Carlos Hermosilla

T cell responses in calves to a primary Eimeria bovis infection: phenotypical and functional changes

The study aimed to characterize T cell responses in calves to a primary E. bovis infection. For this purpose, peripheral blood lymphocytes (PBL) were isolated from six infected calves and three controls during prepatency (Day 12 post infection (p.i.), patency (Day 25 p.i.) and postpatency (Day 35 p.i.). In addition, lymphocytes were isolated from various lymphatic organs (lnn. cervicales superficiales, lnn. jejunales craniales, lnn. jejunales caudales, lnn. caecales, lnn. colici, Peyers patches (PP) and spleen) at necropsy (Day 35 p.i.). FACS analyses determined the proportions of CD4+-, CD8+-, CD2+-, and gammadelta+-T cells. Proliferative responses of the cells after stimulation with Concanavalin A (Con A) and an E. bovis-merozoite I antigen (EbAg) were measured. Furthermore, in situ hybridization experiments were performed for the detection of IL-2 and IL-4 mRNA in histological sections of lymphatic organs. Proportions of CD4+-, CD8+- and CD2+-expressing PBL were significantly increased 12 days p.i. in infected calves. While the proportions of CD4+- and CD8+-PBL declined until day 25 p.i. and finally reached control values, proportions of activated PBL (CD2+-T cells) remained at a high level throughout the observation period. Those of gammadelta+-PBL, in contrast, remained unaffected. The proportions of CD4+-, gammadelta+- and CD2+-T cells in lymphatic organs were significantly increased in comparison to uninfected controls, when determined 35 days p.i. Concerning the proportions of CD8+-T cells of the organs, however, there were no differences between the groups. PBL and cells from lymphatic organs except those from the PP showed strong proliferative response to the mitogen Con A, without a significant difference between the groups. Reactions to EbAg in contrast differed significantly between controls and E. bovis infected calves. Proliferation responses of PBL of infected animals were highest 12 days p.i.; subsequently they decreased and 35 days p.i. they were found within the ranges of controls. Lymphocytes isolated from lymphatic organs of infected animals reacted significantly stronger than lymphocytes from control animals, whereby most marked differences occured with cells from lymph nodes draining E. bovis infested parts of the intestine and from the spleen. These reactions were accompained by an increased transcription of the IL-2 gene but not of the IL-4 gene in gut associated lymphnodes of infected calves when compared with infected controls. The data suggest strong antigenic stimuli by developing first generation schizonts, and of predominant involvement of (CD4+) Th1 cells in the course of a primary E. bovis infection of calves.

The intriguing host innate immune response: novel anti-parasitic defence by neutrophil extracellular traps

The capacity of polymorphonuclear neutrophils (PMN) and other leucocytes of the innate immune system to expel their DNA in a controlled process into the extracellular environment to trap and kill pathogenic microorganisms led to a paradigm shift in our comprehension of host leucocyte-pathogen interactions. Formation of neutrophil extracellular traps (NETs) has recently been recognized as a novel effector mechanism of the host innate immune response against microbial infections. Meanwhile evidence has arisen that NET formation is a widely spread mechanism in vertebrates and invertebrates and extends not only to the entrapment of microbes, fungi and viruses but also to the capture of protozoan and metazoan parasites. PMN produce NETs after stimulation with mitogens, cytokines or pathogens in a controlled process which depends on reactive oxygen species (ROS) and the induction of the Raf-MEK-ERK-mediated signalling pathway cascade. NETs consist of nuclear DNA as a backbone decorated with histones, antimicrobial peptides, and PMN-specific granular enzymes thereby providing an extracellular matrix capable of entrapping and killing invasive pathogens. This review is intended to summarize parasite-related data on NETs. Special attention will be given to NET-associated mechanisms by which parasites, in particular apicomplexa, might be hampered in their ability to reproduce within the host cell and complete the life cycle.

First autochthonous case of canine ocular Dirofilaria repens infection in Germany

THE genus Dirofilaria includes two species of veterinary importance: Dirofilaria immitis, a parasite of the cardiovascular system, and Dirofilaria repens, a parasite of the subcutaneous tissue of dogs, cats and other carnivores (Pampiglione and others 1991, 1995, Favia and others 1996, Pampiglione and Rivasi 2000). Both species are usually found in areas with warm climates. This short communication describes the first case of obviously autochthonous D repens infection in a dog in Germany. A two-year-old, female German longhair pointer, which had spent its entire life in the southern part of Baden Wuerttemberg in Germany, was admitted to a small animal clinic in July 2004. Clinical examination revealed conjunctivitis in the left eye. A granulomatous nodule, 5 x 3 mm in size, was present in the dorsal bulbar conjunctiva; other parts of the eye were not affected. The conjunctival nodule was removed surgically, and a milky-white nematode was recovered from the extracted tissue. The dog was serologically negative by commercial tests for antibodies to Leishmania species (FLUOLEISH; MegaCor) and Ehrlichia canis (FLUOEHRLICHIA; MegaCor), and no circulating D immitis antigen was found (DiroCheck; Synbiotics, FASTest HW Antigen; MegaCor). No Babesia canis DNA was detected in a sample of blood by PCR, and no microfilariae were observed in blood samples using the Knott method (Sloss and Kemp 1978). The isolated worm was a gravid, female nematode 15 cm long, with prominent longitudinal cuticular ridges. The vulva was located in the anterior part of the body, and the uteri were filled with embryonated eggs and microfilariae (Fig 1a). Free microfilariae were unsheathed, 7·5 μm wide and 230 μm long, and generally showed a coiled body; the anterior end was rounded and the caudal end was hook-like in appearance (Fig 1b). Based on the morphologies of the female nematode and the microfilariae, the worm was identified as D repens. PCR was performed on extracted nematode DNA using highly repetitive nucleotide sequences of D repens DNA (Chandrasekharan and others 1994) and highly conserved nucleotide sequences of D immitis DNA (Favia and others 1996) as primers (Table 1). The PCR reaction mixture was incubated at 94°C for two minutes and was then subjected to 35 cycles of 30 seconds at 94°C, 30 seconds at 60°C and 45 seconds at 72°C, and then kept for 5 minutes at 72°C. The amplification products were separated electrophoretically on 2 per cent agarose gel and visualised under ultraviolet light. The PCR produced an amplification product of an expected size, 175 base pairs, for D repens, and was negative for D immitis (Fig 2). Subcutaneous infections with D repens in dogs and cats have been reported from southern European countries such as Spain (Cancrini and others 2000), Italy (Pampiglione and others 1995, Cancrini and others 2003) and Greece (Vakalis and Himonas 1997). To date, the most northern autochthonous D repens infections of dogs have been reported from Tessin, Switzerland (Bucklar and others 1998), the Aosta Valley, Italy (Tarello 2003) and Tours, France (Chauve 1997). To the best of the authors’ knowledge, autochthonous D repens infections have not previously been observed in central or northern Europe. The case described here seems to be the northernmost autochthonous D repens infection recorded so far. Limiting factors in the geographical spread of D repens are the availability of mosquitoes as intermediate hosts, and suitable environmental temperatures. A minimum average temperature of 18°C is necessary for the development of D immitis larvae in mosquitoes (Abraham 1988). In the geographical area where the dog lives, susceptible mosquitoes (Aedes, Anopheles and Culex species) are abundant (Dahl and others 1999), and the average temperature during the summer months varies between 14·6°C in September and 18·3°C in July (Anon 2005). Veterinary Record (2006) 158, 134-135

Neutrophil Extracellular Traps as Innate Immune Reaction against the Emerging Apicomplexan Parasite Besnoitia besnoiti

Besnoitia besnoiti infection in cattle is an important emerging protozoan disease in Europe causing economic losses and severe clinical signs, such as generalized dermatitis, orchitis, and vulvitis in affected animals. Neutrophil extracellular trap (NET) formation was recently demonstrated as an important effector mechanism of PMN acting against several invading pathogens. In the present study, interactions of bovine PMN with tachyzoites of B. besnoiti were investigated in this respect in vitro. For the demonstration and quantification of NETs, extracellular DNA was stained by Sytox Orange or Pico Green. Fluorescent illustrations as well as scanning electron microscopy analyses (SEM) showed PMN-promoted NET formation rapidly being induced upon contact with B. besnoiti tachyzoites. Co-localization of extracellular DNA with histones, neutrophil elastase (NE) and myeloperoxidase (MPO) in parasite entrapping structures confirmed the classical characteristics of NET. Exposure of PMN to viable, UV attenuated and dead tachyzoites showed a significant induction of NET formation, but even tachyzoite homogenates significantly promoted NETs when compared to negative controls. NETs were abolished by DNase treatment and were reduced after PMN preincubation with NADPH oxidase-, NE- and MPO-inhibitors. Tachyzoite-triggered NET formation led to parasite entrapment as quantitative assays indicated that about one third of tachyzoites were immobilized in NETs. In consequence, tachyzoites were hampered from active invasion of host cells. Thus, transfer of tachyzoites, previously being confronted with PMN, to adequate host cells resulted in significantly reduced infection rates when compared to PMN-free infection controls. To our knowledge, we here report for the first time B. besnoiti-induced NET formation. Our results indicate that PMN-triggered extracellular traps may represent an important effector mechanism of the host early innate immune response against B. besnoiti which may lead to diminishment of initial parasite infection rates during the acute infection phase.

Eimeria bovis-triggered neutrophil extracellular trap formation is CD11b-, ERK 1/2-, p38 MAP kinase- and SOCE-dependent

Eimeria bovis is an important coccidian parasite that causes high economic losses in the cattle industry. We recently showed that polymorphonuclear neutrophils (PMN) react upon E. bovis sporozoite exposure by neutrophil extracellular trap (NET) formation. We focused here on the molecular mechanisms that are involved in this process. The sporozoite encounter led to an enhanced surface expression of neutrophil CD11b suggesting a potential role of this receptor in E. bovis-mediated NETosis. Antibody-mediated blockage of CD11b confirmed this assumption and led to a significantly decreased sporozoite-triggered NET. In addition, E. bovis-induced NETosis was found to be Ca2+-dependent since the inhibition of store-operated calcium entry (SOCE) significantly diminished NET. Furthermore, NADPH oxidase, neutrophil elastase (NE) and myeloperoxidase (MPO) were confirmed as key molecules in sporozoite-triggered NETosis, as inhibition thereof blocked parasite-triggered NET. PMN degranulation analyses revealed a significant release of matrix metalloprotease-9 containing granules upon sporozoite exposure. We further show a significantly enhanced phosphorylation of ERK1/2 and p38 MAPK in sporozoite-exposed PMN indicating a key role of this signaling pathway in E. bovis-mediated NETosis. Accordingly, ERK 1/2 and p38 MAPK inhibition led to a significant decrease in NET formation. Finally, we demonstrate that sporozoite-induced NETosis is neither a stage-, species-, nor host-specific process.

Microarray-based transcriptional profiling of Eimeria bovis-infected bovine endothelial host cells

Within its life cycle Eimeria bovis undergoes a long lasting intracellular development into large macromeronts in endothelial cells. Since little is known about the molecular basis of E. bovis-triggered host cell regulation we applied a microarray-based approach to define transcript variation in bovine endothelial cells early after sporozoite invasion (4 h post inoculation (p.i.)), during trophozoite establishment (4 days p.i.), during early parasite proliferation (8 days p.i.) and towards macromeront maturation (14 days p.i.). E. bovis infection led to significant changes in the abundance of many host cell gene transcripts. As infection progressed, the number of regulated genes increased such that 12, 45, 175 and 1184 sequences were modulated at 4 h, 4, 8 and 14 days p.i., respectively. These genes significantly interfered with several host cell functions, networks and canonical pathways, especially those involved in cellular development, cell cycle, cell death, immune response and metabolism. The correlation between stage of infection and the number of regulated genes involved in different aspects of metabolism suggest parasite-derived exploitation of host cell nutrients. The modulation of genes involved in cell cycle arrest and host cell apoptosis corresponds to morphological in vitro findings and underline the importance of these aspects for parasite survival. Nevertheless, the increasing numbers of modulated transcripts associated with immune responses also demonstrate the defensive capacity of the endothelial host cell. Overall, this work reveals a panel of novel candidate genes involved in E. bovis-triggered host cell modulation, providing a valuable tool for future work on this topic.

Monocyte- and macrophage-mediated immune reactions against Eimeria bovis

Innate immune reactions conducted by macrophages may affect the outcome of primary infections and are crucial for the transition to adaptive immune responses. In bovine coccidiosis little is known on early monocyte/macrophage-mediated responses. We therefore investigated in vivo, in vitro and ex vivo reactions of monocytes and macrophages against Eimeria bovis, one of the most pathogenic Eimeria species in cattle. Macrophages significantly infiltrate the gut mucosa of E. bovis-infected calves, particularly after challenge infection. Furthermore, peripheral monocytes of infected animals, as precursor cells of macrophages, exhibited enhanced ex vivo phagocytic and oxidative burst activities. Enhanced levels of both activities were found early after infection and towards the end of first merogony. In vitro exposure of macrophages to sporozoites led to phagocytosis of the pathogen, whilst monocytes failed to do so. Phagocytosis occurred independently of the viability of the sporozoites, indicating that active invasion by the parasites was negligible. Phagocytosis occurred in the absence of immune serum, but could clearly be enhanced by addition of immune serum, suggesting macrophage-derived antibody-dependent cytotoxicity. Furthermore, co-culture of macrophages with sporozoites and stimulation with merozoite I antigen induced distinct levels of cytokine and chemokine gene transcription. Thus, the transcription of genes encoding for IFN-gamma, IL-12, TNF-alpha, IL-6, CXCL1, CXCL8, CXCL10 and COX-2 was upregulated after sporozoite encounter. In contrast, soluble merozoite I antigen only induced the gene transcription of IL-6 and IL-12 and failed to upregulate IFN-gamma and TNF-alpha gene transcripts. In monocytes, IFN-gamma and CXCL10 were found upregulated, all other immunoregulatory molecules tested were not affected. In summary, our results strongly suggest that macrophage-mediated, innate immune reactions play an important role in the early immune response to E. bovis infections in calves.

Harbour seal (Phoca vitulina) PMN and monocytes release extracellular traps to capture the apicomplexan parasite Toxoplasma gondii.

Extracellular traps (ETs) are composed of nuclear DNA as backbone adorned with histones, cytoplasmic antimicrobial peptides/proteins which are released from a range of vertebrate and invertebrate host immune cells in response to several invading pathogens. Until now this ancient novel innate defence mechanism has not been demonstrated in any marine mammal. Interactions of harbour seal (Phoca vitulina)-PMN and -monocytes with viable tachyzoites of Toxoplasma gondii were investigated in this respect in vitro. For the demonstration and quantification of harbour seal PMN- and monocyte-derived ETs, extracellular DNA was stained with Sytox Orange. Fluorescence assays as well as scanning electron microscopy (SEM) analyses demonstrated PMN- and monocyte-promoted ET formation rapidly being induced upon contact with T. gondii-tachyzoites. The co-localisation of extracellular DNA decorated with histones (H3), neutrophil elastase (NE) and myeloperoxidase (MPO) in parasite entrapping structures confirmed the classical characteristics of PMN- and monocyte-promoted ETs. Exposure of harbour seal PMN and monocytes to viable tachyzoites resulted in a significant induction of ETs when compared to negative controls. Harbour seal-ETs were efficiently abolished by DNase I treatment and were reduced after PMN and monocytes pre-incubation with the NADPH oxidase inhibitor diphenilane iodondium. Tachyzoites of T. gondii were firmly entrapped and immobilised within harbour seal-ET structures. To our best knowledge, we here report for the first time on T. gondii-induced ET formation in harbour seal-PMN and -monocytes. Our results strongly indicate that PMN- and monocyte-triggered ETs represent a relevant and ancient conserved effector mechanism of the pinniped innate immune system as reaction against the pathogenic protozoon T. gondii and probably against other foreign pathogens occurring in the ocean environment.

Angiostrongylus vasorum in Great Britain: a nationwide postal questionnaire survey of veterinary practices

The lungworm, Angiostrongylus vasorum, was first reported in indigenous dogs in southwestern England in 1980 and has since been recorded in Wales, southeastern England and, more recently, in the West Midlands, northern England and Scotland. The nationwide distribution of the parasite was evaluated using a postal questionnaire sent to 3950 small animal practices during 2009. Information was sought on the location of each practice, awareness of the parasite locally, number of cases diagnosed over the past year and whether diagnosis was based on clinical signs alone or supported by additional tests. 1419 practices returned a usable response, the majority being located in a city/town. Nearly one-third of responding practices were aware of the parasite locally, 20.7 per cent had seen at least one confirmed case and 0.3 per cent >20 confirmed cases over the past year. The most widely used tests were faecal examination and any type of imaging. Existing clusters of infection were detected in southeastern England and south Wales; infection was also found to be widespread in central England, though patchy in northern England and Scotland. Using distribution of clinical cases as an indicator of parasite distribution, this study confirmed that A. vasorum has spread beyond traditional UK endemic foci.

NADPH oxidase, MPO, NE, ERK1/2, p38 MAPK and Ca2+ influx are essential for Cryptosporidium parvum-induced NET formation.

Cryptosporidium parvum causes a zoonotic infection with worldwide distribution. Besides humans, cryptosporidiosis affects a wide range of animals leading to significant economic losses due to severe enteritis in neonatal livestock. Neutrophil extracellular trap (NET) formation has been demonstrated as an important host effector mechanism of PMN acting against several invading pathogens. In the present study, C. parvum-mediated NET formation was investigated in human and bovine PMN in vitro. We here demonstrate that C. parvum sporozoites indeed trigger NET formation in a time-dependent manner. Thereby, the classical characteristics of NETs were demonstrated by co-localization of extracellular DNA with histones, neutrophil elastase (NE) and myeloperoxidase (MPO). A significant reduction of NET formation was measured following treatments of PMN with NADPH oxidase-, NE- and MPO-inhibitors, confirming the key role of these enzymes in C. parvum-induced NETs. Additionally, sporozoite-triggered NETosis revealed as dependent on intracellular Ca(++) concentration and the ERK 1/2 and p38 MAPK-mediated signaling pathway. Moreover, sporozoite-triggered NET formation led to significant parasite entrapment since 15% of the parasites were immobilized in NET structures. Consequently, PMN-pre-exposed sporozoites showed significantly reduced infectivity for epithelial host cells confirming the capability of NETs to prevent active parasite invasion. Besides NETs, we here show that C. parvum significantly up-regulated CXCL8, IL6, TNF-α and of GM-CSF gene transcription upon sporozoite confrontation, indicating a pivotal role of PMN not only in the bovine and human system but most probably in other final hosts for C. parvum.