2nd International Conference on Innate Immunity and Immune System Diseases July 21-22, 2016 Berlin, Germany

Mastitis is an inflammation of the mammary gland commonly caused by bacterial infection. Regardless of the extensive management practices, it represents one of the most economically important health traits for milk production. Pentraxins are a superfamily of conserved molecules with immune functions such as complement activation and opsonization. PTX3 is the prototypic long pentraxin and is produced by different cell populations after pro-inflammatory stimuli (TLR ligands, IL-1β and TNFα). Some studies have demonstrated the up-regulation of PTX3 mRNA during ruminant mastitis but its role is still unknown. To better understand the role of PTX3 we investigated its pattern of expression in a model of S. aureus intramammary infection in goat. Because no data on goat PTX3 are available, we first studied PTX3 pattern of expression in a wide panel of goat healthy tissues. PTX3 mRNA was expressed at high level in bone marrow, mammary gland, aorta, pancreas, skin and lungs. Then we focus our attention to the mammary gland; six healthy goats were infused with PBS in the right udder and with S. aureus in the left udder and mammary biopsies for immunohistochemistry and RNA extraction were collected 30 hours post infection. Immunohistochemistry revealed that in non-infected udders PTX3 was mainly expressed in the apical cytoplasmic portion of mammary epithelial cells (MEC) and in macrophages; whereas during S. aureus infection PTX3 was up-regulated in MEC and in the secretum. Moreover, PMNs recruited during infection were variably intensely positive. PTX3 mRNA expression was low in healthy udders compared to the infected ones, indeed this molecule is commonly induced after pro-inflammatory stimulation. Finally, we investigated the modulation of PTX3 during field mammary infection, comparing its expression in healthy and S. aureus infected goats (blood leukocytes, milk somatic cells, milk fat globules). PTX3 mRNA was significantly up-regulated in circulating PMN and milk somatic cells. In circulating monocytes and in milk fat globules we observed a statistically non-significant tendency of increase expression in S. aureus infected animals. These observations could reveal a role of PTX3 as a key player in the immune response of mammary gland to S. aureus infection and it could represent a marker of mastitis in goat that can be used for a fast and early diagnosis of intramammary infection in the field.

Neutrophils are the most important host innate immune defenders which freely circulate in blood and recruited to inflammation area. Neutrophils are short lived polymorphonuclear granulocytes (PMN) and play an important role activity to the inflammation area in vertebra hosts and they kill the pathogens with various defence strategies (phagocytosis, degranulation and netosis). During netosis, the neutrophil nuclear and granule contents are given to extracellular region to fight with pathogens. It is reported that neutrophil extracellular traps (NETs) exist in several bacteria, fungi, viruses and protozoa. There are no information on netosis reaction to T.gondii in cat which are definite hosts and intermediate hosts of toxoplasmosis. Hereby, it is intended to demonstrate NETs reaction in cat’s neutrophils confronted with T.gondii tachyzoites in vitro. Whole blood of cat layered with Percoll solutions (72, 63, 54 and 45%) for neutrophil purification. Then, NETs activity was observed using fluorescence microscopy. Myeloperoxidase, neutrophil elastase and histone (H3) shown as co-localise in fine DNA networks released from cat PMN after reacting with T.gondii tachyzoites. The results suggest cat PMN create NETs to T.gondii tachyzoites and netosis is important mechanism for fighting with T.gondii in cats. According to Authors knowledge, it is the first report that NETs develop in cat neutrophils against to T.gondii tachyzoites.

Sami GOKPINAR has completed PhD education and postdoctoral studies in Kirikkale University, Turkey. He has studied Parasitology Department at the University Kirikkale, Turkey.

Toxoplasma gondii infected a lot of intermediate hosts including equids. Serum antibodies against to T.gondii have been found in donkeys in the world. Neutrophil extracellular traps (NETs) are one of the major fighting mechanism of polymorph nuclear neutrophils (PMN) against pathogens. NETs provides extracellular entrapment and killing of pathogens including parasites in organism. The aim of the study was to detect NETs formation of polymorph nuclear neutrophils (PMN) of donkey encounter with T.gondii tachyzoites. For the first time, we demonstrate extracellular traps released from donkey PMN after confronted with T. gondii tachyzoites in vitro. Myeloperoxidase, neutrophil elastase and histones (H3) were observed in fine NETs structures triggered by T.gondii using fluorescence microscopy. Fluorometric analysis revealed that NETs amount increased depending on incubation time.

Elena Aleksandrovna Kotenkova has completed her PhD from VNIIMP and published more than 35 papers. She is the Biochemist in Experimental Clinical Research Laboratory.

Tissue specific proteins and peptides were extracted from Sus scrofa thymus, spleen and lymph nodes (TSL) by physiological saline on distilled water (DW) and deuterium depleted water (DDW). Study of TSL+DW and TSL+DDW was carried out on Wistar rats with cytostatic induced immunodeficiency model Animals were randomly divided in 4 Groups: Group-1: Intact (n=10); Group-2: Treated with DW (n=10), Group-3: Treated with TSL+DW (n=10), Group-4: Treated with TSL+DDW (n=10). Lymphocytes and monocytes counts in Group-2: Decreased by 31.9% (p<0.05) and 40.4% (p<0.05) compared with Group-1 while granulocytes count increased by 48.3% (p<0.05). Lymphocytes and monocytes counts in Group-3 and Group-4 increased by 21.7% (p<0.05) and 38.1% (p<0.05), 24.8% (p<0.05) and 14.3% (p<0.05) compared with Group-2 while granulocytes count decreased by 20.2% (p<0.05) and 25.4 % (p<0.05). CD4 count in Group-2 decreased by 49.1% (p<0.05) compared with Group-1 while CD3 count increased by 19.0% (p<0.05). CD3/CD4 index was 2.69, which was higher in intact animals more than 2 times. CD4 in Group-3 increased by 67.4% (p<0.05) compared with Group-2 while CD3 was higher Group-2 by 32.7% (p<0.05). CD3/CD4 index was 2.13, that was lower Group-2 by 20.8% (p<0.05). CD4 in Group-4 did not increase while CD3 was higher in Group-2 by 18.5% (p<0.05). CD3/CD4 index was 3.10, that was higher in Group-2 by 15.2% (p<0.05). Revealed data confirmed TSL influence on immunity. Pathways activation depended on solubilizing agent. Presumably, TSL+DDW may stimulate both B-cells and T-cells differentiation while TSL+DW primary stimulate CD3 and CD4 T-lymphocytes differentiation.

Kader YILDIZ has studied Parasitology Department at the University Kirikkale, Turkey. She has completed PhD education from Ankara University, Turkey.

Toxoplasma gondii, a zoonotic parasite, possess broad hosts range. Sheep is easily infected with T.gondii and the parasite causes some important pathology dependent on the stage of pregnancy at which the ewe becomes infected. Whereas cattle is poor host of the parasite epidemiologically, cattle can be successfully infected with T.gondii, but this parasite is eliminated perhaps due to innate immunity. Innate immune system providing a quick defence against to pathogens enters the body is essential for survivor of multicellular organisms. Primary role of innate immune system is restrict pathogens in infections area and thus prevent to spread into the organism. Innate immunity is a complex mechanism involving several instruments. Neutrophil is major component of innate immune system and fight pathogens with different strategies in organism. One of them, netosis is an important effector mechanism of the host early immune response against pathogens. The aim of the study was to comparison of neutrophil extracellular traps (NETs) formation of polymorph nuclear neutrophils (PMN) of cattle and sheep confronted with T.gondii tachyzoites. And also aim to determine the effects of incubation time and different parasites ratio on NETs development in cattle and sheep PMN. Sheep and cattle experiments were started at the same day under the same experimental conditions. Netosis was observed in cattle and sheep PMN after confronted with T. gondii tachyzoites in vitro. NETs amount was measured using fluorometer. NETs amount increased depending on tachyzoites intensity and incubation time. Fluorescence microscopic analyses revealed that myeloperoxidase, neutrophil elastase and histones (H3) were detected in NETs structures from sheep and cattle PMN after encounter with T.gondii.