






































Type of the Paper (Article


 

  

 

 
Cluj Vet J 2024, vol 29, issue 1 http://clujveterinaryjournal.ro 

Research Article 

Correlating Eosin Fluorescence Patterns and Basophilic Alterations in a DEN-Induced HCC 
Murine Model Through Confocal Microscopy  
 
Romelia Pop 1*, Dragoș Hodor1, Cornel Cătoi1, Teodora Mocan2,3, Lucian Mocan2,4 and Alexandru-Flaviu Tăbăran1 

 
 1 Department of Pathology, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary 

Medicine of Cluj-Napoca, Calea Manaștur, 400372 Cluj-Napoca, Romania; romelia.pop@usamvcluj.ro; dra-
gos.hodor@usamvcluj.ro; cornel.catoi@usamvcluj.ro; alexandru.tabaran@usamvcluj.ro; 

2 Nanomedicine Department, Regional Institute of Gastroenterology and Hepatology “Octavian Fodor”; 
3 Department of Physiology, University of Medicine and Pharmacy, “Iuliu Hatieganu”; teodora.mocan@umfcluj.ro 
4 3-rd Surgery Clinic, University of Medicine and Pharmacy, 400162 Cluj-Napoca, Romania; lu-

cian.mocan@umfcluj.ro  
*Correspondence: romelia.pop@usamvcluj.ro; Tel: +40744263975 

Abstract: Hepatocellular carcinoma is a major global health concern and a leading cause of cancer-related mortality. 
This study employed chemically induced murine models to simulate human hepatocellular carcinoma, utilizing 28 
albino Swiss mice with the administration of a single dose of the complete carcinogen diethylnitrosamine (DEN) at 
100mg/kg, followed by serial euthanasia. The focus was on understanding early hepatocellular alterations and distin-
guishing precursor changes from incidental changes. The assessment of hepatocellular alteration foci relied on hema-
toxylin and eosin (H&E) staining. However, the research introduced a novel approach by exploiting the fluorescence 
properties of eosin, a component of the H&E stain. This approach was applied to investigate basophilic alteration foci 
indicating alterations in cellular composition exhibiting notably heightened RNA staining. To achieve this, confocal 
laser scanning microscopy was employed, correlating changes in fluorescence intensity with tinctorial alterations. The 
histological examination revealed that basophilic foci displayed unique nodular lesions with intense basophilic cyto-
plasm. Nuclear alterations, including hyperchromasia and basophilia, contributed to understanding cellular and nu-
clear changes in these foci. Statistical analysis highlighted a discernible reduction in eosin fluorescence intensity within 
basophilic foci compared to normal hepatic tissue. 

 
Keywords: hepatocellular carcinoma; animal model; basophilic foci; histology, eosin, confocal microscopy 

1. Introduction 

Hepatocellular carcinoma (HCC) ranks as the fifth most prevalent neoplasm 
in humans worldwide and stands as the primary cause of cancer-related mortality 
in specific regions [1]. This reality drives continuous research directed at formu-
lating innovative therapies and diagnostic methods. The investigation into HCC 
mechanisms and the assessment of potential treatments heavily rely on animal 
models. Apart from spontaneous models involving animals like dogs and wood-
chucks, chemically induced rodent models take a central position in HCC re-
search [2-5]. The administration of the carcinogenic and mutagenic compound 
Diethylnitrosamine (DEN) (CH3-CH2)2N-N=O) through parenteral or oral routes 
in experimental models animals is particularly utilized for its significant transla-
tional value in understanding the complex profile of aggressive forms of hepato-
cellular carcinoma (HCC) in humans [6]. DEN-induced hepatic tumors in rodents, 
through various mechanisms such as epigenetic and genetic changes, typically 
undergo a sequential progression. This starts with an initial phase of inflamma-
tion and necrosis, leading to long-term hepatocellular hyperplastic and dysplastic 

Received: 14 December 2023 
Accepted: 15 March 2024 
Published: 9 April 2024 

DOI:10.52331/nqpej009 

 

 

 
Copyright: © 2024 by the authors. 
Submitted for possible open access 
publication under the terms and 
conditions of the Creative Commons 
Attribution (CC BY) license 
(http://creativecommons.org/licenses
/by/4.0/). 

mailto:romelia.pop@usamvcluj.ro;
mailto:dragos.hodor@usamvcluj.ro;
mailto:dragos.hodor@usamvcluj.ro;
mailto:alexandru.tabaran@usamvcluj.ro
mailto:teodora.mocan@umfcluj.ro


Cluj Vet J 2024, vol 29, issue 1 17 of 37 
 

changes, defined as "alteration foci," ultimately resulting in neoplastic transformation, including hepa-
tocellular adenoma and carcinoma [7,8]. The hepatocellular "alteration foci" are identified as prene-
oplastic changes, characterized by their distinctive staining patterns on the conventional hematoxylin 
and eosin (HE) stain. While both acidophilic and basophilic foci are referenced in the context of hepa-
tocellular alteration, in cases of HCC induced by DEN, the "basophilic foci" emerge as the primary 
preneoplastic phenotype changes in hepatocytes. Despite being mechanistically complex, the hepato-
cellular basophilic phenotype observed in alteration foci in DEN-induced HCC is attributed to meta-
bolic shifts within cells. This reflects a progressive transition from anabolic to catabolic glucose metab-
olism, indicative of the Warburg effect, to sustain irregular cell proliferation [9]. A significant problem 
in comprehending the progression of chemically induced hepatocellular carcinomas in rodents is dis-
tinguishing early hepatocellular alterations that serve as precursors to carcinomas from those that are 
incidental or secondary phenomena [10]. The conventional method for defining and assessing hepa-
tocellular alteration foci [11] traditionally relies on H&E staining. This classic staining technique, em-
ployed in all regulatory toxicological studies in animal models, utilizes hematoxylin (C16H14O6) to stain 
anionic components (like DNA and RNA). Simultaneously, the xanthene pigment eosin Y 
(C20H6Br4Na2O5) is applied to stain cationic compounds (such as proteins). Eosin Y binds electrostati-
cally to the carboxylic and phenolic groups of arginine, histidine, lysine, and tryptophan residues [12]. 
Interestingly, Eosin is produced through the bromination of fluorescein, displaying a fluorescence 
photoactivity similar to its parent compound [13]. This unique fluorescence characteristic of eosin has 
been recently utilized as a diagnostic tool for quantifying liver injury [14]. Moreover, eosin serves as a 
fluorescent pH indicator, and certain derivatives are employed as reactive fluorescent labels. Eosin Y 
and fluorescein are applied as benchmarks for quantum yield, photosensitizing agents, laser dyes, and 
labeling biological specimens [15]. The present work aims to systemically assess the changes in the 
eosin fluorescence pattern in the preneoplastic, basophilic hepatic nodules obtained in a DEN-induced 
HCC murine model.2. Materials and Methods 

Animals 

The study involved 28 albino Swiss mice, young males, with an average weight of around 25g. 
Mice of the Swiss strain were purchased according to the regulations in force from the Iuliu Hațieganu 
UMF Animal Facilities Cluj-Napoca. These mice were housed in plastic cages with unrestricted access 
to food and water, under conditions of 22–23°C temperature, 55% humidity, and a 12-hour light/dark 
cycle. They were provided with standard pelleted rodent feed manufactured by Cantacuzino National 
Insitute for Medical-Military Research and Development. The research protocol received approval 
from DSVSA (National Veterinary Medicine Authority no. 267/12.07.2021). All procedures related to 
the use of laboratory animals adhered to the guidelines and European regulations outlined in EU Di-
rective 2010/63/EU and Romanian law 43/2014. 

Experimental design 

The procedure for inducing liver carcinoma was modified from the method outlined by Sun et al. 
in 2012 [16], involving the following steps. The mice were assigned to four groups (Control, G1, G2, 
and G3), each with seven mice. Before each injection, mice were weighed to ensure accurate calculation 
of the diethylnitrosamine (DEN) dose. A single intraperitoneal dose of diethylnitrosamine at 100mg/kg 
was administered. The progression of hepatic alterations and HCC was monitored over time by sys-
tematically euthanizing animals at 2 months (G1), 5 months (G2), and 8 months (G3) post-DEN ad-
ministration. The mice were euthanized with prolonged exposure to isoflurane. The spontaneous 



Cluj Vet J 2024, vol 29, issue 1 18 of 37 
 

deaths were not recorded during this experiment.The analysis of eosin fluorescence was carried out 
on the first and second sacrificed groups in which the presence of basophilic foci was observed.. 

Histopathological analysis 
The liver was sampled for subsequent analysis and fixed in 10% formalin for 48 hours. Samples 

were fixed, dehydrated, and clarified in ethyl alcohol and xylene. Paraffin infiltration at 58°C for 5 
hours was done, and 2 μm-thin sections were obtained. For staining, slides underwent xylene immer-
sion, ethanol solutions, hematoxylin, and eosin Y. Dehydration included ethanol immersions, and 
clearing was done with xylene. Finally, Permount and coverslips were applied for microscopic obser-
vation of stained tissue sections. [17]. Histological examination of the samples was conducted using 
an Olympus BX51 microscope, and bright field images were captured using an Olympus SP350 digital 
camera, and processed using the Olympus cellSens software. The assessment of liver histopathology 
was performed based on the International Harmonization of Nomenclature and Diagnostic (IN-
HAND) histological criteria standards for rodents [11.]Chemical and Reagents 

 N-Nitrosodiethylamine solution was purchased from Sigma (Lot# 049K1613). H&E staining kit 
was acquired from Abcam (H&E Staining Kit ab245880). 

Confocal scanning laser microscopy (CSLM) analysis 
Confocal fluorescent pictures were captured utilizing a Zeiss LSM 710 confocal laser scanning 

module installed on an Axio Observer Z1 Inverted Microscope. To visualize cell structures, a 488 ex-
citation laser line was employed to detect Eosine (BP 493-625 nm emission). All pictures were taken 
with a Plan Apochromat 63× (1.4; oil immersion, DIC M27) Zeiss objective. Standard ZEN software 
provided by the Zeiss system manufacturer was utilized for image merging, processing, and analysis. 
Fluorescence values were denoted in arbitrary units (AU). 

Eosine quantification by CLSM 
The assessment of Eosine expression in the liver followed a methodology similar to our previous 

work [18]. Quantitative analysis of the confocal images was automated using the point-by-point fluo-
rescence quantification functions within the ZEN software. To ensure consistent quantification condi-
tions and maintain high reproducibility, CLSM image acquisition was standardized and upheld 
throughout the experiment (acquisition time 30 s, lasers' output power 20%, pinhole diameter 53 μm, 
master gain 420, digital gain 15 Each image section measured 512 × 512 pixels (135× 135μm), covering 
an area of 18.225 μm² for every scanned microscopic field. Mean fluorescence was assessed on five 
basophilic foci by examining two fields (obx63) per focus (totaling 10 fields with basophilic foci). In a 
similar matter, the perilesional liver for each basophilic focus was assessed in 2 histological fields from 
the same histological slides. Additionally, measurements on 10 fields from an uninjected mouse serv-
ing as the control were conducted Statistical analysis 

Statistical evaluations were conducted utilizing RStudio version 4.3.1. For datasets distribution it 
was used the Shapiro-Wilk normality test. To assess the intensity of the Eosine in the Kruskal-Wallis 
and with Dunn’s post-hoc analysis with Bonferroni correction was utilized. 

3. Results 

Modifications associated with DEN administration were noted in the second and third groups, 
occurring approximately 5 and 8 months following administration. Pathologically, the first group (sac-
rificed at 2 months) showed no visible macroscopic changes, while the second group displayed distinct 
masses in four out of seven examined livers. The masses were white-beige, well-demarcated, measur-
ing up to 3 mm, with a multifocal or multifocal-coalescing arrangement, distributed in all hepatic lobes 
(up to 15 masses /liver). Similarly, in the third experimental group, masses were detected in five out 



Cluj Vet J 2024, vol 29, issue 1 19 of 37 
 

of seven examined livers. In this group, the masses measure up to 1 cm, maintaining the same multi-
focal or multifocal-coalescing arrangement as in group 3. Intratumoral necrosis was occasionally noted 
in the masses of this group. Histologically, the basophilic foci, (randomly distributed and predomi-
nantly observed within the initial two groups and evaluated in the context of this experiment), consist 
of well-defined, nodular, circular, un-encapsulated lesions characterized by hepatocytes organized in 
interconnected cord-like structures, exhibiting minimal cellular polymorphism. These foci promi-
nently displayspecific tinctorial staining patterns and granularity. The global tissue architecture within 
the basophilic foci experiences a moderate level of disruption. The cellular components in these foci 
may appear darker (basophilic) compared to adjacent tissues. The majority of hepatocytes within the 
basophilic foci exhibit a prominent abundance of granular and intensely basophilic cytoplasm. Note-
worthy histological features encompass nuclear alterations, including occasional nuclear enlargement, 
and heightened chromatic intensity (hyperchromasia). The nuclei within these foci reveal vacuolated 
chromatin, and in some instances, the presence of 1-2 distinct basophilic nucleoli. These observed his-
topathological characteristics collectively contribute to the comprehensive understanding of the cellu-
lar and nuclear alterations inherent in basophilic following DEN administration (Figure 1 image A, B, 
C). 

 

 

Figure. 1. Histological images (A, B and C) and Confocal laser (D, E, and F) of the mice liver, 
comparing the hepatocellular preneoplastic basophilic alteration foci (BF) with the normal liver pa-
renchyma (NL). Image A. Histological micrographs showing a well-demarcated, subscapular hepato-
cellular basophilic focus (BF), characterized by tortuously arranged hepatic chords, with hepatocytes 
showing increased cytoplasmic basophilia relative to adjacent normal parenchyma, without signifi-
cant compression of the bordering tissue. Image B. The hepatocytes of the basophilic focus, are 



Cluj Vet J 2024, vol 29, issue 1 20 of 37 
 

characterized by bands of localized, clumped basophilic bodies in the peripheral cytoplasm with clear 
intervening areas ("tigroid" pattern).  Image B. Hepatocytes from the control group, showing normal 
patterns of cytoplasmic staining. Image D. Confocal images of the basophilic focus (BF) presented in 
image A, showing reduced overall fluorescence compared with the adjacent hepatic tissue (NL). Im-
age E. Confocal images of the hepatocytes from the basophilic focus, showing a reduced cytoplasmic 
fluorescence compared with the fluorescence observed in the adjacent, normal liver (image F). CLSM 
objective 10 (image D) and x63×/1.4 Oil Plan Apochromatic objective (images E and F); H&E images, 
ob x10 (images A) and x 100 (images B and C). Scale bar=20μm.  

Concerning the CLSM manifestation of Eosin expression, a discernible reduction in fluorescence 
intensity was evident when comparing normal hepatic tissue to basophilic foci. Within the confines of 
a morphological normal liver, the fluorescence intensity of Eosin exhibits a visibly elevated profile as 
opposed to the diminished Eosin fluorescence encountered within basophilic foci, thereby indicating 
a discernible alteration in Eosin fluorescence within the latter. (Figures 1 Image D,E,F, and Figure 2,). 

 

 

Figure. 2. Confocal laser scanning microscopy micrographs of liver tissue, presenting the basophilic 
foci (images A and B) and their fluorescence spectra (image E) compared with the normal adjacent 
liver parenchyma (images C, and D) and its fluorescent spectra (image F). The fluorescence inten-
sity distribution histograms (images E and F) show the altered expression of the Eosin/fluorescein 
within the hepatocytes of the basophilic foci compared with normal hepatic tissue. The Eosin/flu-
orescein expression = red channel. Images B and D compare the fluorescence expression of Eo-
sin/fluorescein as a heatmap in the basophilic foci (B) compared with normal tissue hepatic (D). 
Warmer colors indicate higher Eosin/fluorescein expression intensities. 63×/1.4 Oil Plan Apochro-
mate objective. 

 



Cluj Vet J 2024, vol 29, issue 1 21 of 37 
 

Statistically, the Shapiro-Wilk test indicates that the data for all groups are normally distributed. 
Posthoc Dunn's test with Bonferroni correction revealed significant differences between the "Baso-
philic foci" group and both the "Normal liver (control)" and "Normal liver (perilesional)" groups, but 
not between the latter two. Specifically, the comparison of basophilic foci with normal liver (control) 
yielded a P-value of 0.00485, while the comparison with normal liver (perilesional) also resulted in a 
P-value of 0.00485. Despite the data's normality, the Kruskal-Wallis test revealed significant differ-
ences among the groups' medians, with post-hoc Dunn's test confirming significant disparities be-
tween the "Basophilic foci" group and both "Normal liver (control)" and "Normal liver (perilesional)" 
groups, albeit not between the latter two. 

 

Figure 3. Image A. represets Comparative Analysis of Eosin Median Intensity Across Liver Tis-
sue Conditions in Mice. Box plot A representation of the median intensity of eosin staining 
across three distinct liver tissue conditions in mice: Basophilic foci (BF), Normal liver (control, 
NLC), and Normal liver (perilesional, NLPL). The eosin median intensity is markedly lower in 
Basophilic foci (BF) compared to Normal liver (control, NLC), suggesting a diminished central 
tendency of eosin intensity in BF. In contrast, the median intensity in Normal liver (perilesional, 
NLPL) is elevated, indicating a higher eosin affinity or increased density of staining in perile-
sional compared to control tissues. These observations may reflect differential eosinophilic ac-
tivity or structural variations among the hepatic conditions examined. Image B represents Var-
iability in Eosin Staining Intensity Among Liver Tissue Conditions in Mice. This box plot illus-
trates the variability, as measured by standard deviation, of eosin staining intensity within three 
different liver tissue conditions in mice: Basophilic foci (BF), Normal liver (control, NLC), and 
Normal liver (perilesional, NLPL). The standard deviation is substantially lower in the Baso-
philic foci (BF) group, suggesting more homogeneity in staining intensity, whereas the Normal 
liver (perilesional, NLPL) exhibits a higher standard deviation, indicating greater variability in 
eosin uptake or distribution. The Normal liver (control, NLC) shows intermediate variability. 
These differences in standard deviation may imply distinct histological characteristics or differ-
ential responses to staining due to the underlying pathology of the tissue."  

 



Cluj Vet J 2024, vol 29, issue 1 22 of 37 
 

 

Figure 4. Image A represents Evaluation of Maximum Eosin Staining Intensity in Diverse Liver 
Tissue States in Mice. The box plot showcases the maximum intensity of eosin staining within three 
liver tissue states: Basophilic foci (BF), Normal liver (control, NLC), and Normal liver (perilesional, 
NLPL). The Basophilic foci (BF) group exhibits a lower range of maximum intensity, suggesting a 
restricted eosin presence or a lesser degree of staining peak. In contrast, the Normal liver (perile-
sional, NLPL) displays a substantially higher maximum intensity, indicating intense eosinophilic 
engagement or staining concentration, potentially reflective of heightened inflammatory or morpho-
logical alterations in perilesional tissue. Image B represents Assessment of Minimum Eosin Staining 
Intensity Across Varied Liver Conditions in Mice. The box plot depicts the minimum intensity of 
eosin staining for Basophilic foci (BF), Normal liver (control, NLC), and Normal liver (perilesional, 
NLPL) conditions in murine hepatic tissue. The minimum intensity levels are notably higher in the 
Basophilic foci (BF) and Normal liver (perilesional, NLPL) groups compared to the Normal liver 
(control, NLC), indicating a greater baseline level of eosin staining in these conditions. This could 
suggest a floor effect of eosinophil infiltration or binding affinity in the BF and NLPL tissues." 

 

4. Discussion 

In rodent toxicity experiments involving specific xenobiotics, cellular changes known as baso-
philic foci are observable alterations in liver tissue. These changes, visible under stains like H&E, 
reveal unique characteristics in terms of cell type, staining patterns, and texture, offering insights 
into liver morphological variations linked to hepatocarcinogenesis [19]. Basophilic foci typically 
appear as circular or ovoid lesions, differing noticeably from normal liver tissue in staining reac-
tions and cellular appearance. The differentiation of basophilic foci types, based on staining attrib-
utes, hepatocyte size, and texture, aids in understanding the complex cellular transformations 
within the liver. While maintaining lobular architecture, these foci may vary in portal areas and 
central veins depending on their size. Compression of sinusoids within the foci can affect the de-
tection of typical parenchymal plates, and increased cellular numbers may lead to the development 
of tortuous hepatic cords [11]. 



Cluj Vet J 2024, vol 29, issue 1 23 of 37 
 

Early stages of hepatocarcinogenesis induced by DEN administration involve molecular 
changes highlighted by Watanabe et al. [20], revealing mRNA networks associated with cancer-
related genes, cell cycle regulation, and cell death. The progression from pre-neoplastic foci to dis-
tinct phenotypes such as basophilic, eosinophilic, or clear cell foci is linked to metabolic turnover, 
with specific molecular events driving this transition. Elevated insulin growth factor 2 (IGF-2) levels 
and downstream signaling play key roles in shaping glycogen storage phenotypes, ultimately lead-
ing to the basophilic phenotype [21]. Initially, exposure to DEN elevates insulin IGF-2 levels, initi-
ating downstream signaling that reduces glucose-6-phosphatase (G6Pase) activity, resulting in gly-
cogen storage phenotypes. Moreover, IGF signaling activates the Ras/Raf mitogen-activated signal-
ing cascade, promoting cell proliferation [22]. Over time, the foci transition from anabolic to cata-
bolic glucose metabolism to support cell proliferation, culminating in the development of the baso-
philic phenotype. Some altered hepatocyte foci (AHF) exhibit mutations in Hras and Braf onco-
genes, potentially providing a growth advantage, as these molecular alterations are more frequently 
observed in late-stage neoplastic lesions [23]. Braf mutations induce ERK1/Akt hyperphosphoryla-
tion, leading to the induction of pro-survival/pro-proliferative complement component C5/C5a in 
basophilic foci [24]. Consequently, AHF are generally considered putative preneoplastic lesions in 
chemically induced models, although the full significance of morphologically similar lesions in hu-
man hepatocarcinogenesis remains incompletely understood. 

The development of hepatocellular carcinoma (HCC), involving initiation, promotion, and pro-
gression, is influenced by interactions among genetic, epigenetic, and environmental factors. Initi-
ation often arises from genetic mutations induced by various carcinogens, such as aflatoxin, hepa-
titis B or C viruses, alcohol, metabolic disorders, and inflammation, causing DNA damage [26,27]. 
Chronic liver diseases create a conducive environment for HCC promotion, leading to the accumu-
lation of genetic abnormalities and preferential selection of growth-advantaged cells. Subsequent 
genetic modifications in the promotion phase activate oncogenes, facilitate cell growth, and deacti-
vate tumor suppressor genes, collectively contributing to unregulated cell multiplication [28,29]. As 
tumors progress, angiogenesis is triggered for continued growth, enabling infiltration into adjacent 
tissues and dissemination to distant organs, involving alterations in adhesion, migration, and inva-
sion. HCC strategically evades the immune system, avoiding detection and elimination by the 
body's innate defense mechanisms [30,31]. 

Traditional staining methods, like hematoxylin-eosin, and selective fluorescence reactions, no-
tably with eosin Y, provide valuable tools for histological examination. Eosin Y's versatility extends 
beyond histology, serving as a fluorescent dye for various cellular structures, highlighting its utility 
in diverse applications [13,32]. By comparing optical images of basophilic alteration foci stained by 
H&E with their confocal fluorescence spectra, a correlation between the loss of fluorescence inten-
sity and loss of hepatocyte tinctorial affinity for eosin (defined as "basophilia") is observed. These 
findings are significant from a diagnostic perspective, employing confocal laser scanning micros-
copy (CLSM) as a tool in toxicological pathology, aiding in establishing the link between tinctorial 
changes of basophilic foci (preneoplastic changes) and their fluorescence spectral shifts 

5. Conclusions 

Following this study, we managed to demonstrate the presence of alterations in the fluores-
cence spectrum of eosin when bound to proteins in both normal and tumoral liver tissues. This 
highlights how fluorescence spectroscopy can potentially complement histopathological observa-
tions and unveil information that aligns with classical H&E staining methods. Given its inherent 



Cluj Vet J 2024, vol 29, issue 1 24 of 37 
 

sensitivity to changes in the biomolecular composition across diverse cell and tissue types, fluores-
cence microscopy can offer information surpassing that of individual conventional diagnostic tech-
niques. Anticipating widespread applications, we envision that this fluorescence imaging approach 
will prove valuable in diverse fields, including cell biology, biomedical analysis and diagnosis, and 
the chemical identification of various components within cells and tissues. 

 
Author Contributions: RP, AFT, and TM carried out the HCC protocol. RP and AFT carried out the histological and 
CLSM assessment and drafted and reviewed the manuscript. DH was involved in the data analysis and statistics. CC 
and LM reviewed the manuscript. All authors read and approved the final manuscript. 

Funding: The authors wish to acknowledge funding granted by the National Authority for Scientific Research and In-
novation Romania, CNCS-UEFISCDI, cod PN-III-P2-2.1-PED-2021-0073 and PN-III-P2-2.1-PED-2019-0997. 

Institutional Review Board Statement: The experimental protocol has been approved by DSVSA (National Veterinary 
Medicine Authority no. 267/12.07.2021). All procedures involving the use of laboratory animals followed the guidelines 
and European norms 337 established by EU Directive 2010/63/EU and Romanian law 43/2014 

Data Availability Statement: The data that support the findings of this study are available from the Department of 
Veterinary Pathology, University of Agriculture Science and Veterinary Medicine. Data are, however available from the 
authors upon reasonable request and with the permission of the Department of Veterinary Pathology, University of 
Agriculture Science and Veterinary Medicine. 

Acknowledgments: Not applicable  

Conflicts of Interest: The authors declare no conflict of interest. 

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