Horst Zahner

Neutrophil extracellular trap formation as innate immune reactions against the apicomplexan parasite Eimeria bovis.

Eimeria bovis infections are under immunological control and recent studies have emphasized the role of early PMN-mediated innate immune responses in infected calves. Neutrophil extracellular traps (NETs) have recently been demonstrated to act as a killing mechanism of PMN against several pathogens. In the present study, the interactions of bovine PMN with sporozoites of E. bovis were investigated in this respect in vitro. For demonstration and quantification of NET formation, extracellular DNA was stained by Sytox Orange. Fluorescence images after Sytox Orange staining as well as scanning electron microscopy (SEM) showed NET formation to occur upon contact with E. bovis sporozoites. Exposure of PMN to viable sporozoites induced stronger NET formation than to dead or homogenized parasites. NET formation was abolished by treatment with DNase and could be reduced by diphenylene iodonium, which is described as a potent inhibitor of NADPH oxidase. After sporozoite and PMN co-culture, extracellular fibres were found attached to sporozoites and seemed to trap them, strongly suggesting that NETs immobilize E. bovis sporozoites and thereby prevent them from infecting host cells. Thus, transfer of sporozoites, previously being confronted with PMN, to adequate host cells resulted in clearly reduced infection rates when compared to PMN-free controls. NET formation by PMN may therefore represent an effector mechanism in early innate immune reactions against E. bovis. This is the first report indicating Eimeria-induced NET formation.

Cutaneous leishmaniosis in a horse in southern Germany caused by Leishmania infantum.

This report describes a case of cutaneous leishmaniosis in a horse in southern Germany. Diagnosis is based on histopathology, immunohistochemistry and electron microscopy. The protozoan was identified as Leishmania infantum via PCR and restriction fragment length polymorphism. The horse did not show specific Leishmania antibodies. The lesions healed completely within 6 months without any specific treatment. Since neither the infected horse nor its dam had ever left their rural area, autochthonous infection in Germany cannot be excluded. Factors possibly influencing the epidemiological situation are discussed.

HSP70-controlled GFP expression in transiently transformed schistosomes☆

Among the parasitic helminths schistosomes are of high medical and economic importance. Despite of the world-wide relevance of this parasite, very little is known about the cellular mechanisms controlling its development and concerning the host-parasite interaction. Within the last decade a great effort has been made in this blood fluke to identify genes which play important roles during these processes. However, molecular analysis was limited by the fact, that neither function nor regulation of candidate genes could be investigated in this organism due to the lack of transformation protocols. Here, we present the strategy of ballistic gene transfer to introduce and characterize transgenes in different schistosome life stages. As a transformation vector, the heat shock protein 70 (hsp70) gene promoter and terminator from Schistosoma mansoni were cloned and fused to the green fluorescent protein (GFP) reporter gene. In a first attempt, the hsp70--GFP vector was successfully tested in a eukaryotic cell line. Thereafter, adult male schistosomes and sporocysts were transformed with this vector, and GFP expression was demonstrated using molecular and microscopical methods. PCR, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses confirmed the presence, transcription and translation of the transgene in adults. Confocal laser scanning microscopy revealed GFP-activity at various sites along the surface of the worms after hs induction and within sporocysts. These results suggest diverse roles for hsp70 during the development of schistosomes. Furthermore, the results demonstrate the feasibility of this method and open the perspective to analyze a variety of molecular functions in schistosomes.

Eimeria infections in cows in the periparturient phase and their calves: oocyst excretion and levels of specific serum and colostrum antibodies.

Faecal Eimeria oocyst excretion and levels of antibodies to first generation merozoite antigen of E. bovis in sera and colostra were followed in 86 and 70 cow-calf pairs in northern (group EF) and central Germany (group H), respectively, over periods of 3 weeks before to 3 weeks after calving in cows and from birth to an age of 63 days in calves. Oocysts were found in 30 and 7.7% of cows in groups EF and H, respectively. They belonged to 10 (group EF) and four Eimeria spp. (group H) with E. bovis, E. ellipsoidalis, E. auburnensis and E. zuerni as the most frequently occurring species. Prevalence and intensity of oocyst excretion varied with time resulting in peak values around the date of parturition, particularly in the case of E. bovis. Peak values at the time of parturition were also seen in case of strongyle egg excretion. Seven (group H) and nine Eimeria spp. (group EF) were found in the calves. The predominant species E. ellipsoidalis, E. zuerni, E. bovis and E. auburnensis were detected for the first time earlier after birth (3-5 weeks) than the others. The prevalence of Eimeria infections increased to 67.1% (group EF) and 50.1% (group H) 9 weeks after birth. Specific IgM and IgA antibody levels (the latter only determined in group EF) in cow sera remained almost constant throughout the observation period, whereas IgG(1) and IgG(2) levels were reduced at the time of parturition. Levels of specific antibodies in sera and colostra were significantly correlated. Except IgM antibodies, significant inverse correlations were found in cows between intensity of infection with E. bovis and specific serum IgG (group H) and IgG(2) (group EF) antibodies. Antibodies to E. bovis were detected in calves sera only after colostrum intake with significant correlations between levels in calves sera and colostra. Levels decreased, starting within the first week of life (most conspicuously in case of IgM and IgA) until the third week. Subsequently, but except IgG(1) antibody concentrations increased until the end of the observation period. Interrelations between antibody levels and the total amount of E. bovis oocysts excreted by the calves until the ninth week of life varied with the age of the animals. Inverse relationships in the first 3 weeks of life as suggested by negative correlation coefficients could not be proven statistically. Thus, there is no unambiguous proof for immunoprotection of calves against E. bovis via maternal immunity. Considering antibody levels in the 3-9 weeks old calves significant direct correlations with E. bovis oocyst excretion were found in case of IgM, IgG(2) and IgA, reflecting an active immune response of young calves to coccidial infection.

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.

Eimeria bovis in cattle : colostral transfer of antibodies and immune response to experimental infections

IgM, IgG1, and IgG2 antibodies toEimeria bovis first-generation merozoite antigens were determined by enzyme-linked immunosorbent assay and Western blotting in naturally infected cows and in their offspring before and after the uptake of colostrum. In addition, calves were examined following experimental primary and challenge infections. Neonate calves received maternal antibodies via colostrum. All isotypes determined were transmitted, but only IgG1 was concentrated in the colostrum and it occurred at significantly increased levels in sera from the calves as compared with those from the respective dams. Recognition patterns (Western blotting) displayed by related maternal serum and colostrum and those shown by calves that had ingested colostrum were very similar, but marked variations occurred between individual pairs. Experimental infection of 15-week-old calves with 0.7×105 oocysts caused strong protective immunity against a challenge with 1×105 oocysts. In contrast, animals that had undergone a weak intercurrent infection were not protected. Experimental infections induced a considerable increase in IgG1 and IgG2 antibody levels, whereas IgM values increased only slightly. The spectrum of merozoite antigens recognized by the sera increased markedly after experimental infection, although high individual variations were found in the calves. However, there was no correlation between the levels of any specific antibody or the recognition patterns and the status of immunity to a severe challenge.

Experimental chemotherapy of filariasis: comparative evaluation of the efficacy of filaricidal compounds in Mastomys coucha infected with Litomosoides carinii, Acanthocheilonema viteae, Brugia malayi and B. pahangi

Eleven types/classes of compound with antifilarial activity were comparatively evaluated in Mastomys coucha infected with Litomosoides carinii, Acanthocheilonema viteae, Brugia malayi or B. pahangi. The paper deals with the efficacy of (i) predominantly microfilaricidal compounds [diethylcarbamazine, levamisole, avermectins (ivermectin, milbemycin), nitrofurans (nitrofurantoin, hydroxymethylnitrofurantoin, nifurtimox, furazolidone, furapyrimidone), organophosphorals (metrifonate, haloxon), and aminophenyl-amidines], (ii) predominantly macrofilaricidal compounds [suramin, benzimidazoles (flubendazole, mebendazole, oxfendazole, ciclobendazole, albendazole, cambendazole, fenbendazole), and arsenicals (thiacetarsamide, Mel PH, R7/45)], and (iii) micro- and macrofilaricidal compounds [benzazole derivatives (CGP 20376 and other benzothiazoles) and nitrophenylamines (amoscanate, CGP 6140)]. Minimum effective doses against microfilariae and minimum curative doses against adult filariae as well as detailed data on dose-efficacy relationships are reported for the various drugs. The results obtained in M. coucha are compared with those published for other experimental in vivo filarial systems, thus attempting to describe a general status of in vivo antifilarial activity of the compounds.

Prevalence, risk factors and economic importance of infestations with Sarcoptes scabiei and Haematopinus suis in sows of pig breeding farms in Hesse, Germany

Abstract.  A cross‐sectional survey was performed in 110 randomly selected pig‐breeding farms of southern Hesse, Germany to estimate the prevalence of ectoparasite infestations and to find possible risk factors. Ear scrapings of, if available, 10 sows per farm were examined for Sarcoptes scabiei var. suis (De Geer) (Acaridida: Sarcoptidae) by the potassium hydroxide digestion method, and a total of 2754 sows was inspected for skin lesions and infestations with Haematopinus suis (L.) (Anoplurida: Haematopinidae). Data on farm profiles and sows were collected by a questionnaire. In total, 19.1% and 2.5% of the sows were found to be infested with S. scabiei or H. suis, respectively. The percentage of mite or louse infestation was significantly higher in sows showing pruritus than in those without skin lesions. Both ectoparasite infestations were related neither to the age of sows nor their reproduction status, nor to the time interval to last ectoparasite treatment. Using farms as the unit of analysis, the estimated prevalence of mange mite and louse infestations was 45.4% and 14.5%, respectively. There was no significant association between the presence of S. scabiei and H. suis in the farms. Risk factors for S. scabiei infestation were mixed housing of dry and nursing sows in the same unit (vs. separate housing) and straw bedding (vs. strawless). For louse infestation, only mechanical cleaning of stable units (vs. additional use of disinfection methods) and pasturing of gilts and dry sows were identified as risk factors. The economic loss by S. scabiei infestation in the study population was assessed at €4200 per affected farm and year on average.

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.