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Öğe Extracellular Trap Formation by Donkey Polymorphonuclear Neutrophils Against Toxoplasma gondii(Elsevier Science Inc, 2019) Yildiz, Kader; Gokpinar, Sami; Sursal, Neslihan; Babur, Cahit; Ozen, Dogukan; Azkur, Ahmet KursatToxoplasma (T.) gondii is an obligatory intracellular apicomplexan parasite. The donkey is one of the intermediate hosts of T. gondii. There are almost no data about T. gondii infection in donkeys, apart from a few seroprevalence reports. The aim of the study was to detect the formation of extracellular traps by donkey polymorphonuclear neutrophils (PMNs) when exposed to T. gondii tachyzoites in vitro. Tachyzoites were observed to be entrapped within extracellular traps (NET) structures. Myeloperoxidase, neutrophil elastase (NE), and histone (H3) were observed in NET structures. NE and reactive oxygen species activity increased with time and was higher in the PMN-tachyzoite cocultures. Some tachyzoites were seen close localization to the nucleus of neutrophils. Degranulation and extracellular traps were observed simultaneously in some donkey neutrophils after incubation for 1 hour. The tachyzoite invasion rate decreased in PMN-tachyzoite cocultures in comparison to the controls. NETs can negatively affect the viability of entrapped tachyzoites in addition to their action of physical entrapment. NE may play a key role in the viability of T. gondii in donkeys. The amount of extracellular DNA increased with incubation time in the PMN-tachyzoite cocultures (P < .001). This is a first report regarding the formation of donkeys NETs after exposure to T. gondii tachyzoites in vitro. Unlike sheep and cattle PMN, degranulation and extracellular traps developed in the same neutrophil. Further studies focusing on signaling pathways may shed light on what determines the behavior of donkey neutrophils after exposure to T. gondii. (C) 2018 Elsevier Inc. All rights reserved.Öğe Feline Giardiasis In Turkey: Prevalence And Genetic And Haplotype Diversity Of Giardia Duodenalis Based On The Beta-Giardin Gene Sequence In Symptomatic Cats(ALLEN PRESS INC, 2020) Sursal, Neslihan; Simsek, Emrah; Yildiz, KaderGiardia duodenalis is a common zoonotic protozoan parasite with a broad host distribution. The main objectives of the present study were to determine the prevalence of giardiasis and to reveal the genetic and haplotype diversity of G. duodenalis in symptomatic cats in Turkey. Fecal samples were collected from cats (n = 102) with diarrhea that were admitted to different pet clinics in the Central Anatolia region of Turkey. All samples were analyzed by microscopic examination (ME), rapid immunochromatographic test (ICT), and PCR targeting the beta-giardin (bg) loci of the parasite. Phylogenetic, haplotype, and network analyses of G. duodenalis based on the bg gene were carried out. Overall, G. duodenalis was detected in 70/102 (68.6%) of the cats with diarrhea by ME (38/102, 37.3%), ICT (51/102, 50%), and PCR (30/102, 29.4%). According to sequence analyses of the bg gene region, all isolates were identified as G. duodenalis assemblage B. Haplotype analyses revealed 2 known and 8 novel haplotypes for G. duodenalis assemblage B. This study provides first prevalence and genetic and haplotype diversity data on G. duodenalis assemblage B from cats in Turkey.Öğe Neutrophil isolation from feline blood using discontinuous Percoll dilutions(Georg Thieme Verlag Kg, 2018) Sursal, Neslihan; Cakmak, Ayse; Yildiz, KaderObjective: Some studies have performed in vitro neutrophil isolation from feline blood. The major limiting factor for these studies is the small volume of blood that can be collected without development of potentially life-threatening complications. In the present study we attempted neutrophil isolation from feline venous blood samples using discontinuous Percoll gradients. Material and methods: Blood was collected from the cephalic vein of clinically healthy adult cats. The blood samples were layered on Percoll dilutions (72 %, 63%, 54% and 45%). After centrifugation, the feline polymorphonuclear leukocytes (PMN) accumulated as a band between 72-63% Percoll dilutions. The total cell count was calculated using light microscopy counts. The percentage of the neutrophils was determined microscopically after staining with Diff-Quik stain. Neutrophil viability was evaluated with a 0.01 % Trypan blue assay. The activation was determined based on intact cell morphology in the isolated neutrophils. Results: The mean PMN number was 22 x 10(5) per ml (minimum-maximum: 20-26 x 10(5)ml). Neutrophil homogeneity was > 95% in the cell suspensions. The viability of isolated neutrophils was > 98%. The technique did not result in neutrophil activation. Conclusion and clinical relevance: Discontinuous Percoll gradients (72%, 63 %, 54% and 45%) can be used to isolate neutrophils from blood samples of cats. The technique was simple to perform and neutrophil activation was minimal.Öğe Occurrence and First Molecular Characterization of Cryptosporidium felis in a Cat in Turkey(KAFKAS UNIV, VETERINER FAKULTESI DERGISI, 2020) Sursal, Neslihan; Simsek, Emrah; Yildiz, KaderCryptosporidium species are highly prevalent and significant zoonotic protozoan parasites that cause severe diarrhea in many hosts. To date, cryptosporidiosis has been molecularly reported from humans and different animal hosts in Turkey. The aim of this case report is to reveal the molecular characterization based on small subunit (SSU) rRNA gene sequence of Cryptosporidium oocysts that were microscopically found in a three-month age female cat with severe diarrhea. SSU rRNA gene of Cryptosporidium was amplified for sequence and phylogenetic analyses. According to sequence analysis, Cryptosporidium spp. oocysts were characterized as C. felis (isolate name: ANK_1; accession number: MN394123). ANK_1 isolate showed a range from 99.1% to 100% identity with the most of C. felis sequences available in GenBank. However, it also exhibited high genetic distance (1.5-4.3%) with some of C. felis sequences. The intraspecific genetic differences among all SSU rRNA sequences of C. felis isolates included in the data set were determined ranged from 0.0% to 6.9%. As a conclusion, we provided the first microscopic and molecular evidence of the occurrence of C. felis in a house cat in Turkey. Although C. felis has a relatively low zoonotic risk to public health when compared with C. parvum, infected cats should not be neglected for zoonotic transmission due to close relationships between cats and humans.Öğe Role of NETs in the difference in host susceptibility to Toxoplasma gondii between sheep and cattle(Elsevier Science Bv, 2017) Yıldız, Kader; Gökpınar, Sami; Gazyağcı, Aycan Nuriye; Babur, Cahit; Sursal, Neslihan; Azkur, Ahmet KürşatThe main aim of this study was to compare extracellular traps (NETs) formation by polymorphonuclear neutrophils (PMNs) of cattle and sheep when exposed to T. gondii tachyzoites in vitro. The effects of parasite concentrations and different incubation periods on NETs development in cattle and sheep PMNs were studied. The effect of NET structures on host cell invasion by tachyzoites was also studied. This is the first report of NETs development by sheep and cattle PMNs against T. gondii in vitro. T. gondii-induced extracellular DNA production from PMNs was dependent on tachyzoite concentrations and incubation time in both sheep and cattle. Many nuclear and cytoplasmic changes were observed in sheep and cattle PMNs after exposure to T. gondii tachyzoites. The typical appearance of NETs, with MPO, NE and histone (H3) attached to extracellular DNA, was observed. Tachyzoites were entrapped within this structure. Myeloperoxidase (MPO) activity was higher in the cattle PMNtachyzoite co-cultures than sheep. NETs structures released from sheep PMNs caused mechanical immobilisation of T. gondii tachyzoites, however, NET structures released from cattle PMNs may be lethal to tachyzoites. Bovine MPO may have a lethal effect on T. gondii tachyzoites in vitro during a 3 h incubation. Besides other mechanisms that effect on host susceptibility to T. gondii in sheep and cattle, extracellular traps formation as a part of immunological reactions may be play a role in host susceptibility to T. gondii.
