key: cord-103715-53v1qoyc authors: Bauman, Neda; Ilić, Andjelija; Lijeskić, Olivera; Uzelac, Aleksandra; Klun, Ivana; Srbljanović, Jelena; Ćirković, Vladimir; Bobić, Branko; Štajner, Tijana; Djurković-Djaković, Olgica title: Computational image analysis reveals the structural complexity of Toxoplasma gondii tissue cysts date: 2020-05-21 journal: bioRxiv DOI: 10.1101/2020.05.21.108118 sha: doc_id: 103715 cord_uid: 53v1qoyc Toxoplasma gondii is an obligate intracellular parasite infecting up to one third of the human population. The central event in the pathogenesis of toxoplasmosis is the conversion of tachyzoites into encysted bradyzoites. A novel approach to analyze the structure of in vivo-derived tissue cysts may be the increasingly used computational image analysis. The objective of this study was to quantify the geometrical complexity of T. gondii cysts by morphological, particle, and fractal analysis, as well as to determine if and how it is impacted by parasite strain, cyst age, and host factors. Analyses were performed on 31 images of T. gondii brain cysts of four type-2 strains (the reference Me49 strain and three local isolates, named BGD1, BGD14, and BGD26) using ImageJ software package. The parameters of interest included diameter, circularity, relative particle count (RPC), fractal dimension (FD), lacunarity, and packing density (PD). Although cyst diameter varied widely, its negative correlation with RPC was observed. Circularity was remarkably close to 1, indicating that the shape of the brain cysts was a perfect circle. RPC, FD, and PD did not vary among cysts of different strains, age, and derived from mice of different genetic background. Conversely, lacunarity, which is a measure of heterogeneity, was significantly lower for BGD1 strain vs. all other strains, and higher for Me49 vs. BGD14 and BGD26, but did not differ among Me49 cysts of different age, and derived from genetically different mice. This study is the first application of fractal analysis in describing the structural complexity of T. gondii cysts. Despite all the differences among the analyzed cysts, most parameters remained conserved. Fractal analysis is a novel and widely accessible approach, which along with particle analysis may be applied to gain further insight into T. gondii cyst morphology. 3 to determine if and how it is impacted by a number of parasite or host factors. Additionally, morphological and particle analyses of T. gondii cysts were applied to gain further insight into the cyst 73 shape uniformity, as well as to a possible correlation between the cyst size and the number of parasites. Perimeter Area y Circularit   circularity values close to 1.0, whereas elongated shapes have very low circularity (close to 0.0). Cysts were transferred to black background images for subsequent particle analysis. For particle analysis, images were first enhanced using histogram equalization. During this relative particle count (RPC) was determined following the local Bernsen auto-thresholding, watershed further segmented by a watershed algorithm for two reasons: a) it seems that, due to a low local border clearly separated particle areas. In that manner, all of the cyst cross-section images were subjected to an 148 automated processing and the obtained particle numbers, N p , were expected to be proportional to the Fig 2C. The FD was obtained as a negative slope of the best-fit regression line: Each FD calculation is accompanied by the correlation coefficient, , describing a goodness of 2 r 193 fit of the regression line, in a particular case. In the cumulative mass method, a probability of finding a certain pixel number within a given Lacunarity of a fractal set represents the ratio of the second-order moment (variance) to the In addition to the mentioned parameters, we defined another parameter measuring the 207 percentage of space covered by bradyzoites, and termed it 'packing density' (PD). The two-dimensional 208 analysis was performed on the cyst cross-sectional areas captured on microscope. Both particle analysis 209 and fractal analysis provided the number of black pixels in a considered cross section, N B . The total number of pixels in a cross section, N T , was known in all cases, which allowed the packing density 211 estimation as 212 . (7) The 3A; Fig 4A,B) . Lacunarity, on the other hand, differed among the strains, in that it was significantly lower 265 in the BGD1 strain in comparison to the three other ones (p≤0.001), as well as in BGD14 and BGD26 vs. Me49 (Tukey p=0.044 and p=0.033, respectively), but not between BGD14 and BGD26 (p=0.99) (Fig 3B) . In an attempt to improve the understanding of the nature of the tissue cyst structural The analyzed cysts widely differed in diameter, with the cysts obtained from the experiment 299 being smaller, albeit not significantly, compared to historical (archived) cysts. This may be due to a bias 300 towards larger, more interesting cysts kept in a laboratory photo archive. Advances in the life cycle of Toxoplasma gondii Genetic analysis of tachyzoite to bradyzoite differentiation mutants 350 in Toxoplasma gondii reveals a hierarchy of gene induction Dynamics of Toxoplasma gondii differentiation Toxoplasma development -turn the switch on or off? 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Vet S2 Data -Fractal dimension and lacunarity of tissue cysts (n=31) S3 Data -Binary images of T. gondii cysts segmented by watershed algorithm (n=31)