPI and DNase I were applied to unveil role of NADPH oxidase (NOX) and characterize DNA-backbone composition of E. ninakohlyakimovae-triggered caprine NETosis. Scanning electron microscopy (SEM)- and immunofluorescence-analyses demonstrated that caprine PMN underwent NETosis upon contact with sporozoites and oocysts of E. ninakohlyakimovae, ensnaring filaments which firmly entrapped parasites. Detailed co-localization studies of E. ninakohlyakimovae-induced caprine NETosis revealed presence of PMN-derived DNA being adorned with nuclear H3 and NE corroborating molecular characteristics of NETosis. E. ninakohlyakoimovae-induced caprine NETosis was found to be NOX-independent since DPI inhibition led to a slight decrease of NETosis. Exposure of caprine PMN to vital E. ninakohlyakimovae sporozoites as well as SOA resulted in up-regulation of IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in stimulated PMN. Since vital E. ninakohlyakimovae-sporozoites induced caprine NETosis, this effective entrapment mechanism might reduce initial sporozoite epithelial host cell invasion during goat coccidiosis ultimately resulting in less macromeront formation and reduced merozoites I production.

Analysis of potential risk factors of caprine coccidiosis.

Caprine coccidiosis represents a major concern in goat industry worldwide causing high economic losses, mainly due to poor weight gains, treatment costs and even death of kids. Therefore, analyses on risk factors involved in the establishment and outcome of this disease are important to improve infection status and herd management. In Portugal, approximately 400,000 goats are reared every year. The majority of them are allocated in the southern part of the country. In the current study, a representative number of animals (382 goats) and herds (37 herds) from Alentejo were monitored. Faecal samples from five young and five adult goats of each farm were collected rectally and examined individually by using the modified McMaster technique for oocyst counts (oocyst per gram of faeces - OPG). Additionally, a questionnaire on the husbandry conditions and anti-parasitic treatments was performed. Overall, a prevalence of Eimeria infections of 99% and 100% was detected on animal and herd level, respectively. Eimeria arloingi and E. ninakohlyakimovae as the two most pathogenic caprine species also constituted the most prevalent ones (34.91% and 29.90%, respectively), both in the young and the adult goat groups. As expected, OPG vales were significantly higher (p = 0.031) in young than in adult goats. Furthermore, risk factor analyses revealed young age, higher total number of animals on the farm and outdoor kidding as strongly associated with a higher risk of caprine coccidiosis. Overall, the high prevalence of pathogenic Eimeria species on Portuguese goat farms in combination with the occurrence of related risk factors strengthen the importance of coccidiosis control in caprine industry.

Ophthalmic Thelazia callipaeda infections: first feline and new canine imported cases in Germany.

The first case of feline ocular Thelazia callipaeda infection and two new canine imported infections in West Germany are here described. The three animals had a history of recent travel to/from other countries. The young adult cat imported from Spain presented an intermittent unilateral ocular discharge. During in-depth ophthalmic examination, a single alive nematode was removed from the conjunctival compartment of the affected eye. Referring to the canine cases, an adult female dog originated from Kenya presented epiphora and mucous whitish-grey discharge of the right eye. During flushing of the nasolacrimal duct two small, thin and long nematodes were removed. Furthermore, a male Borzoi racing dog with regular visit to racing tracks in different countries presented ocular mucous discharge. At ophthalmologic examination, two transparent-whitish vital nematodes were removed. All nematode specimens of the three cases were morphologically identified as adult T. callipaeda parasites. The animals were treated orally with milbemycin oxime (2.0 mg/kg; cat) or milbemycin oxime/praziquantel (0.5 mg/kg and 5.0 mg/kg; dogs) twice with 1-week interval resulting in complete resolution of symptoms. The repeated introduction of patent T. callipaeda-infected animals, especially from southern and eastern endemic countries, will ease the establishment of ophthalmic thelaziosis in Northern Europe. The male fruit fly, Phortica variegata, an intermediate host of T. callipaeda, is endemic within European countries. Considering the clinical and zoonotic relevance of ophthalmic thelaziosis, enhanced disease awareness of European medical and veterinarian doctors and in-depth eye examination for proper detection of T. callipaeda are crucial for appropriate anthelmintic treatments and to limit spreading of the infection.

The V1016G mutation of the voltage-gated sodium channel (VGSC) gene contributes to the insecticide resistance of Aedes aegypti from Makassar, Indonesia.

Aedes aegypti represents one of the main vectors of at least five relevant arthropod-borne viral infections in humans (i.e., Rift Valley fever, Dengue fever, Zika, chikungunya, and yellow fever) worldwide. Ae. aegypti control strategies are mostly based on using chemical insecticides (i.e., organophosphates, pyrethroids, carbamates, and organochlorines) and reducing larval sources. Furthermore, monitoring the growth activity and mapping the geographical distribution of insecticide resistance are mandatory, as recommended by the WHO. Accordingly, we conducted a study on the possible mechanism by which Ae. aegypti develops resistance to several frequently used chemical insecticides (i.e., λ-cyhalothrin, bendiocarb, cyfluthrin, deltamethrin, malathion, and permethrin) in the city of Makassar, Sulawesi, Indonesia. The results showed the progression of resistance toward the examined insecticides in Ae. aegypti populations in Makassar. The mortality rate of Ae. aegypti was less than 90%, with the highest resistance recorded against 0.75% permethrin. The molecular evaluation of the voltage-gated sodium channel gene (VGSC) showed a significant correlation of the V1016G gene mutation in the tested 0.75% permethrin-resistant Ae. aegypti phenotypes. Nevertheless, the F1534C point mutation in the VGSC gene of Ae. aegypti did not show a significant correlation with the phenotype exhibiting insecticide resistance to 0.75% permethrin. These results indicate that Ae. aegypti mosquitoes in Makassar City have developed resistance against the frequently used insecticide permethrin, which might spread to less-populated regions of Sulawesi. Therefore, we call for further entomological monitoring of insecticide resistance not only on Sulawesi but also on other closely located islands of the Indonesian archipelago to delay the spread of Ae. aegypti insecticide resistance.

Epidemiological survey and risk factor analysis on Eimeria infections in calves and young cattle up to 1 year old in Colombia.

A large-scale cross-sectional epidemiological study was conducted to evaluate prevalence, species diversity, and associated risk factors of Eimeria infections in 55 cattle farms across seven states of Colombia, including subtropical and tropical regions. In total, 1333 fecal samples from young animals (< 1 year of age) were examined at a single sampling date from August 2016 to December 2016. Flotation and McMaster techniques were conducted for parasitological investigation. Excreted Eimeria oocysts were allowed to sporulate in vitro and thereafter identified to species level based on morphological and morphometric characteristics. The overall Eimeria prevalence was 75.5% (1006/1333), with no difference observed between age categories. In total, 13 different Eimeria species were identified. The most prevalent species was E. bovis (33.5%), followed by E. auburnensis (12.5%) and E. zuernii (11.9%). Analysis of extrinsic associated risk factors revealed the floor type, feeding system, watering system, and herd size as significant (p < 0.05) risk factors for Eimeria spp. infections. Based on these data, it can be assumed that bovine coccidiosis infections occur ubiquitously in the country and might play an important role especially in its subclinical form by affecting production parameters in conventional cattle management systems.

Associated risk factors influencing ovine Eimeria infections in southern Spain.

Eimeria infections are common in sheep industry worldwide nonetheless very little is known on infection dynamics and associated risk factors influencing the outcome of clinical ovine coccidiosis. The aim of the present work was to analyse intrinsic (e. g. host age) and extrinsic (flock size, farm size and animal density) risk factors influencing dynamics of Eimeria spp. infections in sheep farms of the semi-arid region of Murcia in southern Spain. Overall, 64 ovine flocks in Murcia were surveyed and a total of 396 faecal samples were collected between May and July 2015. Flotation and McMaster techniques were performed for parasitological investigation. Excreted oocysts were identified on the basis of morphological as well as morphometric characteristics. In all analysed samples Eimeria spp. oocysts (100%) were found and 11 different Eimeria species were identified. Most prevalent species was E. ovinoidalis (97%) followed by E. parva/E.pallida (97%), E. weybridgensis (90.9%) and E. crandallis (89.4%), respectively. Concomitant infections with more than one Eimeria species were observed in all analysed samples. Analysis of intrinsic/extrinsic risk factor revealed age, herd/farm size and animal density to be the main factors significantly influencing Eimeria oocysts per gram of faeces counts (OPG) and overall prevalence of Eimeria spp. infections in sheep.

Occurrence of endoparasites in wild Antillean manatees (Trichechus manatus manatus) in Colombia.

The recognized impact of parasites in wildlife populations demands surveillance of endangered species like the Antillean manatees (Trichechus manatus manatus) in Colombia. We conducted a parasitological survey in four rescued sea cows in order to document the parasite diversity of this sirenian in the Caribbean wetland of Colombia and contribute to the molecular characterization of its trematodes. The flukes Chiorchis fabaceus, Nudacotyle undicola and the protozoans Eimeria manatus and E. nodulosa were identified in analysed faecal samples. For C. fabaceus and N. undicola, partial regions of ribosomal RNA genes were amplified and sequenced in order to infer their phylogenetic relations. The current study constitutes a new sirenian host (T. manatus manatus) record for the genus Eimeria and the trematode N. undicola.

Fatty and hydroxycarboxylic acid receptors: The missing link of immune response and metabolism in cattle.

Fatty and hydroxycarboxylic acids are one of the main intermediates of energy metabolism in ruminants and critical in the milk production of cattle. High production demands on a dairy farm can induce nutritional imbalances and metabolism disorders, which have been widely associated with the onset of sterile inflammatory processes and increased susceptibility to infections. The literature suggests that short-chain fatty acids (SCFA), long-chain fatty acids (LCFA) and hydroxycarboxylic acids are relevant modulators of the host innate inflammatory response. For instance, increased SCFA and lactate levels are associated with subacute ruminal acidosis (SARA) and the activation of pro-inflammatory processes mediated by diverse leukocyte and vascular endothelial cells. As such, free LCFA and the ketone body ß-hydroxybutyrate are significantly increased in the plasma 1-2 weeks postpartum, coinciding with the time period in which cows are more susceptible to acquiring infectious diseases that the host innate immune system should actively oppose. Today, many of these pro-inflammatory responses can be related to the activation of specific G protein-coupled receptors, including GPR41/FFA3 and GPR43/FFA2 for SCFA; GPR40/FFA1 and GPR120/FFA4 for LCFA, GPR109A/HCA2 for ketone body ß-hydroxybutyrate, and GPR81/HCA1 for lactate, all expressed in different bovine tissues. The activation of these receptors modulates the release of intracellular granules [e.g., metalloproteinase-9 (MMP-9) and lactoferrin], radical oxygen species (ROS) production, chemotaxis, and the production of relevant pro-inflammatory mediators. The article aimed to review the role of natural ligands and receptors and the resulting impact on the host innate immune reaction of cattle and, further, to address the most recent evidence supporting a potential connection to metabolic disorders.

Modulation of cholesterol-related sterols during Eimeria bovis macromeront formation and impact of selected oxysterols on parasite development.

Obligate intracellular apicomplexan parasites are considered as deficient in cholesterol biosynthesis and scavenge cholesterol from their host cell in a parasite-specific manner. Compared to fast proliferating apicomplexan species producing low numbers of merozoites per host cell, (e. g. Toxoplasma gondii), the macromeront-forming protozoa Eimeria bovis is in extraordinary need for cholesterol for offspring production (≥ 170,000 merozoites I/macromeront). Interestingly, optimized in vitro E. bovis merozoite I production occurs under low foetal calf serum (FCS, 1.2%) supplementation. To analyze the impact of extensive E. bovis proliferation on host cellular sterol metabolism we here compared the sterol profiles of E. bovis-infected primary endothelial host cells grown under optimized (1.2% FCS) and non-optimized (10% FCS) cell culture conditions. Therefore, several sterols indicating endogenous de novo cholesterol synthesis, cholesterol conversion and sterol uptake (phytosterols) were analyzed via GC-MS-based approaches. Overall, significantly enhanced levels of phytosterols were detected in both FCS conditions indicating infection-triggered sterol uptake from extracellular sources as a major pathway of sterol acquisition. Interestingly, a simultaneous induction of endogenous cholesterol synthesis based on increased levels of distinct cholesterol precursors was only observed in case of optimized parasite proliferation indicating a parasite proliferation-dependent effect. Considering side-chain oxysterols, 25 hydroxycholesterol levels were selectively found increased in E. bovis-infected host cells, while 24 hydroxycholesterol and 27 hydroxycholesterol contents were not significantly altered by infection. Exogenous treatments with 25 hydroxycholesterol, 27 hydroxycholesterol, and 7 ketocholesterol revealed significant adverse effects on E. bovis intracellular development. Thus, the number and size of developing macromeronts and merozoite I production was significantly reduced indicating that these oxysterols bear direct or indirect antiparasitic properties. Overall, the current data indicate parasite-driven changes in the host cellular sterol profile reflecting the huge demand of E. bovis for cholesterol during macromeront formation and its versatility in the acquisition of cholesterol sources.

Prevalence of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Crenosoma vulpis larvae in native slug populations in Germany.

Metastrongyloid parasites represent sparsely studied parasites of dogs and cats in Germany. Recent European surveys indicate that these parasites are spreading in Europe. Actual data on prevalence of Angiostrongylus vasorum in dogs and foxes reveal several endemic foci in Germany. However, actual data on the prevalence of A. vasorum and other metastrongyloid lungworm larvae in a wide range of slug and snail intermediate hosts, such as Arion lusitanicus, are missing for Germany. To fill this gap, we conducted an epidemiological survey on native German slugs in selected regions of Hesse and Rhineland-Palatinate. The focus was on slugs, because in study areas slugs appear to be more abundant than snails. Slugs were collected throughout different seasons of the year in areas that were previously proven to be hyperendemic for A. vasorum fox infections. Overall, a total of 2701 slugs were collected and examined for lungworm larvae via artificial digestion. The number of A. vasorum larvae per slug varied considerably (1-546 larvae per specimen). Some hotspot areas with high A. vasorum prevalence in slugs (up to 19.4%) were identified. The overall A. vasorum prevalence varied with season with largest number of slugs infected in summer (9.1%) and lowest number in winter (0.8%). The current study revealed a total A. vasorum prevalence of 4.7% in slugs based on microscopic analyses. Confirmation of lungworm species was made by specific duplex-real-time PCRs. Hence, these data demonstrate that final hosts are at a permanent risk for A. vasorum infections during all seasons when living in investigated areas. Besides A. vasorum, other lungworm larvae were also detected, such as Crenosoma vulpis (the fox lungworm, 2.3%) and Aelurostrongylus abstrusus (feline lungworm, 0.2%).

Age-related immune response to experimental infection with Eimeria ninakohlyakimovae in goat kids.

Both the immune response developed in ruminants against Eimeria spp. and the ability to bear patent infections seems to be dependent on the age of the host. In the present study we have evaluated the influence of the age in the development of protective immune responses against Eimeria ninakohlyakimovae. For this purpose, 3, 4 and 5-week-old goat kids were infected with sporulated oocysts and subjected to a homologous challenge 3 weeks later. Goat kids primary infected at 6, 7 and 8 weeks of age served as challenge controls, and uninfected animals were used as negative controls. The protective immunity was assessed by clinical, haematological, parasitological, immunological and pathological parameters. Altogether, the results demonstrate that goat kids of either 3, 4 or 5 weeks of age are able to develop patent infections and immunoprotective responses against E. ninakohlyakimovae, as all age groups: (i) released significantly less oocysts after challenge, which was associated to milder clinical signs; (ii) displayed a local immune response, with significant increase of numerous cellular populations; and (iii) had increased levels of IgG and IgM, and mainly of local IgA. Nevertheless, detailed analysis of the data showed some differences between the three age groups, related both to the Eimeria infection outcome and the resulting immune response, suggesting that youngest goat kids are not fully immunocompetent. This finding may be of interest for the design of immunoprophylactic approaches and/or prophylactic/methaphylactic treatments against goat coccidiosis.

Regional report on Angiostrongylus vasorum in Colombia: Genetic similarity to European lineage.

The canine lungworm Angiostrongylus vasorum is considered neglected in South America and was only sporadically reported in dogs and wildlife. Gastropods act as obligatory intermediate hosts for this parasitosis. We here analysed Achatina fulica (African giant snail) populations from 5 regions of Colombia for A. vasorum infections. In total, 609 snails were collected from the departments Antioquia, Valle del Cauca and Putumayo. Angiostrongylus vasorum-infected A. fulica were found in all departments with a total prevalence of 3.9%. Larvae originating from Putumayo were molecularly characterized and identified as the European lineage of A. vasorum. This regional report shows for the first time the presence of A. vasorum in intermediate hosts in Colombia and the European genotype in South America.

Current Status of Aedes aegypti Insecticide Resistance Development from Banjarmasin, Kalimantan, Indonesia.

Aedes aegypti represents the principal vector of many arthropod-borne diseases in tropical areas worldwide. Since mosquito control strategies are mainly based on use of insecticides, resistance development can be expected to occur in frequently exposed Ae. aegypti populations. Surveillance on resistance development as well as testing of insecticide susceptibility is therefore mandatory and needs further attention by national/international public health authorities. In accordance, we here conducted a study on Ae. aegypti resistance development towards several often used insecticides, i.e., malathion, deltamethrin, permethrin, λ-cyhalothrin, bendiocarb, and cyfluthrin, in the periurban area of Banjarmasin city, Kalimantan, Indonesia. Our results clearly showed resistance development of Ae. aegypti populations against tested insecticides. Mortalities of Ae. aegypti were less than 90% with the highest resistance observed against 0.75% permethrin. Collected mosquitoes from Banjarmasin also presented high level of resistance development to 0.1% bendiocarb. Molecular analysis of voltage-gated sodium channel (Vgsc) gene showed significant association of V1016G gene point mutation in resistance Ae. aegypti phenotypes against 0.75% permethrin. However, F1534C gene point mutation did not correlate to Ae. aegypti insecticide resistance to 0.75% permethrin. Irrespective of periurban areas in Kalimantan considered as less densed island of Indonesia, Ae. aegypti-derived resistance to different routinely applied insecticides occurred. Our findings evidence that Ae. aegypti insecticide resistance is most likely spreading into less populated areas and thus needs further surveillance in order to delay Ae. aegypti resistance development.

Bottlenose dolphins (Tursiops truncatus) do also cast neutrophil extracellular traps against the apicomplexan parasite Neospora caninum.

Neutrophil extracellular traps (NETs) are web-like structures composed of nuclear DNA decorated with histones and cytoplasmic peptides which antiparasitic properties have not previously been investigated in cetaceans. Polymorphonuclear neutrophils (PMN) were isolated from healthy bottlenose dolphins (Tursiops truncatus), and stimulated with Neospora caninum tachyzoites and the NETs-agonist zymosan. In vitro interactions of PMN with the tachyzoites resulted in rapid extrusion of NETs. For the demonstration and quantification of cetacean NETs, extracellular DNA was stained by using either Sytox Orange® or Pico Green®. Scanning electron microscopy (SEM) and fluorescence analyses demonstrated PMN-derived release of NETs upon exposure to tachyzoites of N. caninum. Co-localization studies of N. caninum induced cetacean NETs proved the presence of DNA adorned with histones (H1, H2A/H2B, H3, H4), neutrophil elastase (NE), myeloperoxidase (MPO) and pentraxin (PTX) confirming the molecular properties of mammalian NETosis. Dolphin-derived N. caninum-NETosis were efficiently suppressed by DNase I and diphenyleneiodonium (DPI) treatments. Our results indicate that cetacean-derived NETs represent an ancient, conserved and relevant defense effector mechanism of the host innate immune system against N. caninum and probably other related neozoan parasites circulating in the marine environment.