Continuous use of anticoccidial treatments against Eimeria infections has resulted in the develop... more Continuous use of anticoccidial treatments against Eimeria infections has resulted in the development of drug resistance. This study aimed to evaluate the anticoccidial efficacy of a methanolic extract derived from the endemic Canary rue (Ruta pinnata) plant of the Canary Islands, Spain, against Eimeria ninakohlyakimovae using in vitro assays. Freshly unsporulated oocysts were exposed to different concentrations of R. pinnata extract and thereafter evaluated for sporulation inhibition. Additionally, anticoccidial activity was examined by testing the viability of the E. ninakohlyakimovae sporozoites and their ability to infect bovine colonic epithelial cells after incubation with different concentrations of R. pinnata plant extract. The inhibition of oocyst sporulation by the extract was both time and concentration dependent, with certain combinations affording the same levels of sporulation inhibition as formaldehyde used as positive control (P < 0.001). Moreover, concentrations ...
Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-caus... more Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-cause of diarrhoea-induced mortality in children. In contrast to other apicomplexans, C.parvum has minimalistic metabolic capacities which are almost exclusively based on glycolysis. Consequently, C. parvum is highly dependent on its host cell metabolism. In vivo (within the intestine) infected epithelial host cells are typically exposed to low oxygen pressure (1–11% O2, termed physioxia). Here, we comparatively analyzed the metabolic signatures of C. parvum-infected HCT-8 cells cultured under both, hyperoxia (21% O2), representing the standard oxygen condition used in most experimental settings, and physioxia (5% O2), to be closer to the in vivo situation. The most pronounced effect of C. parvum infection on host cell metabolism was, on one side, an increase in glucose and glutamine uptake, and on the other side, an increase in lactate release. When cultured in a glutamine-deficient medium,...
Professional mononuclear phagocytes such as polymorphonuclear neutrophils (PMN), monocytes, and m... more Professional mononuclear phagocytes such as polymorphonuclear neutrophils (PMN), monocytes, and macrophages are considered as the first line of defence against invasive pathogens. The formation of extracellular traps (ETs) by activated mononuclear phagocytes is meanwhile well accepted as an effector mechanism of the early host innate immune response acting against microbial infections. Recent investigations showed evidence that ETosis is a widely spread effector mechanism in vertebrates and invertebrates being utilized to entrap and kill bacteria, fungi, viruses, and protozoan parasites. ETs are released in response to intact protozoan parasites or to parasite-specific antigens in a controlled cell death process. Released ETs consist of nuclear DNA as backbone adorned with histones, antimicrobial peptides, and phagocyte-specific granular enzymes thereby producing a sticky extracellular matrix capable of entrapping and killing pathogens. This review summarizes recent data on protozoa...
To determine whether the human spermatozoon is a sufficient stimulus to trigger the release of ne... more To determine whether the human spermatozoon is a sufficient stimulus to trigger the release of neutrophil extracellular traps (NETs) in a time- and dose-dependent manner. Experimental study. University-based laboratory. Semen samples from four men and blood samples from six healthy female donors. Polymorphonuclear neutrophils (PMN) isolated from peripheral blood were incubated with fresh human spermatozoa for 60, 90, 120, and 180 minutes and at different PMN/sperm concentrations (1:1 [25 × 10(4)], 1:3 [25 × 10(4):75 × 10(4)], 1:6 [25 × 10(4):15 × 10(5)], 1:18 [25 × 10(4):45 × 10(5)]). During coincubation of PMN/sperm, the release of NETs was measured by PicoGreen. Immunofluorescence for histone H3, neutrophil elastase (NE), and myeloperoxidase (MPO) was performed. Different NETs inhibitors were tested: diphenylene iodonium, Suc-Ala- Ala-Pro-Val chloromethyl ketone (CMK), and 4-aminobenzoic acid hydrazide (ABAH) inhibitors of NADPH oxidase, NE, and MPO. Progressive mobility was assessed at increasing doses of neutrophils (1:18 [25 × 10(4):45 × 10(5)], 6:18 [15 × 10(5):45 × 10(5)], 9:18 [252 × 10(4):45 × 10(5)]). The quantity of NETs increased at the ratio of 1:6 after 2 hours and continued to increase subsequently. A ratio of 1:18 showed significant increases in NETs production at all times. Assessment of the inhibitors showed that CMK and ABAH inhibit NETs formation. Scanning and transmission electron microphotographs and immunofluorescence confirmed NETs formation induced by the spermatozoa. After 1 hour, progressive motility diminished in the two groups with the highest proportion of neutrophils and after 2 hours in all groups exposed to neutrophils. We show that the stimulus of the human spermatozoon triggers the release of NETs; this response is dose dependent and increases with exposure time. The motility of affected spermatozoa diminishes, suggesting that this interaction on a larger scale would decrease the probability of successful fertilization.
The apicomplexan parasite Eimeria ovinoidalis is distributed worldwide and causes clinical ovine ... more The apicomplexan parasite Eimeria ovinoidalis is distributed worldwide and causes clinical ovine coccidiosis. As one of the most pathogenic species in sheep, the principal clinical sign is profuse diarrhoea in young animals, which leads to important economic losses in the ovine industry. We here aimed to establish an in vitro culture system for the development of E. ovinoidalis macromeronts, as no suitable systems are currently available for any ovine Eimeria species. Faecal samples containing more than 90% of E. ovinoidalis oocysts were collected from naturally infected lambs and ewes in Murcia Region (Spain). E. ovinoidalis oocysts were collected, left to sporulate in potassium dichromate and stored at 4°C until further studies were conducted. Moreover, a suitable excystation protocol was effectively established, resulting in the release of viable sporozoites, which were allowed to infect primary bovine umbilical vein endothelial cells (BUVEC) and permanent bovine colonic epitheli...
Nonesterified fatty acids (NEFAs) are involved in proinflammatory processes in cattle, including ... more Nonesterified fatty acids (NEFAs) are involved in proinflammatory processes in cattle, including in the increased expression of adhesion molecules in endothelial cells. However, the mechanisms underlying these effects are still unknown. The aim of this study was to assess the effects of NEFAs on the intracellular calcium (Ca(2+) i) influx, nitric oxide production, and ICAM-1 and IL-8 expression in primary bovine umbilical vein endothelial cells (BUVECs). Myristic (MA), palmitic (PA), stearic (SA), oleic (OA) and linoleic acid (LA) rapidly increased Ca(2+) i. The calcium response to all tested NEFAs showed an extracellular calcium dependence and only the LA response was significantly inhibited until the intracellular calcium was chelated. The EC50 values for MA and LA were 125 μM and 37 μM, respectively, and the MA and LA effects were dependent on calcium release from the endoplasmic reticulum stores and on the L-type calcium channels. Only the calcium response to MA was significantl...
International journal for parasitology. Parasites and wildlife, 2015
Baleen and sperm whales, belonging to the Order Cetartiodactyla, are the largest and heaviest exi... more Baleen and sperm whales, belonging to the Order Cetartiodactyla, are the largest and heaviest existent mammals in the world, collectively known as large whales. Large whales have been subjected to a variety of conservation means, which could be better monitored and managed if physiological and pathophysiological information, such as pathogen infections, could already be gathered from free-swimming animals instead of carcasses. Parasitic diseases are increasingly recognized for their profound influences on individual, population, and even ecosystem health. Furthermore, a number of parasite species have gained importance as opportunistic neozoan infections in the marine environment. Nonetheless, traditional approaches to study parasitic diseases have been impractical for large whales, since there is no current routine method for the capture and handling of these large animals and there is presently no practical method to obtain blood samples remotely from free-ranging whales. Therefor...
The present study represents the first report on the gastrointestinal parasite fauna infecting th... more 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, Zalop...
Eimeria ninakohlyakimovae is an important coccidian parasite of goats which causes severe hemorrh... more Eimeria ninakohlyakimovae is an important coccidian parasite of goats which causes severe hemorrhagic typhlocolitis in young animals, thereby leading to high economic losses in goat industry worldwide. The first merogony of E. ninakohlyakimovae occurs within host endothelial cells (ECs) of the lacteal capillaries of the villi of the distal ileum resulting in the formation of macromeronts (up to 170μm) within 10-12days post-infection (p.i.) and releasing >120,000 merozoites I. The E. ninakohlyakimovae-macromeront formation within highly immunoreactive host endothelial cells (ECs) should rely on several regulatory processes to fulfill this massive replication. Here host EC-parasite interactions were investigated to determine the extent of modulation carried out by E. ninakohlyakimovae in primary caprine umbilical vein endothelial cells (CUVEC) during the first merogony. Gene transcription of the adhesion molecule E-selectin and the cytokine TNF-α were significantly enhanced in the ...
Continuous use of anticoccidial treatments against Eimeria infections has resulted in the develop... more Continuous use of anticoccidial treatments against Eimeria infections has resulted in the development of drug resistance. This study aimed to evaluate the anticoccidial efficacy of a methanolic extract derived from the endemic Canary rue (Ruta pinnata) plant of the Canary Islands, Spain, against Eimeria ninakohlyakimovae using in vitro assays. Freshly unsporulated oocysts were exposed to different concentrations of R. pinnata extract and thereafter evaluated for sporulation inhibition. Additionally, anticoccidial activity was examined by testing the viability of the E. ninakohlyakimovae sporozoites and their ability to infect bovine colonic epithelial cells after incubation with different concentrations of R. pinnata plant extract. The inhibition of oocyst sporulation by the extract was both time and concentration dependent, with certain combinations affording the same levels of sporulation inhibition as formaldehyde used as positive control (P < 0.001). Moreover, concentrations ...
Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-caus... more Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-cause of diarrhoea-induced mortality in children. In contrast to other apicomplexans, C.parvum has minimalistic metabolic capacities which are almost exclusively based on glycolysis. Consequently, C. parvum is highly dependent on its host cell metabolism. In vivo (within the intestine) infected epithelial host cells are typically exposed to low oxygen pressure (1–11% O2, termed physioxia). Here, we comparatively analyzed the metabolic signatures of C. parvum-infected HCT-8 cells cultured under both, hyperoxia (21% O2), representing the standard oxygen condition used in most experimental settings, and physioxia (5% O2), to be closer to the in vivo situation. The most pronounced effect of C. parvum infection on host cell metabolism was, on one side, an increase in glucose and glutamine uptake, and on the other side, an increase in lactate release. When cultured in a glutamine-deficient medium,...
Professional mononuclear phagocytes such as polymorphonuclear neutrophils (PMN), monocytes, and m... more Professional mononuclear phagocytes such as polymorphonuclear neutrophils (PMN), monocytes, and macrophages are considered as the first line of defence against invasive pathogens. The formation of extracellular traps (ETs) by activated mononuclear phagocytes is meanwhile well accepted as an effector mechanism of the early host innate immune response acting against microbial infections. Recent investigations showed evidence that ETosis is a widely spread effector mechanism in vertebrates and invertebrates being utilized to entrap and kill bacteria, fungi, viruses, and protozoan parasites. ETs are released in response to intact protozoan parasites or to parasite-specific antigens in a controlled cell death process. Released ETs consist of nuclear DNA as backbone adorned with histones, antimicrobial peptides, and phagocyte-specific granular enzymes thereby producing a sticky extracellular matrix capable of entrapping and killing pathogens. This review summarizes recent data on protozoa...
To determine whether the human spermatozoon is a sufficient stimulus to trigger the release of ne... more To determine whether the human spermatozoon is a sufficient stimulus to trigger the release of neutrophil extracellular traps (NETs) in a time- and dose-dependent manner. Experimental study. University-based laboratory. Semen samples from four men and blood samples from six healthy female donors. Polymorphonuclear neutrophils (PMN) isolated from peripheral blood were incubated with fresh human spermatozoa for 60, 90, 120, and 180 minutes and at different PMN/sperm concentrations (1:1 [25 × 10(4)], 1:3 [25 × 10(4):75 × 10(4)], 1:6 [25 × 10(4):15 × 10(5)], 1:18 [25 × 10(4):45 × 10(5)]). During coincubation of PMN/sperm, the release of NETs was measured by PicoGreen. Immunofluorescence for histone H3, neutrophil elastase (NE), and myeloperoxidase (MPO) was performed. Different NETs inhibitors were tested: diphenylene iodonium, Suc-Ala- Ala-Pro-Val chloromethyl ketone (CMK), and 4-aminobenzoic acid hydrazide (ABAH) inhibitors of NADPH oxidase, NE, and MPO. Progressive mobility was assessed at increasing doses of neutrophils (1:18 [25 × 10(4):45 × 10(5)], 6:18 [15 × 10(5):45 × 10(5)], 9:18 [252 × 10(4):45 × 10(5)]). The quantity of NETs increased at the ratio of 1:6 after 2 hours and continued to increase subsequently. A ratio of 1:18 showed significant increases in NETs production at all times. Assessment of the inhibitors showed that CMK and ABAH inhibit NETs formation. Scanning and transmission electron microphotographs and immunofluorescence confirmed NETs formation induced by the spermatozoa. After 1 hour, progressive motility diminished in the two groups with the highest proportion of neutrophils and after 2 hours in all groups exposed to neutrophils. We show that the stimulus of the human spermatozoon triggers the release of NETs; this response is dose dependent and increases with exposure time. The motility of affected spermatozoa diminishes, suggesting that this interaction on a larger scale would decrease the probability of successful fertilization.
The apicomplexan parasite Eimeria ovinoidalis is distributed worldwide and causes clinical ovine ... more The apicomplexan parasite Eimeria ovinoidalis is distributed worldwide and causes clinical ovine coccidiosis. As one of the most pathogenic species in sheep, the principal clinical sign is profuse diarrhoea in young animals, which leads to important economic losses in the ovine industry. We here aimed to establish an in vitro culture system for the development of E. ovinoidalis macromeronts, as no suitable systems are currently available for any ovine Eimeria species. Faecal samples containing more than 90% of E. ovinoidalis oocysts were collected from naturally infected lambs and ewes in Murcia Region (Spain). E. ovinoidalis oocysts were collected, left to sporulate in potassium dichromate and stored at 4°C until further studies were conducted. Moreover, a suitable excystation protocol was effectively established, resulting in the release of viable sporozoites, which were allowed to infect primary bovine umbilical vein endothelial cells (BUVEC) and permanent bovine colonic epitheli...
Nonesterified fatty acids (NEFAs) are involved in proinflammatory processes in cattle, including ... more Nonesterified fatty acids (NEFAs) are involved in proinflammatory processes in cattle, including in the increased expression of adhesion molecules in endothelial cells. However, the mechanisms underlying these effects are still unknown. The aim of this study was to assess the effects of NEFAs on the intracellular calcium (Ca(2+) i) influx, nitric oxide production, and ICAM-1 and IL-8 expression in primary bovine umbilical vein endothelial cells (BUVECs). Myristic (MA), palmitic (PA), stearic (SA), oleic (OA) and linoleic acid (LA) rapidly increased Ca(2+) i. The calcium response to all tested NEFAs showed an extracellular calcium dependence and only the LA response was significantly inhibited until the intracellular calcium was chelated. The EC50 values for MA and LA were 125 μM and 37 μM, respectively, and the MA and LA effects were dependent on calcium release from the endoplasmic reticulum stores and on the L-type calcium channels. Only the calcium response to MA was significantl...
International journal for parasitology. Parasites and wildlife, 2015
Baleen and sperm whales, belonging to the Order Cetartiodactyla, are the largest and heaviest exi... more Baleen and sperm whales, belonging to the Order Cetartiodactyla, are the largest and heaviest existent mammals in the world, collectively known as large whales. Large whales have been subjected to a variety of conservation means, which could be better monitored and managed if physiological and pathophysiological information, such as pathogen infections, could already be gathered from free-swimming animals instead of carcasses. Parasitic diseases are increasingly recognized for their profound influences on individual, population, and even ecosystem health. Furthermore, a number of parasite species have gained importance as opportunistic neozoan infections in the marine environment. Nonetheless, traditional approaches to study parasitic diseases have been impractical for large whales, since there is no current routine method for the capture and handling of these large animals and there is presently no practical method to obtain blood samples remotely from free-ranging whales. Therefor...
The present study represents the first report on the gastrointestinal parasite fauna infecting th... more 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, Zalop...
Eimeria ninakohlyakimovae is an important coccidian parasite of goats which causes severe hemorrh... more Eimeria ninakohlyakimovae is an important coccidian parasite of goats which causes severe hemorrhagic typhlocolitis in young animals, thereby leading to high economic losses in goat industry worldwide. The first merogony of E. ninakohlyakimovae occurs within host endothelial cells (ECs) of the lacteal capillaries of the villi of the distal ileum resulting in the formation of macromeronts (up to 170μm) within 10-12days post-infection (p.i.) and releasing >120,000 merozoites I. The E. ninakohlyakimovae-macromeront formation within highly immunoreactive host endothelial cells (ECs) should rely on several regulatory processes to fulfill this massive replication. Here host EC-parasite interactions were investigated to determine the extent of modulation carried out by E. ninakohlyakimovae in primary caprine umbilical vein endothelial cells (CUVEC) during the first merogony. Gene transcription of the adhesion molecule E-selectin and the cytokine TNF-α were significantly enhanced in the ...
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Papers by Carlos Hermosilla