Partridge and embryonated partridge egg as new preclinical models for candidiasis

Candida albicans (C. albicans) is the most common cause of candidiasis in humans and animals. This study was established to a new experimental infection model for systemic candidiasis using partridge and embryonated partridge egg. First, we tested the induction of systemic candidiasis in partridge and embryonated partridge egg. Finally, interaction between virulence factors of C. albicans and Bcl-2 family members was predicted. We observed that embryonic infection causes a decrease in survival time and at later embryonic days (11–12th), embryos showed lesions. Morphometric analysis of the extra-embryonic membrane (EEM) vasculature showed that vascular apoptotic effect of C. albicans was revealed by a significant reduction in capillary area. In immunohistochemistry assay, low expression of Bcl-2 and increased expression of Bax confirmed apoptosis. The gene expression of Bax and Bcl-2 was also altered in fungi-exposed EEM. Ourin silico simulation has shown an accurate interaction between aspartic proteinase, polyamine oxidase, Bcl-2 and BAX. We observed that the disease was associated with adverse consequences, which were similar to human candidiasis. Acquired results support the idea that partridge and embryonated partridge egg can be utilized as appropriate preclinical models to investigate the pathological effects of candidiasis.

Recently, the chicken egg and embryo have also been considered as the laboratory models for experimental investigations [7][8][9] . To reduce the use of mammalian or non-mammalian animals in medical experimentation, alternative in vivo and in vitro models are increasingly necessary options.
Candida albicans (C. albicans) is the most common cause of fungal infections in humans 10,11 . It generally colonizes in regions such as the genitourinary tract, gastrointestinal track, lower respiratory and oropharynx. Candidemia is the fourth most common cause of nosocomial bloodstream infections in the USA and in Europe and has a significant impact on hospitalization cost and patient outcome 12,13 . An increasing incidence of fungal infections with Candida spp. has been noted in immunocompromised patients such as those staying in ICUs, postsurgical and neutropenic patients 14,15 . The systemic candidiasis has also occurred in patients with malignant diseases in recent years [16][17][18] . Different reports in the literature have documented that systemic candidiasis in premature infants is frequently fatal or associated with significant morbidity in survivors [19][20][21] . In this respect, introduction of newer in vivo preclinical model for precise studying of the candidiasis, its pathogenesis and treatment is an important and valuable issue nowadays.
In recent years, the partridge industry has experienced remarkable development and chukar partridge has been widely raised industrially in many parts of the world 22,23 . As a research subject, chukar partridge has certain advantages as follows: Modest size of the bird simplifies housing in laboratory animal facilities and reduced feed costs. The bird handling is easy and fulfilling its nutritional requirement is not a difficult task or expensive. The chukar partridge is not very sensitive to environmental conditions and it can be easily reared in floor and cage system. Furthermore, the modest size of breeding adults facilitates breeding in the laboratory. The female partridges generally lay 3-5 eggs per week and under favorable environments, they produce for long periods. Their eggs can be grown in large batches in incubators, and allow the effects of particular substances on embryonic development to be studied. The partridge embryo is also an amniote and it presents significant experimental advantages for the study of amniotes similar to those of humans. Unlike mammillaria, partridge embryo can be easily studied and manipulated as they monitor by removing a small section of the eggshell. This has made it possible to follow their developmental stages via time-lapsed video-microscopy [24][25][26][27][28] . Therefore, we chose the chukar partridge and embryonated partridge egg so that they could be used as laboratory models in areas that are easy to access.
On the other hand, in the past decade, some species of Phasianidae family (e.g. Japanese Quail), have become important experimental animals for scientific researches. They are used extensively in physiology, nutrition, genetics, embryology, toxicology, pathology and endocrinology researches 29,30 . Chukar partridge has also classified into the Phasianidae family. Considering this property, a major advantage that seems to be very promising is the fact that it is possible to achieve a new experimental model for scientific investigations.
In this context, the objective of this study was to establish new experimental infection models for systemic candidiasis and its pathogenesis using partridge and embryonated partridge egg. The present study was aimed to answer the following questions:

Results
The results are explained in terms of A) clinical and histopathological signs and lesions following systemic candidiasis in partridge model, B) vascular analysis, IHC and qPCR results from the EEM vasculature and C) in silico results.
(A) Clinical and histopathological signs and lesions following systemic candidiasis in partridge model Clinical signs. The infected partridges began to exhibit the clinical signs on the second day of infection. The early symptoms manifested with multiple adverse health outcomes which included depression, lethargy and ruffled feathers ( Fig. 1I-a). Following the progression of the disease, some infected birds were unable to stand and maintained their posture with drooping of the wings (Fig. 1I-b). Neurological disturbance such as spasmodic tremors in the head was also seen in four sick birds, mainly on the fourth day after infection. No mortality occurred during the experimental period in the infected group; however, two birds were culled due to severe debilitation on days 3 and 4 following infection. No clinical signs were observed in any of the control birds.
Cloacal temperature. The normal cloacal temperature of partridge chicks, before the inoculation of C.
albicans, was 41.3 ± 0.3 °C. An increase in the cloacal temperature was a primary response to infection. The temperature began to increase from normal value 30 h after infection. It continued to increase for another 24 h (≥ 42 °C) and remained almost constant for 3-4 days post-infection. The mentioned temperature (≥ 42 °C) was not observed in any of the control birds. It was also significant after statistical analysis (control bird = 41.29 ± 0.17, infected birds = 41.95 ± 0.07, p = 0.002).
Gross findings. Gross lesions associated with systemic candidiasis were observed in the kidneys, liver, lungs, heart and brain. Congestion was noticed in some internal organs including the kidneys, liver and lungs of the affected birds. The kidneys and liver were swollen. In some affected birds, hydropericardium with white necrotic foci occurred in myocardium (Figs. 1I-c,d). Congestion of the brain surfaces was evident in birds showing neurological disturbance (Fig. 1I-e). The alimentary tract of most cases was devoid of food ( Fig. 1I-f). Muscular atrophy was observed in debilitated birds; however, the skeletal structure appeared normal.
Histopathological findings. Histological lesions were observed in the kidneys, liver, spleen, lungs, pancreas, heart and brain of the infected birds. Varying degrees of congestion were detected in some internal organs including the kidneys, liver and lungs ( Fig. 3I-a, c and e). In some cases, granulomas were evident in the lungs (Fig. 3I-e), spleen ( Fig. 3I-b) and brain (Fig. 3II-c to f). Histologically, granulomas were composed of both mycelial and yeast forms of C. albicans with areas of necrosis and inflammatory cell infiltration (particularly the macrophages). Lesions of the liver included congestion, hepatocellular degeneration, necrosis, central vein dilatation, bile duct hyperplasia and vascular thrombosis (Fig. 3I-c and d). The pancreas showed degeneration and necrosis in the exocrine portion ( Fig. 3I-f). Myocardial fiber necrosis with infiltration of mononuclear cells, particularly the macrophages, was noticed in myocardium ( Fig. 3II-a and b). In the brain, gliosis, edema, neuronal ischemic cell change and granuloma formation in the cerebrum, molecular/granular layer of the cerebellum and midbrain were observed (Fig. 3II-c to f).

Susceptibility of the partridge embryo model to systemic candidiasis.
To confirm the susceptibility of the partridge embryo model to C. albicans by human origin, we chose the 10th embryonic day. We observed that embryonic infection causes a decrease in survival time, and at later embryonic days (the 11-12th) (B) Vasculature analysis, IHC and qPCR results from EEM vasculature As described previously, the EEM partridge modelwas used to evaluate the effect of C. albicans on vascular apoptosis. The results were as follows: Mean capillary area. The response to C. albicans inoculation onto the partridge EEM is presented in Immunohistochemistry results. In order to prove vascular apoptosis in the partridges' EEM vasculature, H&E and IHC staining were performed to detect apoptotic cells and their components. As illustrated in Fig. 3III, in C. albicans infected group, Bcl-2 on leukocytes, endothelial and stromal cells wereless intensely stained compared with Bax. Gene expression results. The expression levels of apoptotic-regulator genes in C. albicansinfected EEM were determined by qPCR. Our results showed increased expression of Bax genes and reduced expression of Bcl-2, VEGF-A and VEGF-R2 (Fig. 2II). In silico assay, as mentioned earlier, was applied to investigate the interaction between C. albicans virulence factors (ASP, PAO and TRY) and apoptotic-regulator proteins (Bcl-2 and Bax). The results are presented below.
Structural parameters of simulated proteins. The bioinformatics tools were applied to construct the structures of the apoptotic-regulator proteins (Bcl-2 and Bax, Gallus gallus) ( Fig. 4I-a and b). The qualities of constructed models were checked by Ramachandran plots (Fig. 4I-c and d). Ramachandran results for Bcl-2 and Bax were respectively as follows: 88.1% and 87.2% of the residues located in most favored regions, 9.4% and 9.9% of the residues located in additional allowed regions and 2.5% and 2.3% of the residues located in generously allowed regions. The Ramachandran plot is a fundamental tool in the analysis of protein structure. It locates the amino acid residues of the simulated models Ramachandran 31 . A good quality Ramachandran plot has over 85-90% in the most favored regions. Ramachandran plots of simulated proteins (Bcl-2 and BAX) have 88.1% and 87.2% of residues in the most favored regions; therefore, these data exhibited that good quality models were simulated.
The structural parameters of the simulated Bcl-2 were as follows: molecular weight = 21,422.02, total number of atoms = 2954, total number of amino acids = 194, number of negatively charged residues (Asp + Glu) = 22 and the number of positively charged residues (Arg + Lys) = 17.
The conformational parameters of the simulated Bax were as follows: molecular weight = 21,666.16, total number of atoms = 3085, total number of amino acids = 196, number of negatively charged residues (Asp + Glu) = 19 and number of positively charged residues (Arg + Lys) = 17.

Molecular docking.
After constructing the 3D structures of Bcl-2 and Bax, active binding pockets were determined using CASTp program. The volumes of the pockets were 6391.543 and 7094.497 for Bcl-2 and Bax, respectively.
The dockings of ASP, PAO and TRY with Bcl-2 and Bax were performed using the HEX program. The PDB file format of receptors and ligands were uploaded in HEX for energy minimization and structure refinement and then, the highest scoring conformations (lowest energy) were selected. The results are described below: The docking between ASP and Bcl-2 was successful, with a significant score (E-total score: − 564.43 kcal/ mol). The 3D docking result is demonstrated in Fig. 5I-a. When searching for binding residues on the surface of ASP, we detected that VAL, GLU, SER, VAL, ASN, ARG, GLU, MET, ALA, GLN, MET, SER, GLY, GLN, LEU, HIS, LEU and THR were important for the interaction with Bcl-2.
After docking, the affinity between PAO and Bax was also high (E-total score: − 526.59 kcal/mol) ( Fig. 5I-d) and the protein-protein interaction successfully occurred through the binding residues including ASP, LYS, LEU, ASP, GLN, ASP, GLN, ALA, PHE, ASN, ASP, MET, ILE, ASP, GLY, LEU and VAL.
The analysis of data showed that TRY interacted with Bcl-2 and Bax with low affinity (E-total score: − 143.62 and − 160.53 kcal/mol, respectively) ( Fig. 5I-e and f).

Affinity of C. albicans virulence factors for Bcl-2 family from different origin.
As pointed out earlier, cross-dockings were done with 5JSN and 5W5X, instead of their specific ligands, to evaluate the affinity of ASP, PAO and TRY for Bcl-2 family proteins that originated from a different species (Human). The results are shown in Fig. 5III and Table 1. The analysis of data revealed that after separating the original ligands from 5JSN and 5W5X, the ASP, PAO and TRY still tended to interact with them. The 5JSN and 5W5X are crystallographic structures of human Bcl-2 and Bax, which interacted with specific inhibitor or activator, respectively. When searching for binding residues on the surfaces of 5JSN and 5W5X faced toward their inhibitor or activator, various residues including ASP, PHE, SER, ARG, ARG, TYR, ARG, ARG, ASP, PHE, ALA, GLU, MET, SER, SER, GLN, LEU as well as SER, GLU, SER, LEU, LYS, ARG, ILE, GLY, ASP, GLU, LEU, ASP, SER, ASN, MET were predicted, respectively ( Fig. 5III-a and b). When comparing these results, we found that several of these residues are known to interact with ASP, PAO and TRY and are located near the binding sites of 5JSN and 5W5X with ASP, PAO and TRY, suggesting that ASP, PAO and TRY may alter the activity of 5JSN and 5W5X similar to that of their specific inhibitor or activator. Similar residues, which are located at the binding sites of 5JSN and 5W5X faced toward their ligands, are illustrated in Fig. 5III-c to h (residues in white color).

Self-docking.
We assessed the accuracy of the results by a self-docking stage (validation stage). Among the various conformations, the best-docked conformation was selected. Self-docking results for 5JSN and 5W5X are listed in Table 1, and the best conformation for each docking is demonstrated in Fig. 5II. As predicted, the accuracy of the results was proved with an appropriate binding energy. As revealed in Fig. 5II-c and d, the specific ligands of the 5JSN and 5W5X were docked with their receptors by correct orientations.   Table 2.

Discussion
In this work, we demonstrate that the partridge and embryonated partridge egg are new and effective preclinical infection models for evaluating systemic candidiasis. Moreover, some mechanisms, which could be associated with the pathogenesis of the organism, were assessed. Various disorders and lesions were noticed following systemic candidiasis in partridge.
The clinical signs were seen over a 48-96 h period in the partridge model. It was a variety of adverse health consequences including depression, lethargy, decreased feed and water intake and growth depression. Five days post-infection, the analysis of body weight revealed that C. albicans caused severe growth depression. The next indicator of the systemic candidiasis in the partridge model was neurological disturbance. Several types of neural disturbances were observed in the Galliformes: 1) extreme opisthotonus with spasmodic tremors, 2) extreme torticollis with cranial rotation and 3) extreme torticollis which resulted in the head being drawn in a medialventral direction 32 . A spasmodic tremor was seen in the head of some partridge birds on day 4 post-infection. Birds with head tremors exhibited the microscopic appearance of C. albicans in different parts of the brain.
Various gross abnormalities and microscopic lesions in multiple tissues were also indicators of the candidiasis in the partridge model. Based on the results of this study, it is deduced that C. albicans can affect the kidneys, liver, spleen, lungs, pancreas, heart and brain of the partridge model. We noticed gross and microscopic lesions in these organs, however, further data may be provided on alterations in other organs.
Our results are in agreement with the clinical presentation of bloodstream candidiasis in humans. It generally presents as fever and worsening of clinical status, especially in a patient with risk factors who is not responding to appropriate therapy. When C. albicans disseminates in humans, various organs are usually involved with the kidney, liver, spleen, myocardium and eye being the most common, and less frequently the other organs (e.g. lung, joint and bone). Candida infects both meninges and brain parenchymal tissue. The organism induces diffuse encephalopathy with diminished consciousness in which the predominant lesion is micro abscess. The severity of the illness and its profile (acute or chronic) may be conditioned by the size of the inoculums. In the partridge model (our work), 5 × 10 5 cfuof C. albicans produces neurocandidiasis on the fourth day after infection.
Complex cellular and molecular processes may be related to the pathogenesis of C. albicans. In the current research, details of the vascular apoptotic effect of C. albicans were assessed through in vivo (EEM partridge model) and in silico investigations. Some highlights of our findings on the vascular alteration are herein discussed.
Morphometrical analysis of the partridge's EEM vasculature has revealed that C. albicans affects the vascular plexus negatively. The method applied to assess the vascular alteration effect of C. albicans was the calculation of the mean capillary area in the captured images. To date, this method has been widely used in vascular analysis 7,33,34 .
The next aspect to discuss is the considerable alteration in the expression of Bcl-2 and Bax proteins following C. albicans treatment. The pathways and mechanisms by which C. albicans induced apoptotic activity in vessels have not been identified explicitly. However, regarding the IHC results, which revealed that Bcl-2 was less intensely and Bax was more intensely stained in C. albicans treated EEM, we suggest that alteration in the Bcl-2 family members could be one of the mechanisms involved in the vascular apoptotic activity of C. albicans. This assumption was also supported by our in silico study in which C. albicans virulence factors bound to the active site of Bcl-2 and Bax proteins.    The results of our study verified the low expression of Bcl-2, VEGF-A and VEGF-R2 and increased expression of Bax genes in the fungus-exposed group. It seems that Akt/PKB signaling pathway is involved in this process and consequently the VEGF-A caused a decrease in the expression of Bcl2 and increased the expressions of Bax gene 35 .
We also exploit a docking assay to reveal more details about the apoptotic activity of C. albicans. Docking assay has recently been considered as a valuable technique to elucidate the interaction between receptor and ligand [36][37][38] . It is well known that the Bcl-2 family members are critical targets for apoptotic and anti-apoptotic agents 39,40 . In this regard, an important highlight in our study is the interaction of ASP, PAO and TRY with Bcl-2 and Bax proteins. Our data suggest the modulation of Bcl-2 family members via the binding of C. albicans virulence factors, which must be validated by further experiments.
Another highlight of the protein-protein interaction is the quantitative aspect of binding affinity between C. albicans virulence factors and Bcl-2 family proteins. Based on docking results, which revealed the lowest scoring energy for ASP (− 564.43 and − 610.11) compared to PAO (− 507.77 and − 526.59) and TRY (− 143.62 and − 160.53), it is predicted that the affinity of ASP for Bcl-2 and Bax is higher than that of PAO and TRY. Therefore, ASP can be regarded as a hopeful target for designing anti-apoptotic agents to alleviate the devastating outcomes of candidiasis.
As explained in the materials and methods section, cross-docking was performed to appraise the possible interaction of ASP, PAO and TRY with Bcl-2 family members of Homo sapiens (5JSN and 5W5X). Concerning their successful interaction, it is worthwhile to note that the virulence factors of C. albicans are able to selectively interact with Bax and Bcl-2 proteins originating from species other than the birds (Gallus gallus).
To the best of the authors' knowledge, this is the first study to target different aspects of systemic candidiasis with the help of the partridge model. Our results show that C. albicans of human origin not only causes systemic candidiasis in the partridge model, but also exhibits adverse clinical consequences, which were similar to human candidiasis. Therefore, partridge and embryonated partridge egg are powerful and attractive preclinical models to study the pathogenesis of candidia spp. because of cost, ease of handling and technical feasibility. The acquired results also indicate that C. albicans alters the normal growth of vessels and changes the expression of apoptoticregulator proteins and genes. The presented data on vascular alteration activity of C. albicans permit us to suggest that this activity is one of the important pathways in the pathogenicity of organism. In this investigation, we also employed the partridge's EEM model for apoptotic studies. This model offers a promising approach to assess the role of various mechanisms, which are associated with the pathogenesis of the diseases.  Center (PTCC), Iran. It was obtained from a human patient and, based on antigenic properties, is categorized to serotype A. This isolate produces D-arabinolactone oxidase, DNA topoisomerase, aspartic proteinases, aspartyl proteinase, estrogen-binding protein, lanosterol synthase, 2, 3-oxidosqualenelanosterol cyclase, phenethyl alcohol, polyamine oxidase and tryptophol. The inoculum was prepared from the mentioned strain in Sabouraud's dextrose broth (Oxoid, Thermo Fisher Scientific, Basingstoke, UK) at 42 °C for 18 h 32 . The broth culture was then centrifuged (2 min at 2500 g) and the remaining pellet was washed three times with sterile phosphate buffered saline (pH 7.4). Finally, the cell suspension was adjusted to an optical density of 10 6 cfu/ml 41 . The cell numbers in inoculum were confirmed by plating serial dilutions on Sabouraud's dextrose agar plates.

This section is explained in terms of
Birds and housing conditions. Day-old chukarpartridge chicks (Alectoris chukar) were obtained from a commercial breeder farm (Hasanzadeh Partridge Farm, Yazd, Iran). Upon arrival at the Animal Research Center of Shahid Bahonar University, Kerman, Iran, the birds were housed in an electrically-heated battery cage (Belderchin Damavand Co. PLC-DQSH, Tehran, Iran) at 34 °C for 10 days and then were held in two floor pens. The partridges were kept 30 days for acclimation. The temperature was controlled and gradually reduced from 34 °C on day 1-29 °C on day 30 and remained constant throughout the study. During the first week of brooding, a photoperiod of 23 h/day was maintained and then a photoperiod of 20 h/day was used. The temperature and photoperiod are necessary for raising and managing partridge chick especially during the first 5 weeks of life. Poor or inadequate temperature and photoperiod is stressful for bird and causing a problem in partridge management. Therefore, temperature and photoperiod were supplied for partridge chicks according to recommended procedures 28 .Food and water were provided ad libitum. The food was formulated according to the nutritional requirements of the partridge chicks from which all medications had been omitted. The ingredients of the diet are presented in Table 3  At 30 days of age, twenty-four partridge chicks were selected on the basis of their overall appearance and body weight uniformity. Blood samples were randomly collected aseptically from the brachial vein of ten birds and serological tests (Hemagglutination-inhibition tests for H9N2 avian influenza subtype and Newcastle diseases) as well as bacteriological cultures were done to assess the pre-challenge health status of the partridges 42 . All sampled birds showed negative results in antibody tests. Negative bacteriological cultures were also obtained. The birds were randomly separated into two different groups of 12 birds each. The partridges of the first group were injected intravenously (in brachial vein) with 0.5 ml of prepared suspension of C. albicans. The birds of the second group were used as the control and were intravenously inoculated with sterile phosphate buffered saline. Infectious dose was chosen based on our preliminary investigation and experimental study inducing systemic candidiasis in Galliformes 32 . The birds were monitored twice daily during the experimental period (5 days after inoculation). Each bird was evaluated individually and efforts were made so that just one person www.nature.com/scientificreports/ could be involved in the clinical evaluation of the birds in order to avoid subjectivity in the analysis. Cloacal temperature and body weight were also recorded. Cloacal temperature was measured four times daily using a digital medical thermometer (FT15/1, Beurer Company, Ulm, Germany, with a range between 35.5 and 42 °C, measurement accuracy ± 0.1 °C). Body weight was measured by a digital scale (Sartorius TE212, Germany, with a range up to 200 g, measurement accuracy ± 0. 01 g). On day 35 of the experiment, the birds were necropsied and tissue samples were taken for pathological investigations. After being fixed in 10% neutral buffered formalin, serial sections of paraffin-embedded tissues were prepared and processed routinely for hematoxylin and eosin (H&E) as well as periodic acid-Schiff (PAS) staining. Tissues of the kidneys, liver, spleen, lungs, pancreas, heart and brain were examined.

Induction of systemic candidiasis in partridge embryo model. Fertile partridge eggs, of the breed
Alectoris chukar, were incubated in an electrical incubator (General Cocks, Cocksmachine Company, Tehran, Iran) at 37.5 °C and 60% relative humidity. Prior to the fungal inoculation, eggs were checked for embryonic development by candling. On the10th day of incubation, C. albicans was inoculated into the chorioallantoic sac and embryos were allowed to develop for a few more days. For C. albicans inoculation, embryo viability was confirmed prior to inoculation by candling, and infertile and nonviable embryos were removed. For all embryos, the air cell was marked, and the egg-shell was disinfected with 70% ethanol. A 1-2-mm diameter region of the shell just above the air cell marking was penetrated using an electric drill, and the chorioallantoic sac was inoculated via a 1-ml syringe with a 1-inch, 22-ga needle. The 10th day of the embryonic stage was chosen based on our preliminary experiment. On the other hand, on the10th day of incubation, the partridge's chorioallantoic sac is large enough to inoculate an appropriate volume of fungus. The embryos were examined at 3 different time intervals: 24, 48 and 72 h after inoculation. Finally, the embryos were humanely killed by cooling 43 . The eggs were opened at the blunt end and embryos were removed to study any gross abnormalities on the external body surface The experiment was repeated for a total of two trials to ensure the repeatability of the experiment.

(B) Effect of C. albicans on vasculature
In this study, we used the EEM partridge model for evaluating the effect of C. albicans on vasculature and apoptotic-related proteins. In recent years, the EEM of Gallus gallus has provided a valuable model for in vivo evaluation of the vascular toxicity of the agents 44,45 . It is also used as an alternative host model for fungal pathogens 43,46 . Hence, we used the EEM partridge model to investigate the effect of C. albicans on the vasculature and apoptotic-related proteins. This effect was assessed via morphometric analysis of vascular pattern from the partridge EEM. Immunohistochemistry (IHC) and quantitative real-time PCR (qPCR) assays were also performed to evaluate the expressions of apoptotic-regulator proteins and genes following fungus treatment. The details are as follows: Experimental design and image acquisition. Fertile partridge eggs were incubated at 37.5 °C and 60% relative humidity. On the 10th day, C. albicans was inoculated onto the EEM. After infection, the shell was cut with scissors and a window of 15 × 15 mm was opened to allow image acquisition. High-resolution images (5312 × 2988 pixels) were captured from the EEM vasculature. Afterwards, an area of approximately 10 × 10 mm of partridge's EEM was removed and subjected to IHC staining. Vascular pattern analysis. Image analysis was performed using computerized software such as MAT-LAB (Mathworks Matlab R2015a) and ImageJ 1.48 (National Institutes of Health, Bethesda, Maryland, USA). Initially, a defined area (125 mm2) of the EEM was extracted and the contrast was improved (Fig. 2III-a). Effort has been made to select a constant area in each image. The extracted area was converted to a binarized format ( Fig. 2III-b). Then, the areas without any branch vessels were selected for analysis. Five such areas per case were identified and the percentage of the areas containing black pixels were quantified (Fig. 2III-c). The black pixels of the images indicate the red color, or blood, in the original images. The mean of all areas calculated in each image is described as the mean capillary area (MCA) 33 .
Immunohistochemistry assay. The samples of the partridge's EEM were fixed in 10% buffered formalin and embedded in paraffin. Tissue sections were made by the microtome (Slee Germany) and IHC staining was performed for Bcl-2 (mouse monoclonal antibody, American, ID number: PDMO16-lotH147) and Bax (Zytomed Germany, ID number: 502_17990) markers. The expression levels of Bcl-2 and Bax were assessed by counting the stained cells and calculating the mean in 10 high-power fields (400 ×).
Gene expression. The effect of C. albicans on the apoptotic-regulator genes, in vessels, was evaluated by qPCR assessment of relative expression levels of Bax, Bcl-2, VEGF-A and VEGF-R2 genes. Briefly, the total RNA of the partridge EEMs was isolated using the RNeasy mini kit (Qiagen, USA). A nano-drop was used to evaluate the quality of samples (ND-1000, Thermo Scientific Wilmington, DE, USA) and the cDNA was synthesized using a total of 500 ng RNA by RT reagent kit (Takara, Clontech) according to the protocol. Finally, qPCR was performed using a SYBR Green assay (SYBR Premix Ex Ta II; Japan). The specific primers and reference gene sequences are listed in Bellow. The simulated proteins were further assessed using Ramachandran plot at PROCHECK server https ://servi cesn.mbi.ucla.edu/PROCH ECK/)31.

Protein-protein interactions.
On the protein surfaces, the active binding pockets were predicted based on computational geometry theories using CASTp server (http://sts.bioe.uic.edu/castp /). Following the pocket identification, the interactions of ASP, PAO and TRY with Bcl-2 and Bax proteins were assessed via HEX 8.0.0 software (http://hex.loria .fr/dist/index .php). This software is introduced in the Critical Assessment of Predicted Interactions (CAPRI, http://capri .ebi.ac.uk/) as a high-quality docking program based on the fast Fourier transform approach 54 .
The cross-dockings were also performed to evaluate the affinity of ASP, PAO and TRY in a way that Bcl-2 and Bax proteins are linked to specific inhibitor or activator. For this purpose, the molecular structures of 5JSN (crystal structure of Bcl-2 in complex with specific inhibitor) and 5W5X (crystal structure of Bax in complex with specific activator) were selected from the Protein Data Bank (Fig. 5III-a and b). After the separation of the ligands from the coordinates of the receptors, the affinity among ASP, PAO and TRY with 5JSN and 5W5X were assessed. Eventually, a validation step was made by self-dockings among 5JSN, 5W5X and their specific ligands. This step was considered as the key of docking accuracy. Molecular dynamics simulation. Molecular dynamics simulation was performed using the GROMACS 5.4.1 package http://www.groma cs.org/ to assess the conformational property of simulated proteins (ASP, PAO, TRY, Bcl-2 and Bax) and their interactions in the simulated body environment. Briefly, the docked structures of ASP, PAO and TRY with Bcl-2 and Bax were subjected to the GROMOS 54A7 force field and the water model SPC was used as the solvent. The solvated system was defined in a charge neutralized system by adding ions (Na or Cl ions). The cubic solvent box was considered and solvated by explicit water using the gmx solvate algorithm. The system was minimized for 50,000 steps using the steepest descent algorithm and then simulation www.nature.com/scientificreports/ was performed at 310 K and 1 bar under periodic boundary conditions. The v-rescale and Parrinello-Rahman algorithms were used for temperature and pressure coupling, respectively 55,56 . Electrostatic interactions were calculated by the particle-mesh Ewald method 57 . During the production phase, conformers were stored every 0.002 ps. The stability of the structure during the simulations was investigated by calculating the radius of gyration using the tool gmx gyrate of the GROMACS package. The h-bonds, Van der Waal's interactions, solvent accessible surface area and density of structures were also analyzed.
Statistical analysis. Statistical analysis was performed using SPSS version 20. The Fisher's exact and repeated measurement tests were used to determine the significant differences in lesion occurrence and the body weight between experimental groups, respectively. The T test was applied to assess the significant differences in the MCA values. A p value of < 0.05 was considered statistically significant. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.