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.

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.

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.

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.

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.

Parasite prevalence in Mycobacterium spp. infected dairy goats in the region of Murcia (south-east Spain).

Eighty-four Murciano-Granadina dairy goats slaughtered in the region of Murcia (South-East Spain) were necropsied to evaluate parasitic infections. The majority of the animals (94.0%) were parasitised and multiple infections were present. Twenty-one parasite species were found, including 18 nematode species (Muellerius capillaris, Dictyocaulus filaria, Neostrongylus linearis, Cystocaulus ocreatus, Teladorsagia circumcincta, T. occidentalis, T. trifurcata, Marshallagia marshalli, Camelostrongylus mentulatus, Trichostrongylus capricola, Nematodirus abnormalis, N. filicollis, N. spathiger, T. vitrinus, T. colubriformis, Trichuris spp., Chabertia ovina and Skrjabinema ovis); one trematode species (Dicrocoelium dendriticum); one arthropod species (Oestrus ovis); and one protozoa genus (Eimeria spp.). Additionally, 17.85% of the animals were Mycobacterium spp. positive. Therefore, comparison between parasite prevalence, intensity and abundance in tuberculosis-positive and -negative animals was performed. Statistically significant differences between the prevalence of lungworms and gastrointestinal nematodes in Mycobacterium spp. infected and free goats were found. The paper discusses this co-infection between Mycobacteriaceae and endoparasites.

Une étude a été conduite sur 84 chèvres laitières de race Murciano­Granadina abattues dans la région de Murcia (sud-est de l'Espagne) afin d'évaluer la présence d'infections parasitaires. Dans leur grande majorité (94,0 %), les chèvres étaient infestées de parasites et le nombre de pluri-infections était important. Au total, 21 espèces de parasites ont été observées, dont 18 espèces de nématodes (Muellerius capillaris, Dictyocaulus filaria, Neostrongylus linearis, Cystocaulus ocreatus, Teladorsagia circumcincta, T. occidentalis, T. trifurcata, Marshallagia marshalli, Camelostrongylus mentulatus, Trichostrongylus capricola, Nematodirus abnormalis, N. filicollis, N. spathiger, T. vitrinus, T. colubriformis, Trichuris spp., Chabertia ovina et Skrjabinema ovis), une espèce de trématode (Dicrocoelium dendriticum), une espèce d'arthropode (Oestrus ovis) et des membres d'un genre de protozoaire (Eimeria spp.). En outre, la présence de Mycobacterium spp. a été détectée chez 17,85 % des chèvres examinées. Par conséquent, une étude comparative a été réalisée pour déterminer la prévalence, l'intensité et l'abondance des parasites chez les animaux ayant donné des résultats respectivement positifs et négatifs à la détection de la tuberculose. Des différences significatives au plan statistique ont été constatées entre les chèvres infectées et indemnes de Mycobacterium spp., respectivement, pour ce qui concerne la prévalence des vers pulmonaires et des nématodes gastro-intestinaux. Les auteurs font le point sur cette co-infection par des mycobactéries et des endoparasites.

Tras ser sacrificadas, ochenta y cuatro cabras lecheras murciano­granadinas de la región de Murcia (sudeste de España) fueron sometidas a necropsia para determinar la presencia de eventuales parasitosis. La mayoría de los animales (un 94,0%) presentaban parásitos y múltiples infecciones. Se descubrieron veintiuna especies de parásito: 18 especies de nemátodo (Muellerius capillaris, Dictyocaulus filaria, Neostrongylus linearis, Cystocaulus ocreatus, Teladorsagia circumcincta, T. occidentalis, T. trifurcata, Marshallagia marshalli, Camelostrongylus mentulatus, Trichostrongylus capricola, Nematodirus abnormalis, N. filicollis, N. spathiger, T. vitrinus, T. colubriformis, Trichuris spp., Chabertia ovina y Skrjabinema ovis); una especie de tremátodo (Dicrocoelium dendriticum); una especie de artrópodo (Oestrus ovis) y un género de protozoo (Eimeria spp.). Además, el 17,85% de los ejemplares eran positivos para Mycobacterium spp. A partir de ahí se procedió a comparar la prevalencia, intensidad y abundancia de parásitos en los animales positivos para la tuberculosis con respecto a los negativos. Se observaron diferencias estadísticamente significativas entre las cabras que estaban infectadas por Mycobacterium spp. y las que no lo estaban en cuanto a la prevalencia de nemátodos pulmonares y gastrointestinales. Los autores examinan esta infección concomitante entre micobacteriáceas y endoparásitos.

Besnoitia besnoiti infections activate primary bovine endothelial cells and promote PMN adhesion and NET formation under physiological flow condition.

Besnoitia besnoiti is an obligate intracellular and emerging coccidian parasite of cattle that mainly infects host endothelial cells during acute infection. We here analyzed early innate immune reactions of B. besnoiti-infected primary bovine umbilical vein endothelial cells (BUVEC). B. besnoiti infections significantly activated BUVEC since the gene transcripts of several adhesion molecules (P-selectin, intercellular adhesion molecule 1(ICAM-1)), chemokines (CXCL1, CXCL8, CCL5), and of COX-2 were significantly upregulated during in vitro infection. Overall, the highest upregulation of most transcripts was observed at 24 or 48 h post infection (p.i.). Enhanced adhesion molecule expression in infected host cells was confirmed by PMN adhesion assays being performed under physiological flow conditions revealing a significantly increased PMN adhesion on B. besnoiti-infected BUVEC layers at 24 h p.i. Furthermore, we were able to illustrate neutrophil extracellular traps (NETs) being released by PMN under physiological flow conditions after adhesion to B. besnoiti-infected BUVEC layers. The present study shows that B. besnoiti infections of primary BUVEC induce a cascade of pro-inflammatory reactions and triggers early innate immune responses.

Metabolic signatures of Besnoitia besnoiti-infected endothelial host cells and blockage of key metabolic pathways indicate high glycolytic and glutaminolytic needs of the parasite.

Besnoitia besnoiti is an obligate intracellular and emerging coccidian parasite of cattle with a significant economic impact on cattle industry. During acute infection, fast-proliferating tachyzoites are continuously formed mainly in endothelial host cells of infected animals. Given that offspring formation is a highly energy and cell building block demanding process, the parasite needs to exploit host cellular metabolism to meet its metabolic demands. Here, we analyzed the metabolic signatures of B. besnoiti-infected endothelial host cells and aimed to influence parasite proliferation by inhibitors of specific metabolic pathways. The following inhibitors were tested: fluoro 2-deoxy-D-glucose and 2-deoxy-D-glucose (FDG, DG; inhibitors of glycolysis), 6-diazo-5-oxo-L-norleucin (DON; inhibitor of glutaminolysis), dichloroacetate (DCA; inhibitor of pyruvate dehydrogenase kinase which favorites channeling of glucose carbons into the TCA cycle) and adenosine-monophosphate (AMP; inhibitor of ribose 5-P synthesis). Overall, B. besnoiti infections of bovine endothelial cells induced a significant and infection rate-dependent increase of glucose, lactate, glutamine, glutamate, pyruvate, alanine, and serine conversion rates which together indicate a parasite-triggered up-regulation of glycolysis and glutaminolysis. Thus, addition of DON, FDG, and DG into the cultivation medium of B. besnoiti infected endothelial cells led to a dose-dependent inhibition of parasite replication (4 µM DON, 99.5 % inhibition; 2 mM FDG, 99.1 % inhibition; 2 mM DG, 93 % inhibition; and 8 mM DCA, 71.9 % inhibition). In contrast, AMP had no significant effects on total tachyzoite production up to a concentration of 20 mM. Together, these data may open new strategies for the development of therapeutics for B. besnoiti infections.

Targeting essential Eimeria ninakohlyakimovae sporozoite ligands for caprine host endothelial cell invasion with a phage display peptide library.

Eimeria ninakohlyakimovae is an important coccidian parasite of goats which causes severe diarrhoea in young animals. Specific molecules that mediate E. ninakohlyakimovae host interactions and molecular mechanisms involved in the pathogenesis are still unknown. Although strong circumstantial evidence indicates that E. ninakohlyakimovae sporozoite interactions with caprine endothelial host cells (ECs) are specific, hardly any information is available about the interacting molecules that confer host cell specificity. In this study, we describe a novel method to identify surface proteins of caprine umbilical vein endothelial cells (CUVEC) using a phage display library. After several panning rounds, we identified a number of peptides that specifically bind to the surface of CUVEC. Importantly, caprine endothelial cell peptide 2 (PCEC2) and PCEC5 selectively reduced the infection rate by E. ninakohlyakimovae sporozoites. These preliminary data give new insight for the molecular identification of ligands involved in the interaction between E. ninakohlyakimovae sporozoites and host ECs. Further studies using this phage approach might be useful to identify new potential target molecules for the development of anti-coccidial drugs or even new vaccine strategies.

Barriers in implementing research among registered nurses working in the care of the elderly: a multicenter study in Spain.

OBJECTIVE: This study identified barriers to the utilization of research results perceived by nurses who work in nursing homes in Spain. METHODS: An observational, cross-sectional, descriptive, and multicentre study was conducted in 126 nursing homes in different Spanish cities. The BARRIERS to Research Utilization Scale (BARRIERS scale) was used to identify barriers. RESULTS: A total of 756 nurses responded (92.48%). BARRIERS scale variables with the highest scores included Characteristics of the organization (mean=24.89, SD=4.37), followed by Professional features (mean=21.87, SD=4.85). The specific barriers that were rated the highest included "not enough time on the job to implement new ideas" (mean=3.89, SD=0.98), followed by "unknown nursing research" (mean=2.75; SD=1.22) and "Doctors do not cooperate in the implementation" (mean=3.01, SD=1.85). CONCLUSIONS: Geriatric nurses perceive time as the main barrier to implementing the results of research in practice. The number and nature of the barriers are consistent with studies from other countries. Knowledge of the barriers is crucial for institutions and educators to instigate measures that improve the implementation of nursing research, especially in an area like elderly care. To our knowledge, this is the first study conducted among geriatric nurses in Spain.

Gastrointestinal parasite fauna of Emperor Penguins (Aptenodytes forsteri) at the Atka Bay, Antarctica.

In general, the knowledge on parasites infecting Antarctic birds is scarce. The present study intends to extend the knowledge on gastrointestinal parasites of Emperor Penguins (Aptenodytes forsteri) at the Atka Bay, Antarctica. Fecal samples of 50 individual Emperor Penguins were collected at the Atka Bay and analyzed using the sodium-acetate-formaldehyde (SAF) method for the identification of intestinal helminth eggs and/or protozoan parasite stages. In addition, coproantigen ELISAs were performed to detect Cryptosporidium and Giardia infections. Overall, 13 out of 50 penguins proved parasitized (26%). The following stages of gastrointestinal parasites were identified: One Capillaria sp. egg, Tetrabothrius spp. eggs, Diphyllobothrium spp. eggs, and proglottids of the cestode Parorchites zederi. The recorded Capillaria infection represents a new host record for Emperor Penguins. All coproantigen ELISAs for the detection of Cryptosporidium spp. and Giardia spp. were negative. This paper provides current data on parasites of the Emperor Penguin, a protected endemic species of the Antarctica.

Gastrointestinal parasites of free-living Indo-Pacific bottlenose dolphins (Tursiops aduncus) in the Northern Red Sea, Egypt.

The present study represents the first report on the gastrointestinal parasite fauna infecting the free-living and alive Indo-Pacific bottlenose dolphins (Tursiops aduncus) inhabiting waters of the Red Sea at Hurghada, Egypt. A total of 94 individual faecal samples of the examined bottlenose dolphins were collected during several diving expeditions within their natural habitats. Using classical parasitological techniques, such as sodium acetate acetic acid formalin method, carbol fuchsin-stained faecal smears, coproantigen ELISA, PCR and macroscopical analyses, the study revealed infections with 21 different parasite species belonging to protozoans and metazoans with some of them bearing zoonotic and/or pathogenic potential. Four identified parasite species are potential zoonotic species (Giardia spp., Cryptosporidium spp., Diphyllobothrium spp., Ascaridida indet.); three of them are known to have high pathogenic potential for the examined dolphin species (Nasitrema attenuata, Zalophotrema spp. and Pholeter gastrophilus) and some appear to be directly associated with stranding events. In detail, the study indicates stages of ten protozoan species (Giardia spp., Sarcocystis spp., Isospora (like) spp., Cystoisospora (like) spp., Ciliata indet. I and II, Holotricha indet., Dinoflagellata indet., Hexamita (like) spp., Cryptosporidium spp.), seven trematode species (N. attenuata, Nasitrema spp. I and II, Zalophotrema curilensis, Zalophotrema spp., Pholeter gastrophilus, Trematoda indet.), one cestode species (Diphyllobothrium spp.), two nematode species (Ascaridida indet, Capillaria spp.) and one crustacean parasite (Cymothoidae indet.). Additionally, we molecularly identified adult worms of Anisakis typica in individual dolphin vomitus samples by molecular analyses. A. typica is a common parasite of various dolphin species of warmer temperate and tropical waters and has not been attributed as food-borne parasitic zoonoses so far. Overall, these parasitological findings include ten new host records for T. aduncus (i.e. in case of Giardia spp., Sarcocystis spp., Cryptosporidium spp., Nasitrema spp., Zalophotrema spp., Pholeter gastrophilus, A. typica, Capillaria spp., Diphyllobothrium spp. and Cymothoidae indet.). The present results may be used as a baseline for future monitoring studies targeting the impact of climate or other environmental changes on dolphin's health conditions and therefore contribute to the protection of these fascinating marine mammals.

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.

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.

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.

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.

Bovine recombinant IFNgamma induces endothelial cell gene transcription of immunoregulatory molecules and upregulates PMN and PBMC adhesion on bovine endothelial cells.

Interferon gamma (IFNgamma) is an important modulator of immune responses acting on multiple cell types, such as lymphocytes, macrophages or endothelial cells. We investigated the effects of recombinant bovine IFNgamma on bovine umbilical vein endothelial cells (BUVEC) for the level of polymorphonuclear neutrophil cell (PMN)- and peripheral blood mononuclear cell (PBMC)-adhesion as well as the gene transcription of endothelial cell-derived adhesion molecules (E-selectin, P-selectin, VCAM-1, ICAM-1), chemokines (CXCL1, CXCL8, CXCL10, CCL2, CCL5), GM-CSF, iNOS and COX-2 in comparison to TNFalpha-stimulation. IFNgamma strongly induced PMN and PBMC adhesion on BUVEC involving CD4(+), CD8(+) and gammadelta-TCR(+) (WC1(+)) lymphocytes. Furthermore, IFNgamma-stimulation led to a strong upregulation in the transcription of VCAM-1, ICAM-1, CXCL10 and CCL2 genes and to a low to moderate increase in the E- and P-selectin, CXCL1, CXCL8, CCL5, COX-2 and iNOS gene transcripts, but failed to enhance GM-CSF gene transcription. These results indicate that IFNgamma can be considered an important activator of endothelial cells in the bovine system, most probably by influencing the outcome of inflammatory responses through selective upregulation of immunoregulatory molecules.

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.

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.

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.