animals Article Stages of Granulomatous Response Against Histozoic Metazoan Parasites in Mullets (Osteichthyes: Mugilidae) Marta Polinas 1, *, Francesc Padrós 2 , Paolo Merella 1 , Marino Prearo 3 , Marina Antonella Sanna 1 , Fabio Marino 4 , Giovanni Pietro Burrai 1,5 and Elisabetta Antuofermo 1,5 1 Department of Veterinary Medicine, University of Sassari, 07100 Sassari, Italy; paolomerella@uniss.it (P.M.); msanna@uniss.it (M.A.S.); gburrai@uniss.it (G.P.B.); eantuofermo@uniss.it (E.A.) 2 Fish Diseases Diagnostic Service, Facultat de Veterinaria, Universitat Autonoma de Barcelona, 08193 Barcelona, Catalonia, Spain; francesc.padros@uab.cat 3 Fish Disease Laboratory, State Veterinary Institute of Piedmont, Liguria and Aosta Valley, 10154 Torino, Italy; marino.prearo@izsto.it 4 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, 98166 Messina, Italy; fabio.marino@unime.it 5 Mediterranean Center for Disease Control (MCDC), University of Sassari, 07100 Sassari, Italy * Correspondence: mpolinas@uniss.it; Tel.: +39-(079)-229566 Simple Summary: Parasitic diseases represent a common issue in fish and, when histozoic forms are present, this elicits a chronic inflammatory reaction leading to granuloma formation. Despite the large knowledge of granuloma formation due to parasites in visceral organs, little is known about the  development and the evolutive stages of granulomas in naturally infected fish. Mullets (Osteichthyes:  Mugilidae) are a widespread euryhaline fish species that harbor different parasites, thus representing Citation: Polinas, M.; Padrós, F.; a suitable model for the study of parasite-induced granulomas. Combining histopathology and Merella, P.; Prearo, M.; Sanna, M.A.; immunohistochemical tools, we identified three developmental granuloma stages (pre-granuloma, Marino, F.; Burrai, G.P.; Antuofermo, intermediate, and late stage), that ranged from an intact parasite with mild signs of tissue reaction to E. Stages of Granulomatous Response the formation of a structured granuloma. The identified histological patterns could be reliable tools Against Histozoic Metazoan Parasites in the staging of the granulomatous response associated with histozoic parasites and are an attempt in Mullets (Osteichthyes: Mugilidae). to broaden the knowledge of the inflammatory response in different host–parasite systems. Animals 2021, 11, 1501. https:// doi.org/10.3390/ani11061501 Abstract: Histozoic parasite–fish host interaction is a dynamic process that leads to the formation of a granuloma, a specific chronic inflammatory response with discernible histological features. Mullets Academic Editor: Maria José Caballero (Osteichthyes: Mugilidae) represent a suitable model concerning the development of such lesions in the host–parasite interface. The present work aimed to identify granuloma developmental stages from Received: 13 April 2021 the early to the late phase of the infection and to characterize the immune cells and non-inflammatory Accepted: 19 May 2021 components of the granuloma in different stages. For this purpose, 239 mullets were collected from Published: 21 May 2021 4 Sardinian lagoons, and several organs were examined by combining histopathological, bacterio- logical, and immunohistochemical methods. Granulomas associated with trematode metacercariae Publisher’s Note: MDPI stays neutral and myxozoan parasites were classified into three developmental stages: (1) pre-granuloma stage, with regard to jurisdictional claims in characterized by intact encysted parasite and with no or mild tissue reaction; (2) intermediate stage, published maps and institutional affil- with partially degenerated parasites, necrosis, and a moderate number of epithelioid cells (ECs); iations. and (3) late stage, with a necrotic core and no detectable parasite with a high number of ECs and fibroblasts. The three-tier staging and the proposed morphological diagnosis make it conceivable that histopathology could be an essential tool to evaluate the granulomas associated with histozoic parasitic infection in fish. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Keywords: disease staging; wild fish; immunohistochemistry; protozoan; Trematoda This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Animals 2021, 11, 1501. https://doi.org/10.3390/ani11061501 https://www.mdpi.com/journal/animals Animals 2021, 11, 1501 2 of 18 1. Introduction The chronic inflammatory response is a non-specific reaction against a multitude of fac- tors, such as harmful substances, bacteria, mycobacteria, fungi, and parasites, particularly common in fish [1–14]. Chronic inflammation is a complex combination of immune cell action that involves mainly macrophages, epithelioid cells (ECs), fibroblasts, and piscine mast cells (MCs) usually organized as a granulomatous reaction to isolate the pathogens or foreign materials [15,16]. Phagocytosis is a primordial and preserved mechanism of innate immunity in vertebrates, mainly exerted by macrophages, that are considered specialized phagocytes [17,18]. When the noxa overcomes the phagocytic ability of the host, the organ- ism triggers more complex mechanisms to confine the intruder into host tissues, leading to granuloma in affected organs [18]. The size, structure, organization, and composition of these granulomas may present differences according to the causative agent. In natural and experimental infection due to bacterial and particularly mycobacterial and oomycetes species, granulomas have been described as a core of macrophages and bacteria associated with central necrosis, surrounded by a fibrous layer mostly evident in the later stages of infective granulomas [19–21]. Unlike mammals, in fish, the presence of giant cells has been only occasionally described in parasite granuloma [22,23]. It is well known that also some histozoic parasites (e.g., amoebae, myxozoans, larval stages of trematodes, and nematodes) can elicit chronic granulomatous inflammation [24]. More details of the cellular components of granuloma and their distribution have been provided by electron microscopy studies, where ECs have been typically found near or in contact with the parasite body or cyst wall [25]. Although considered transformed macrophages, ECs mainly act to confine the pathogen due to the presence of interdigitation and desmosomes and expression of cytokeratins [2,26–28]. Around EC layers, an outer sheet of fibroblasts has been frequently reported, intermingled with collagen fibers, to further reinforce pathogen incarceration and favor tissue remodeling [29–31]. Moreover, fibroblasts, considered for a long time as plain structural components, have been recently elevated to crucial effectors in the progression and resolution of chronic inflammation in humans, through crosstalk with mast cells and eosinophils [32]. Furthermore, ultrastruc- tural observation showed the presence of MCs near or in contact with fibroblasts, in an apparent degranulation state [33]. A role of MCs in promoting collagen production has been proposed in teleosts, similarly to what was observed in humans in association with parasitic diseases, where these cells are involved in the pathogenesis of fibrotic processes via the secretion of profibrotic substances [34]. Rodlet cells (RCs), although not a regular component in parasite granulomas, increase in the course of infectious diseases, parasitic infections, and environmental pollutants [35,36]. Granuloma formation is a dynamic process, and many times the structure of these specific chronic inflammatory responses can give useful information about the timing, evolution, and prognosis of the disease. Nevertheless, detailed studies on granuloma progression against histozoic parasites have not been carried out, although numerous details of parasite granuloma cellular compo- nents have been reported both in saltwater and freshwater fish [10,30,37]. Among brackish water fish species, the mullets (Osteichthyes: Mugilidae) are widely investigated in par- asitological studies [38,39], so they could represent a suitable model to study different host–parasite interaction systems [40,41]. Moreover, as mullets can harbor trematode parasites with a zoonotic potential (i.e., Heterophyidae), several studies have widely in- vestigated the presence of such parasites also concerning human consumption of raw fish [38,42]. However, only a single study on mullets concerning the lesion development in the host–parasite interface after the infection of Labratrema minimus (Trematoda, Bu- cephalidae) metacercariae has been accomplished [43]. The presence of large resident populations of mullets with already described metazoan parasites in Sardinian lagoons [39] allows a unique opportunity for a detailed study of the complexity and dynamics of the host–parasite relationship. Animals 2021, 11, 1501 3 of 18 The present work aims to characterize the histological features of the granulomatous inflammatory response in visceral organs of mullets naturally infected with trematode and myxozoan parasites. For this purpose, different developmental stages of granuloma, from the early to the late phase of the histozoic parasite infection, were identified using discernible histological features, and afterward, the immune cells and other non-inflammatory components associ- ated with the different stages of the granuloma were characterized by histochemical and immunohistochemical tools. 2. Materials and Methods 2.1. Sampling From July to October 2014, 239 mullets were collected by fishing nets from 4 different coastal lagoons in Sardinia (western Mediterranean Sea): 60 from Calich lagoon (40◦ 350 46.1” N 8◦ 170 59.69” E; Alghero-SS), 60 from San Teodoro lagoon (40◦ 470 51.71” N 9◦ 400 00.05” E; San Teodoro-SS), 60 from Mistras lagoon (39◦ 540 16.09” N 8◦ 270 21.74” E; Cabras-OR), and 59 from Marceddì lagoon (39◦ 420 40.01” N 8◦ 310 06.53” E; Terralba-OR). Identification of Mugilidae species revealed a heterogeneous group composed of Mugil cephalus (11), Chelon labrosus (35), Chelon auratus (83), and Chelon ramada (110), variably combined in the different lagoons. All the specimens were identified at species level following Ben-Tuvia (1986) and Bauchot (1987); fish names conform to FishBase [44–46]. Sampled fish species are reported in Table 1. Table 1. Fish species collected during summer and autumn 2014. Summer Sampling Autumn Sampling CH ST MI MA CH ST MI MA Total Mugil 0 6 0 0 0 5 0 0 11 cephalus Chelon labro- 16 10 0 0 0 1 0 8 35 sus Chelon aura- 1 13 0 29 0 23 0 17 83 tus Chelon 13 1 30 0 30 1 30 5 110 ramada Total 30 30 30 29 30 30 30 30 239 Key: CH = Calich lagoon, ST = San Teodoro lagoon, MI = Mistras lagoon, MA = Marceddì lagoon. 2.2. Parasitological Analysis The mullets were transferred to the Department of Veterinary Medicine, Univer- sity of Sassari, and the internal organs (heart, liver, spleen, and kidney) of a subsample of 12 fish were analyzed for the presence of tissue parasites to identify the main taxa present. Squashes and scrapings of organs were placed on slides, pressed under a cov- erslip, and observed under an optical microscope. The parasites found were trematode metacercariae (TM) and myxozoan plasmodia (MP), and they were identified to the lowest taxonomic level possible according to the observable characters, following the specialistic literature [42,47–51]. 2.3. Bacteriological Analysis A culture test was carried using the anterior kidney of the 239 subjects on a primary isolation medium (blood agar). Colonies present in the plates were cloned and identified by phenotypic (Gram staining, oxidase, growth temperature, different NaCl concentration, pH range), biochemical (API System, bioMérieux), mass spectrometry (MALDI-TOF), and molecular (using generic primers and subsequent sequencing) methods. Animals 2021, 11, 1501 4 of 18 Fresh tissues from sampled organs (liver and spleen) were homogenized and decon- taminated and inoculated in Stonebrink medium tubes and 2 Löwenstein–Jensen medium tubes (Microbiol, Uta, Cagliari, Italy) for mycobacteria culture. Decontamination of the samples was carried out using 1.5% cetylpyridinium chloride, following the standard pro- tocol aimed at eliminating contaminating bacteria, in order to allow mycobacteria growth on specially sown soils [52]. 2.4. Histology and Immunohistochemistry Samples of the liver, spleen, heart, and kidney of the 239 mullets were collected and immediately fixed in 10% buffered formalin for 48 h, dehydrated with increasing alcohol concentrations and xylene in an automatic tissue processor, and paraffin em- bedded. Sections of 3 µm thickness were obtained with a microtome (RM2245, Leica Biosystems, Wetzlar, Germany) and stained with hematoxylin and eosin in an automatic multistainer (ST5020, Leica Biosystems, Wetzlar, Germany). Slides were then evaluated at light microscopy (Nikon Eclipse 80i, Amsterdam, Netherlands). Lesions found in visceral organs (liver, spleen, heart, and kidney) were evaluated according to the parasite class involved [53,54]. A total of 75 well-preserved granulomatous lesions from 47 of the 239 fish were included in this study. All granulomas were selected considering different organs and different classes of parasites involved. Lesions were classified into three developmental stages based on the evaluation of selected histological features (i.e., state of parasites and necrosis) and were investigated to evaluate different structural components (i.e., epithelioid cells and fibroblasts) by immunohistochemistry (IHC). Serial sections were mounted on positively charged slides (Superfrost, Fisher Scientific) for IHC according to a previously developed protocol [55–57]. Briefly, slides were immersed for 20 min in a 98 ◦ C, preheated solution (WCAP, citrate pH 6, BiOptica, Milan, Italy) for antigen unmasking. Tissues were blocked for endogenous peroxidase (Dako REAL Peroxidase-Blocking Solution S2023, Dako, Glostrup, Denmark) and non-specific binding with 2.5% normal horse serum (ImmPRESS reagent kit, Vector Labs, Burlingame, CA, USA). Sections were incubated overnight at 4 ◦ C with the following primary antibodies: anti-cytokeratin AE1–AE3, dilution 1:250 (Dako, Monoclonal mouse Anti-Human Cytokeratin CKAE1/AE3) to evaluate epithelioid cells and anti-Vimentin, dilution 1:500 (Dako, Monoclonal Mouse Anti-Vimentin clone V9) to evaluate fibroblasts. Sections were then incubated with an anti-mouse/rabbit secondary antibody (ImmPRESS reagent kit—peroxidase—MP-7500; Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature and treated with 3,30 -Diaminobenzidine (DAB) chromogen (ImmPACT DAB; Vector Laboratories). Tissues were then counterstained with hematoxylin, dehydrated, and mounted (Eukitt mounting medium; BiOptica, Milano, Italy). Appropriate positive and negative controls were included for each antibody. The presence of collagen, mast cells, and rodlet cells was also evaluated with Masson’s trichrome and Giemsa staining, respectively [58]. To investigate the potential presence of mycobacteria within the granuloma, Ziehl-Neelsen staining was performed. Granulomas were further evaluated for their proximity to the blood vessel and as- signed to one of the following three categories: close to the vessel (granuloma within the thickness of the vascular wall), near the vessel (granuloma in contact, less than 20 microns, with the external part of the vascular wall), far from the vessel (granuloma distance from the vessel >100 microns or not present in the field of view at 200× magnification). Addi- tionally, macrophage aggregates (MAs) associated with granulomas were also evaluated. The 75 granulomas were assigned to the “present” or “absent” categories based on the presence or absence of macrophage aggregates near the granuloma in a field of view at 200× magnification. 2.5. Image Analysis Each lesion was microphotographed and images were analyzed using a Java-based open- source image processing program (https://imagej.net/ImageJ, accessed on 15 May 2020). Animals 2021, 11, 1501 5 of 18 The quantitative evaluation of the collagen area, associated with the lesions, was accom- plished using ImageJ software on microphotographs taken at 200× magnification on slides stained with Masson’s trichrome. In detail, the total area of the granuloma was selected by the “freehand selection” tool, measured, and then isolated from the background with the “clear outside” option. The central area of the lesion, containing parasite or necrotic material, was selected by the freehand tool, measured, and deleted by the “cut” function to avoid erroneous measurements of color values similar to the collagen area. After that, “color threshold” tool settings were adjusted to identify color values corresponding to collagen, and the obtained area was selected and measured. The color threshold measure- ment was repeated three times, and the final collagen area was calculated as the mean of the three consecutive measurements and expressed as a percentage of the total area of the granuloma. Evaluation of cytokeratin and vimentin expression in granulomas was accomplished using ImageJ “Color threshold”, as in the case with collagen. Different from the previous measurement, the color threshold system measured the area free from IHC staining, which was obtained by subtraction from the total area of the granuloma and the remaining measured area. The area positive to cytokeratin or vimentin was then calculated as the mean of three consecutive measurements and expressed as a percentage of the total area of the granuloma. Mast cells and rodlet cells were manually counted using the ImageJ “Multi-point” tool on microphotographs of each granuloma taken at 400× magnification on slides stained with Giemsa and Masson’s trichrome, respectively, and their absolute number was registered. All data obtained from measurements achieved with the ImageJ software were recorded in a database. 2.6. Statistical Analysis Microscopic features of the lesions were analyzed using Stata 11.2 software (StataCorp LP, College Station, TX, USA). Immunohistochemical results were compared using the non-parametric Kruskal–Wallis test with Dunn’s post hoc comparison. The chi-square test (χ2 ) was used to correlate the prevalence of parasites in examined organs. Furthermore, categorical and ordinal variables were compared using the Spearman rho (ρ) rank correla- tion coefficient. A value of ρ approximately equal to 1 indicates a good correlation, a value near 0 indicates a poor correlation, and a negative value indicates an inverse correlation. A p-value < 0.05 was considered significant. 3. Results 3.1. Parasitological Findings The microscopical examination of the fresh mounts of parasites allowed one to ascribe the TM to the Heterophyidae genera Heterophyes, Stictodora, and Phagicola (Ascocotyle) (Figure 1A,B) and the MP to Myxobolus sp. (Myxobolidae) (Figure 1C,D) in the liver, spleen, heart, and kidney (Figure 1). Animals 2021, 11, 1501 6 of 18 Animals 2021, 11, x FOR PEER REVIEW 6 of 19 Figure1.1.(A–D): Figure (A–D):Fresh Freshsmears smearsofofthe theparasites parasitesfound foundininthe theexamined examinedorgans organsofof mugilids. mugilids. (A): (A): De- Detail tail of the circumoral spines of Ascocotyle (Phagycola) sp. metacercaria in the heart (bar 50 μm). (B): of the circumoral spines of Ascocotyle (Phagycola) sp. metacercaria in the heart (bar 50 µm). (B): En- Encysted metacercaria in the heart (bar 100 μm). (C): Cyst of Myxobolus sp. faintly visualized in the cysted metacercaria in the heart (bar 100 µm). (C): Cyst of Myxobolus sp. faintly visualized in the heart of fresh specimens (bar 50 μm). (D): Spores in a melanomacrophage aggregate in the liver heart ofμm). (bar 50 fresh specimens (bar 50 µm). (D): Spores in a melanomacrophage aggregate in the liver (bar 50 µm). 3.2. Bacteriological 3.2. Bacteriological Findings Findings The microbiological The microbiological examination examinationofofallall samples was samples negative was for the negative for presence of path- the presence of ogenic bacteria and mycobacteria. pathogenic bacteria and mycobacteria. 3.3. Histology 3.3. Histology and and Immunohistochemistry ImmunohistochemistryEvaluation Evaluation Amongthe Among the239 239sampled sampledfish, fish,172/239 172/239 (71.96%) (71.96%) presented presentedat atleast least11parasite, parasite,of ofwhich which 78/239 (32.63%) were affected only by 78/239 by TM TM andand 40/239 40/239 (16.73%) only by MP, MP,whereas whereasin in 54/239 fish (22.59%), both classes of parasites were detected. Concerning 54/239 fish (22.59%), both classes of parasites were detected. Concerning organ distribution, organ distribu- tion,were TM TM detected were detected in all examined in all examined organs,organs, with a prevalence with a higher higher prevalence in the(19.6%) in the kidney kidney (19.6%) and heartand heartwhereas (14.2%), (14.2%),MP whereas MP were were more more detected frequently frequently detected in the kidneyin(38.1%) the kidney and (38.1%)(18.8%). spleen and spleenMost(18.8%). Most of the fish of the by affected fisha affected by aofsingle single class class parasite of parasite presented anpresented infection an infection confined confined to a single to a(22.1% organ singlefororgan (22.1% forand metacercariae metacercariae and 14.6% for Myxo- 14.6% for Myxosporea). Mugil cephalus showed a higher prevalence of TM in the heart (54.5%) and in the sporea). kidney for MP Mugil (36.3%) cephalus but without showed a higher any significantofstatistical prevalence TM in theassociation. heart (54.5%) In C. andramada, in the TMkid- were more ney for MPfrequent (36.3%) in thewithout but heart (12.7%), whereasstatistical any significant MP mostly affected the association. Inkidney (57.2%, C. ramada, TM pwere < 0.05). moreThe kidneyin frequent was thethe most heart affected (12.7%), in C.mostly organ MP whereas auratus for both affected theparasite kidney classes (57.2%, (31.3% andThe p < 0.05). 20.4% for metacercariae kidney and Myxosporea, was the most affected organ in C.respectively, p < 0.05). auratus for both In Chelon parasite classes labrosus, MP were (31.3% and 20.4%mostly found in the spleen for metacercariae (31.4%, p < 0.05). and Myxosporea, Details on respectively, p 0.05 p > 0.05 cephalus (54.5%) (36.3%) (27.2%) (36.3%) (27.2%) (45.4%) (36.3%) Chelon 2/35 1/35 5/35 8/35 2/35 11/35 4/35 7/35 p > 0.05 p < 0.05 labrosus (5.7%) (2.8%) (14.2%) (22.8%) (5.7%) (31.4%) (11.4%) (20%) Chelon 12/83 10/83 16/83 2/83 14/83 26/83 17/83 - p < 0.05 p < 0.05 auratus (14.4%) (12%) (19.2%) (2.4%) (16.8%) (31.3%) (20.4%) Chelon 14/110 2/110 9/110 13/110 5/110 17/110 12/110 63/110 p > 0.05 p < 0.05 ramada (12.7%) (1.8%) (8.1%) (11.8%) (4.5%) (15.4%) (10.9%) (57.2%) 34/239 3/239 28/239 40/239 13/239 45/239 47/239 91/239 Total (14.2%) (1.2%) (11.7%) (16.7%) (5.4%) (18.8%) (19.6%) (38.1%) Key: = TM = trematode metacercariae; MP = myxozoan plasmodia. Chi-square test (χ2 ) was used to correlate the prevalence of parasites in each examined organ. Ziehl-Neelsen staining was negative for mycobacteria in all examined organs. The mi- croscopic examination of the organs revealed a multifocal, mild-to-severe granulomatous inflammation around the parasites and three different stages of lesions. Out of 75 lesions, 25 were categorized as stage I, 27 as stage II, and 23 as stage III. Most of the selected fish showed the presence of only one stage of granuloma (68.1%), whereas 23.4% showed the presence of two stages, and in 8.5%, all stages were detectable. Stage I was termed as pre-granuloma stage and characterized by an intact parasite surrounded by a thin cyst wall, in association with scarce inflammatory cells. At this stage, positive labeling for anti-cytokeratin antibody was occasionally expressed by the parasite capsule, whereas immunoreactivity to anti-vimentin antibody was negative (Figure 2A–C). Animals 2021, 11, 1501 8 of 18 Animals 2021, 11, x FOR PEER REVIEW 8 of 19 Figure 2. Figure 2. (A–I): (A–I): Histology Histology andand immunohistochemistry immunohistochemistry panel panel of of granuloma granuloma developmental developmental stages stages in in Mugilidae Mugilidae infected infected organs. Bar 10 μm. Serial section of the same samples in rows (A–C), (D–F) and (G–I). (A–C): organs. Bar 10 µm. Serial section of the same samples in rows (A–C), (D–F) and (G–I). (A–C): Stage I (pre-granuloma Stage I (pre-granuloma stage). Heart. (A): TM encysted surrounded by a thin eosinophilic layer of compressed myocardial cells (HE, 20×). (B): stage). Heart. (A): TM encysted surrounded by a thin eosinophilic layer of compressed myocardial cells (HE, 20×). IHC—positive labeling for the anti-cytokeratin antibody is expressed by parasite capsule (CKAE1/AE3 antibody, 20×). (C): (B): IHC—positive labeling for the anti-cytokeratin antibody is expressed by parasite capsule (CKAE1/AE3 antibody, 20×). IHC—absence of immunoreactivity to anti-vimentin antibody (Vimentin antibody, 20×). (D–F): Stage II (intermediate- (C): stageIHC—absence granuloma). of immunoreactivity Liver. (D): Myxozoan tospores anti-vimentin (arrow)antibody (Vimentin intermingled with antibody, 20×surrounded necrosis and ). (D–F): Stage byIIa (intermediate- few layers of stage epithelioid cells (asterisk) (HE, 40×). (E): IHC—immunoreactivity for the anti-cytokeratin antibody in theby granuloma). Liver. (D): Myxozoan spores (arrow) intermingled with necrosis and surrounded ECalayers few layers (arrow)of epithelioid (CKAE1/AE3 cells (asterisk)20×). antibody, 40× (HE,(F): ). (E): IHC—immunoreactivity IHC—mild signal of immunoreactivityfor the anti-cytokeratin to anti-vimentinantibody antibodyinin the theECfibroblast layers (arrow) layer (arrow) aroundantibody, (CKAE1/AE3 negative20 ×). (F): IHC—mild epithelioid cells (asterisk) signalinofthis stage (Vimentinto immunoreactivity antibody, 20×). (G–I): anti-vimentin antibodyStage in III the(late-stage fibroblast gran- layer uloma). Spleen. (G): Central necrotic core (asterisk). (H): IHC—positive labeling for anti-cytokeratin (arrow) around negative epithelioid cells (asterisk) in this stage (Vimentin antibody, 20×). (G–I): Stage III (late-stage antibody in a thicker layer of ECs in granuloma). this stage Spleen. (G):(asterisk) (CKAE1/AE3 Central necrotic antibody,(H): core (asterisk). 20×).IHC—positive (I): IHC—positive labeling labeling to the anti-vimentin for anti-cytokeratin antibody antibody in a of the outer layer of fibroblasts (arrow). An aggregate of macrophages is occasionally observed in thicker layer of ECs in this stage (asterisk) (CKAE1/AE3 antibody, 20×). (I): IHC—positive labeling to the anti-vimentin proximity to the lesion (arrowhead) (Vimentin antibody, 20×). antibody of the outer layer of fibroblasts (arrow). An aggregate of macrophages is occasionally observed in proximity to the lesion (arrowhead) (Vimentin antibody, 20×). Stage II was termed as intermediate-stage granuloma and characterized by parasites with Stage loss ofIIsomatic details was termed as or partially destroyed intermediate-stage parasites,and granuloma butcharacterized that were stillbyrecogniza- parasites ble in the center of the lesion, and often intermingled with a scarce with loss of somatic details or partially destroyed parasites, but that were to moderate amount still recognizable of in granular the centereosinophilic material of the lesion, and intermingled and often nuclear debriswith (necrosis). a scarceOuter encircling to moderate layers of amount of flattened cells with a moderate granular eosinophilic to abundant material and eosinophilic nuclear debris cytoplasm, (necrosis). an ovoid vesicular Outer encircling layers of nucleus flattenedwith cellsprominent, basophilic, with a moderate and central to abundant nucleoluscytoplasm, eosinophilic were detected around an ovoid the de- vesicular generated parasite and necrosis. Due to these morphological features nucleus with prominent, basophilic, and central nucleolus were detected around and to their strong the and diffuse positive cytoplasmic signal to anti-cytokeratin CKAE1-AE3 antibody, degenerated parasite and necrosis. Due to these morphological features and to their strong these flattened and diffuse cells were identified positive cytoplasmic as signal epithelioid cells (Figure 2D–F). to anti-cytokeratin Moreover, CKAE1-AE3 an external antibody, these layer composed flattened of fusiform cells were identifiedcells with scarcecells as epithelioid eosinophilic cytoplasm, (Figure 2D–F). an ovoid-to-elon- Moreover, an external gated eccentric nucleus layer composed withcells of fusiform dispersed chromatin, with scarce and 1–2 eosinophilic basophilic cytoplasm, an nucleoli, identified ovoid-to-elongated as fibroblasts, eccentric was with nucleus sometimes detected dispersed loosely and chromatin, arranged around ECs. 1–2 basophilic A scarce nucleoli, amount as identified of collagen was occasionally found in this stage, intermingled with fibroblasts. Animals 2021, 11, 1501 9 of 18 Animals 2021, 11, x FOR PEER REVIEW 9 of 19 fibroblasts, was sometimes detected loosely arranged around ECs. A scarce amount of collagen was occasionally found in this stage, intermingled with fibroblasts. Stage III was designated Stage designated as aslate-stage late-stagegranuloma granulomaand and characterized characterized bybya central a centraleo- sinophilic core eosinophilic of necrosis, core withwith of necrosis, parasite remnants parasite only only remnants occasionally visible, occasionally and increased visible, and in- layers oflayers creased epithelioid cells. The of epithelioid external cells. wall was The external wallconstituted by increased was constituted layerslayers by increased of fibro- of fibroblasts strongly blasts strongly positive positive to anti-vimentin to anti-vimentin antibody antibody (Figure (Figure 2G–I).2G–I). Scattered Scattered macrophages macrophages were were detected detected near near parasite parasite cyst cyst walls walls in in stage stage II lesions lesions and/or and/or intermingled with necrotic material or scattered scattered in in the the surrounding surroundingparenchyma parenchyma in in later later stages. stages. Aggregates of macrophages, macrophages, frequently frequently showing showing pigment-laden pigment-laden cyto-cyto- plasm, plasm, were observed observedin inassociation associationwithwiththethe external external layers layers of granulomas of granulomas in 25/75 in 25/75 gran- granulomas ulomas (Figure (Figure 2H,I). 2H,I). At At histological examination,parasites histological examination, parasiteswere weredescribed describedbasedbasedon oncharacteristic characteristicfeatures features of ofthe theparasite parasiteclasses, classes,clearly discernible clearly discernibleat stage I. Trematode at stage I. Trematode metacercariae were were metacercariae detected de- in all examined tected organs,organs, in all examined rangingranging from 150 to 400 from 150microns in diameter, to 400 microns withoutwithout in diameter, body cavity body and cavitysurrounded by a light and surrounded by aeosinophilic capsule. light eosinophilic Oral Oral capsule. and ventral suckers, and ventral identifiable suckers, identifi- by able by the presence of a radially arranged musculature, were often recognizable,well the presence of a radially arranged musculature, were often recognizable, as as as well aascuticle with a thickness of 2–4 microns with occasionally detectable cuticle a cuticle with a thickness of 2–4 microns with occasionally detectable cuticle spines that spines that appeared appearedas asdistinct distincteosinophilic eosinophiliclinear linearstructures structures(Figure (Figure3A). 3A). Figure3. Figure 3. (A–D): (A–D): Histological Histologicalfeatures featuresofofgranuloma granulomastructure structureand andcellular cellularcomponents componentsinin Mugi- Mugilidae lidae infected organs by histozoic parasites. Bar 10 μm. (A): Encysted metacercaria in the heart infected organs by histozoic parasites. Bar 10 µm. (A): Encysted metacercaria in the heart with with evident ventral sucker (arrow). Inset. Magnification of cuticle spines (arrow) on larva surface evident ventral sucker (arrow). Inset. Magnification of cuticle spines (arrow) on larva surface (HE, (HE, 40×). (B): Plasmodium containing spores of myxozoan parasites surrounded by an eosino- 40 ×). (B): philic Plasmodium capsule (arrow) incontaining spores the liver close to aof myxozoan blood parasites (HE, vessel (asterisk) surrounded 20×). (C):byCollagen an eosinophilic fibers in capsule the outer(arrow) inlate-stage layer of the liver close to a blood granuloma vessel(MT, (asterisk) (asterisk) 20×). (D): 20×). cells (HE,Rodlet (C): Collagen (arrows) infibers in the perivascu- outer layer of(MT, lar collagen 100×). granuloma (asterisk) (MT, 20×). (D): Rodlet cells (arrows) in perivascular late-stage collagen (MT, 100×). Histozoic plasmodia of myxozoans were found encysted in the parenchyma of the Histozoic plasmodia heart, liver, spleen, of myxozoans and kidney. were Plasmodia, found encysted containing a variableinnumber the parenchyma of the (until hundreds) heart, liver, spleen, and kidney. Plasmodia, containing a variable number (until hundreds) of mature spores, were round to oval, ranging from 100 to 450 μm, and were delimited by of mature a thin spores, were eosinophilic wallround to 3B). (Figure oval, ranging from 100 to 450 µm, and were delimited by a thinIneosinophilic wall (Figure 3B). granulomas, collagen fibers were recognizable as blue filaments with Masson’s trichrome staining in the external layer of intermediate- and late-stage granulomas (Figure 3C). Animals 2021, 11, 1501 10 of 18 Animals 2021, 11, x FOR PEER REVIEW 10 of 19 In granulomas, collagen fibers were recognizable as blue filaments with Masson’s trichrome staining in the external layer of intermediate- and late-stage granulomas (Figure 3C). Mast Mast cells cells (MCs) (MCs) appeared appeared asas ovoid ovoid cells cells with with distinct distinct borders borders and and abundant abundant cyto- cyto- plasm containing numerous eosinophilic granules, brightly evidenced by plasm containing numerous eosinophilic granules, brightly evidenced by Giemsa Giemsa staining, stain- and ing, aand central nucleus a central (Figure nucleus 4A). 4A). (Figure Figure4. Figure 4. (A–D): (A–D): Histological Histologicalfeatures featuresofofgranuloma granulomastructure structureand andcellular cellularcomponents componentsinin Mugi- Mugilidae lidae infected organs by histozoic parasites. Bar 10 μm. (A): Mast cells (arrows) near the fibroblast infected organs by histozoic parasites. Bar 10 µm. (A): Mast cells (arrows) near the fibroblast layer layer (arrowhead) and the EC layer (asterisk) of a late-stage granuloma (Giemsa, 20×). (B): Same (arrowhead) and the EC layer (asterisk) of a late-stage granuloma (Giemsa, 20×). (B): Same detail detail of figure (A). Positive labeling to the anti-vimentin antibody in MCs (arrows) and fibroblast of figure layer (A). Positive (arrowhead). Thelabeling signal istoabsent the anti-vimentin in the EC layerantibody in MCs (asterisk) (IHC,(arrows) and 20×). (C): fibroblast Mast layer cells (arrow- (arrowhead). The signal is absent in the EC layer (asterisk) (IHC, 20 × ). (C): Mast heads) in the external layer of late-stage granuloma in contact with fibroblasts (arrows) and blue cells (arrowheads) in the external collagen fibers.layer of late-stage Epithelioid granuloma cell layer is visiblein(asterisk) contact with (MT,fibroblasts Oil 100×). (arrows) (D): Sameand blueofcollagen detail figure fibers. Epithelioid (C). Mast cell cells with layer is visible evidenced granules(asterisk) (MT, Oil (arrowheads) in100 ×). (D): contact withSame detail of fibroblasts figure (C). (arrows) andMast col- lagenwith cells fibers. Epithelioid evidenced cell layer granules is visible (asterisk) (arrowheads) in contact(Giemsa, Oil 100×). with fibroblasts (arrows) and collagen fibers. Epithelioid cell layer is visible (asterisk) (Giemsa, Oil 100×). Mast cells also showed a strong cytoplasmic positive signal to anti-vimentin antibody Mast (Figure 4B). cells Inalso bothshowed affected a strong cytoplasmic and unaffected positiveMCs samples, signal to anti-vimentin were occasionally antibody observed (Figure near vascular structures. In the pre-granuloma stage, MCs were closeobserved 4B). In both affected and unaffected samples, MCs were occasionally near to parasites, vascular structures. In the pre-granuloma stage, MCs were close to parasites, whereas in the intermediate- and late-stage granulomas, MCs migrated to the more exter- whereas in the nal intermediate- layers. They were andindeed late-stagemoregranulomas, commonly MCs migrated observed to the more intermingled withexternal layers. fibroblasts and They were indeed sometimes detected more commonly in close contactobserved intermingled to the fibroblasts with fibroblasts membrane, elongatedandin sometimes shape with detected in close granules loosely arranged contact to(Figure the fibroblasts 4C,D). membrane, elongated in shape with loosely arranged granules (Figure 4C,D). Rodlet cells were observed in scarce numbers in all stages, mainly disposed near the Rodlet parasite cells were capsule or inobserved in scarceparenchyma. the surrounding numbers in allThey stages, mainly were disposed near also observed near the the parasite capsule or in the surrounding parenchyma. They were main vascular structure both in parasitized and healthy samples (Figure 3D). also observed near the main The vascular results structure both in parasitized of the structural components and of healthy samples granulomas and (Figure 3D).immune cells, associated Theasresults as well of the structural the description components of histological of granulomas parasite and associated characteristics, immune were combined in acells, gen- as eral scheme of possible developmental stages of granuloma formation (Figure 5). general well as the description of histological parasite characteristics, were combined in a scheme of possible developmental stages of granuloma formation (Figure 5). x FOR PEER REVIEW Animals 2021, 11, 1501 11 11of 19 of 18 Figure 5. Stages Figure 5. Stages and and histological histologicalassociated associatedpatterns patternsofofhistozoic histozoicparasite parasitegranuloma formation granuloma in in formation Mugilidae. SDSD Mugilidae. = standard = stand- deviation; %T.A.G.= mean percentage of the total area of the granuloma. ard deviation; %T.A.G.= mean percentage of the total area of the granuloma. 3.4. Image and Statistical Analysis 3.4. Image and Statistical Analysis Epithelioid cells showed a statistically significant positive correlation with granu- Epithelioid cells showed a statistically significant positive correlation with granuloma loma progressive stages, showing an increase in parallel to the host immune response progressive stages, showing an increase in parallel to the host immune response progression progression (Kruskal–Wallis χ2 (corrected for ties) = 62.686, p = 0.00010; Dunn’s post hoc (Kruskal–Wallis χ2 (corrected for ties) = 62.686, p = 0.00010; Dunn’s post hoc test, pre- test, pre-granuloma vs. intermediate p = 0.000002; pre-granuloma vs. late p = 0.000000; granuloma vs. intermediate p = 0.000002; pre-granuloma vs. late p = 0.000000; intermediate intermediate vs. late p = In vs. late p = 0.000450). 0.000450). In the intermediate the intermediate stage, ECs stage, ECs a covered covered mean area a mean area of of 13.42% 13.42% (± 7.19% SD) of the total area of the granuloma, whereas (± 7.19% SD) of the total area of the granuloma, whereas in the late stage, they reached in the late stage, they reached approximately 1/3 of the total area of the granuloma approximately 1/3 of the total area of the granuloma (mean area 30.85% ± 14.68% SD). (mean area 30.85% ± 14.68% SD). The fibroblast layer increased with granuloma progression (Kruskal–Wallis χ2 (cor- rectedTheforfibroblast layerpincreased ties) = 38.963, = 0.00010;with Dunn’s granuloma post hoc progression (Kruskal–Wallis test, pre-granuloma χ2 (cor- vs. intermediate rected for ties)pre-granuloma p = 0.000842; = 38.963, p = 0.00010; vs. lateDunn’s post hoc test, p= 0.000000, pre-granuloma intermediate vs. late vs.pintermediate = 0.002460). pFibroblasts = 0.000842;covered pre-granuloma vs. late p= 0.000000, intermediate vs. averagely 2.94% (± 3.85% SD) of the total area of the granuloma late p = 0.002460). Fibro- in blasts covered averagely 2.94% (± 3.85% SD) of the stage II, reaching a mean area of 7.15% (± 5.71% SD) in the late stage. total area of the granuloma in stage II, reaching a mean Statistical area ofevidenced analysis 7.15% (± 5.71% SD) in the that collagen hadlate stage. a significant increase in intermediate- Statistical analysis evidenced that collagen and late-stage granulomas (Kruskal–Wallis χ (corrected for ties) 2 had a significant increase in intermediate- = 36.791, p = 0.00010) and late-stage (Dunn’s post hoc granulomas (Kruskal–Wallis test, pre-granuloma vs. late p=χ 0.00000; 2 (corrected for ties) = vs. intermediate 36.791, late pp==0.000231). 0.00010) (Dunn’s Moreover, post hoc test, collagen pre-granuloma showed a statistically vs. late p= 0.00000; significant intermediate association with the vs. fibroblast late p = 0.000231). layer in Moreover, collagen showed the intermediate-stage granuloma a statistically (ρ = 0.6257 significant p < 0.05).association The collagen with the covered layer fibroblast layer a mean in the intermediate-stage granuloma (ρ =0.6257 p < 0.05). area of 0.82% (± 1.43% SD) in the intermediate stage and a mean area of 3.58% (± 2.92%The collagen layer covered a mean area of 0.82% (± 1.43% SD) in the late-stage granuloma. SD) in the intermediate stage and a mean area of 3.58% (± 2.92%MastSD) cells in thewere late-stage observed granuloma. in all stages of the lesion development, showing an increase fromMast cells were observed the pre-granuloma stage, in withall astages meanof the lesion number development, of 0.68 (± 1.86 SD),showing an increase to the intermediate- (2.89 ± from the3.33 pre-granuloma stage, with SD) and late-stage a mean (9.86 granulomas number ± 18.98 of 0.68 (± Statistical SD). 1.86 SD), to the intermedi- analysis of MCs ate- (2.89 ±a 3.33 revealed SD) andincrease significant late-stage in granulomas number in the (9.86 ± 18.98 SD). Statistical pre-granuloma analysis of MCs and intermediate-stage granuloma revealed (Kruskal–Wallis a significant increase χ2 in (corrected number for =29.531, p = 0.00010 ties)pre-granuloma in the (Dunn’s post hoc and intermediate-stage test, pre-granuloma vs. late p = 0.00000; pre-granuloma granuloma (Kruskal–Wallis χ (corrected for ties) =29.531, p = 0.00010 (Dunn’s 2 vs. intermediate p = 0.000773). post hoc Moreover, test, a significant pre-granuloma vs. positive correlation late p = 0.00000; between the vs. pre-granuloma collagen area and intermediate p the number = 0.000773). of mast cells Moreover, was observed a significant positive(ρ =0.2701, correlation p < 0.05). between Evaluation the collagen of theareaassociation and the number between of the number mast cells was of mast cells and observed the granuloma (ρ =0.2701, p < 0.05).proximity Evaluation to vascular structuresbetween of the association revealedthe no significant statistical association. Animals 2021, 11, 1501 12 of 18 Rodlet cells were detected with a mean number of 0.32 (± 0.74 SD) in the pre- granuloma stage, 0.28 (± 0.71 SD) in the intermediate stage, to 0.65 (± 1.19 SD) in the late-stage granuloma. Moreover, conversely to MCs, no statistically significant difference in the number of the RCs was observed in the different stages of host immune response. Macrophages aggregates were found associated in 16% of the pre-granuloma stage, 44.4% in the intermediate stage, and 39.1% in the late-stage granuloma. However, no statistically significant variation was observed between their presence and the different stages of granulomas. Quantification of granuloma’s proximity to vascular structures gave the following results: 33.3% of lesions were considered in the “close” category, 33.3% fell in the “near” cat- egory, and 33.3% in the “far” category. The association between the distance of granulomas from vessels and granuloma stages was not statistically significant (p > 0.05). 4. Discussion Inflammatory lesions associated with TM and MP were detected in (71.9%) of the examined fish, confirming the observation of other authors that reported Mugilidae as a widely parasitized species [38]. The parasitological examination of grey mullets allowed one to identify three genera of TM: Heterophyes sp., Stictodora sp., and Phagicola sp., be- longing to the heterophyid family (Trematoda: Heterophyidae). Heterophyids, which have a fish-eating bird or mammals as the definitive host, are widely encountered para- sites of mullets in Sardinian brackish waters [51,59,60] as well as in other Mediterranean areas [38,42,48]. Pre-granulomas and granulomas associated with metacercariae were detected in all examined organs, with a higher prevalence in the kidney and heart. TM have a wide range of tissue distribution, as they have been reported in almost every organ of mugilids [60–65]. Myxozoan parasites, especially the Myxobolidae family, have been extensively de- scribed in mugilids worldwide [50,66–69]. In the present study, myxozoan plasmodia were detected in all organs, with a higher prevalence in the kidney and spleen. These data are aligned with the literature of Myxobolus spp. infection in many Mugilidae species from the Mediterranean area, which described encysted plasmodia in different tissues [70–75]. Furthermore, albeit the pre-granulomas and granulomas were detected in all examined organs, most fish showed lesions associated with TM and MP only in one organ. This result could reflect the preference of a parasite species for a specific organ, as reported for some species of myxozoan parasites [76]. Parasite infection in fish can be the result of a single contact with intermediate hosts or can be due to multiple exposures to parasites, leading to the formation of younger parasite cysts near to older and almost resolved infection [43]. The granuloma stages distribution in the selected samples reflected these observations and, whereas most of the fish presented a single granuloma stage, probably as a result of a single contact with intermediate hosts, one-third of them resulted in detection of multiple granuloma stages, thus suggesting that these fish were likely subjected to prolonged exposure to the intermediate hosts. The response of the fish immune system after parasite invasion is variable and reliant on a multitude of factors (fish age, infected organ, class of parasite), with a different outcome in terms of damage extent to host tissues [24,77,78]. In the pre-granuloma stage (stage I), parasites were detected encysted in the parenchyma of several organs, generally surrounded by a compact eosinophilic layer, composed of compressed parenchymal cells because of the cyst pressure. The inflammatory response in this phase was absent or mild, and when present, mainly characterized by a limited number of mast cells close to the parasite wall or scattered in the surrounding parenchyma, and by occasional macrophages. This feature has been reported by other authors in early encysted TM [43,64] and histozoic forms of MP [79]. The paucity of the immune response in this early phase could be related to the ability of the parasite to escape the host immune system or to regulate the mounting inflammatory process, as reported for trematode metacercariae and Myxobolus spp., respectively [78]. Animals 2021, 11, 1501 13 of 18 The hallmark of the intermediate-stage granuloma (stage II) was the presence of ep- ithelioid cells that encircled the parasites. These cells were discernible with IHC staining by the strong positive labeling to anti-cytokeratin antibody (CKAE1/AE3), as was reported by studies in other fish species [2,28]. The intermediate stage represented the host im- mune system response directed toward parasite isolation. Encircling and sequestration of parasites by a layer of epithelioid cells were observed in granulomas belonging to both groups of parasites, and it is also a widely reported feature in the course of tissue-dwelling parasite infection [2,80–83]. It is well recognized that the host immune response against parasites differs depending on the location where the infection takes place. In the course of parasitism of luminal organs, such as the intestine, immune cells expel parasites by the local release of vesicle content at the site of parasite infection and by the stimulation of syn- ergist mechanisms (increase in mucous production, stimulation of peristalsis, etc.) [84–87]. On the contrary, in parenchymal organs where a proper elimination of the parasite is not achievable, encapsulation is the mechanism employed to limit parasite spread [10,11,37,88]. Lastly, late-stage granuloma (stage III) represented the late phase of chronic inflam- mation with the highest quantity of immune cells, which led to the resolution of parasite infection. In this stage, due to the presence of central necrosis and the lack of parasite remnants, the results of Ziehl-Neelsen staining excluded the presence of mycobacterial in- fection. Epithelioid cells and fibroblasts, which are recently considered strictly associated with immune response, were found in the largest amount in the late-stage granuloma. Fibroblasts were more prominent and evidenced by a diffuse membrane immunoreactivity to anti-vimentin antibody, a marker for the intermediate filament of mammalian mes- enchymal cells that is preserved in some fish species [89]. Fibroblasts have been frequently reported in association with parasite granulomas in fish tissues, variably intermingled with collagen fibers, to further prevent the spread of parasites [10,30,90,91]. This has been confirmed in the present study since fibroblasts acted to delimitate granuloma expansion and contributed to tissue remodeling by increased production of collagen, especially in intermediate- and late-stage granulomas. In particular, the strong statistical correlation found between fibroblasts and collagen fibers limitedly to intermediate-stage granuloma (p < 0.001) could be explained by the fact that in this phase, both fibroblasts and collagen were present in similar proportions. However, in stage III, even if both categories were numerically increased, collagen production augmented fourfold compared to the twofold rise in fibroblasts. The biological explanation for this difference could be that in late stages, fibroblasts have accomplished their role and their number start to decrease, whereas colla- gen, which represents fibroblast’s final product, is at the highest levels. There is evidence that fibroblasts, although not proper immune cells, make an important contribution in the host response against parasites, by crosstalk with other immune cells, as demonstrated in mammals and fish species [32,92]. An interesting link between teleostean MCs and fibroblasts has been recently evidenced, particularly addressing the role of MCs in pro- moting fibroblast collagen production [11,33]. Mast cell function in the pathogenesis of human fibrosis has been extensively investigated and different mechanisms of their action on fibroblasts have been defined, such as the piecemeal release of cytoplasmic vesicles in the interstitium or via the transfer of molecules by direct cell-to-cell contact (transgranula- tion) [34,93]. This finding is in accordance with what was reported by other authors on the close association of MCs and fibroblasts in parasite granulomas in teleosts [33]. In this work, microscopic examination of stages II and III granuloma revealed the presence of mast cells grouped around the external layer, adherent to fibroblasts, thus leading to the hypothesis that a transgranulation mechanism of MCs towards fibroblasts could be plausible. The statistically significant association of MCs with the collagen layer (ρ = 0.2701, p < 0.05) reinforced the hypothesis that action of MCs could be more specifically addressed to the regulation of fibroblasts in collagen production, rather than to fibroblast recruitment in the site of infection, in intermediate- and late-stage lesions. Animals 2021, 11, 1501 14 of 18 The results of the present work suggest that the role of mast cells as promoters of collagen production may not be the only mechanism in which MCs are involved against histozoic parasites. Indeed, the low number of MCs observed in the pre-granuloma stage and their distribution around the parasite capsule suggest that their action could be involved, synergistically with macrophages, in promoting host response, perhaps by the recruitment of other immune cells at the site of infection. This mechanism, recognized in mammalian mast cells during parasite infection, has been supposed in teleosts, where MCs are recognized as first effectors during parasitism [83]. Mast cell migration from blood vessels to the site of infection has been observed in fish species [94]. In addition to a circulating population, the existence of a local reserve of MCs, which could proliferate in case of parasitism, has been confirmed in mammals and supposed in fish species [11,95]. The latter situation likely represents the present case, where the lack of statistical association between mast cell number and the proximity to blood vessels in the various developmental stages suggests that the number of mast cells associated with granulomas could be the result of the proliferation of a resident population, rather than the consequence of migration from the peripheral bloodstream. This finding is in accordance with what was reported by Dezfuli et al. (2017) [11], which showed the positivity to the proliferating cell nuclear antigen (PCNA) in mast cells of the liver of Gymnotus inaequilabiatus (Valenciennes, 1839) infected by nematode larvae [11]. Another group of immune cells, known as rodlet cells, that represent a unique element of the teleostean immune system [36,96], was found in the present work in a low number. Furthermore, the absence of a recognizable organization around parasite lesions and the lack of any significance at statistical analysis suggested that RCs’ role against histozoic parasites could be less specific and maybe mostly committed to cooperation with other immune cells in the host inflammatory response, rather than a direct action on the parasite, as described in parasite infection of the gills and intestine [97,98]. Macrophage aggregates were inconsistently found at the periphery of the granulomas, sometimes containing brownish cytoplasmic pigments. Aggregates of pigment-laden macrophages (MMAs) are normally present in the liver and spleen of many teleosts and can vary in number and dimension under the influence of several factors, such as age, stress, diseases, and pollution [99]. MMAs have also been reported in the liver, spleen, kidney, and heart around granulomas induced by larvae of helminths [10,11,37]. In the present study, the presence of aggregates of macrophages associated with the granuloma showed a variable presence not statistically correlated with granuloma developmental stages. This result suggests that their presence could be perhaps influenced by other factors, such as age and fish species, as reported by other authors on studies on exposure to pollutants, diet, environmental stress, and fish aging [99,100]. The present study underlined that host immune response against the two groups of parasites revealed a substantial superimposability, leading to a common model of develop- ment of the immune response against trematode metacercariae and myxozoan parasites. 5. Conclusions Parasitic disease is a relevant issue for fish health and represents a challenge between the attempt of parasites to survive and the host’s action aimed to prevent the spread of infection. This study, by combining histopathology with immunohistochemical methods, repre- sents an effort to expand the knowledge of inflammatory reactions associated with parasites in fish organs and their staging. This makes it conceivable that the identified histological patterns could be reliable tools to staging the granulomatous response associated with histozoic parasites and represent an effort to classify granulomas of different origins. Further analyses should be conducted to understand mechanisms of granuloma formation induced by different species of parasites in various experimental models, as well as the development of early detection methods, especially in species of great commercial interest for human consumption. Animals 2021, 11, 1501 15 of 18 Author Contributions: Conceptualization, E.A., F.P., and M.P. (Marta Polinas); Methodology, E.A., F.P., and M.P. (Marta Polinas); data curation, M.A.S., P.M., G.P.B., and M.P. (Marino Prearo); software, G.P.B.; writing—original draft preparation, M.P. (Marta Polinas); writing—review and editing, E.A., F.P., P.M., F.M., and M.P. (Marino Prearo); supervision, E.A. and M.P. (Marino Prearo). All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The conducted experimentation does not fall within the provisions of Legislative Decree No. 26/2014, implementation of the European Directive 2010/63/EU of the European Parliament, as waste material was used, and therefore not regulated by the laws on animal testing. The Ethics Committee of the University of Sassari expressed a favorable ethical opinion (id number 50884). Data Availability Statement: The data presented in this study are available upon request from the corresponding author. Acknowledgments: We thank fishermen from Sardinian lagoons (Italy) (Calich lagoon (Alghero- SS), San Teodoro lagoon (San Teodoro-SS), Mistras lagoon (Cabras-OR), and Marceddì lagoon (Terralba-OR)). Conflicts of Interest: The authors declare no conflict of interest. References 1. Timur, M.; Roberts, R.J. Carrageenin granuloma in the plaice (Pleuronectes platessa); a histopathological study of chronic inflamma- tion in a teleost fish. J. Comp. 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