First Histological Study of the Gastrointestinal Tract and Associated Lymphoid Structures of Harbour Porpoise (Phocoena phocoena)

1. Introduction

Cetacea represent one of the most diverse order of mammals with two distinct suborders: Mysticeti which possess baleen plates in the oral cavity, and Odontoceti which have teeth instead [1].

The anatomy and histology of the gastrointestinal tract (GIT) of cetaceans can be found in various publications [1,2]; however, those are general descriptions in which numerous aspects remain unclear, especially considering the wide diversity of cetacean species. In addition, the study of the normal morphology of the GIT and the cell populations of associated lymphoid tissue in wild cetaceans faces significant methodological limitations, i.e., although stranded individuals provide valuable data, they often present underlying diseases or advanced deterioration, which can significantly alter the morphology of the GIT, compromising the representativeness of the findings. In contrast, individuals obtained through bycatch could be considered a suitable source for the histological characterization of the GIT, since the ultimate cause of death is attributed to fishery interaction, rather than to any underlying pathological condition.

Odontocetes exhibit anatomical peculiarities in their GIT. First, these animals possess a gastric complex consisted of three distinct chambers [3]. The first chamber, the forestomach, continues the oesophagus and performs a mechanical digestion function. Histologically, it is characterized by a stratified keratinized lining epithelium, reflecting the adaptation of this chamber to mechanical digestion, as odontocetes do not chew their food [4]. The main stomach follows the forestomach and serves as the principal chamber for enzymatic and chemical digestion, displaying a histological structure similar to that of terrestrial mammals [1]. Finally, the pyloric stomach is the last chamber and presents a mucosa containing specialized pyloric glands involved in chemical and enzymatic digestion [5]. At the beginning of the intestine, a dilation of the gut, namely the duodenal ampulla, is present, where the hepato-pancreatic duct empties in most species. Although it may be mistaken for a gastric compartment, it represents the most proximal portion of the intestine [1]. One of the most remarkable features in cetaceans of the Delphinidae and Phocoenidae families is the absence of macroscopic differentiation between the small and large intestine [6], with some authors referring to the entire gut as the small intestine [7]. In addition, while mysticetes possess a cecum and a vermiform appendix, these structures are absent in the odontocetes [3]. The intestine continues towards the rectum and the anal canal, where the anal tonsil, a relevant lymphoid tissue, is located [8].

Gut-associated lymphoid tissue (GALT) comprises lymphoid structures in the mucosa and submucosa of the GIT, playing a central role in immune tolerance and pathogen defence [9]. GALT includes Peyer’s patches (organized aggregates mainly found in the ileum), diffuse lymphoid follicles along the intestine, and isolated immune cells such as intraepithelial lymphocytes [10]. In cetaceans, studies in belugas reported diffuse lymphocytes and follicles but no true Peyer’s patches [11]. In young individuals, the distal rectum contains prominent lymphoid structures, that disappear just before the anal canal (“pigskin appearance”), which regress with age, remaining only scattered lymphocytes [8]. More recent studies have confirmed the presence of Peyer’s patches in juvenile cetaceans, especially in the mid to distal gut [12] and highlights age-related involution of GALT in bottlenose dolphins [13].

In addition, GALT includes the anal tonsil, which refers to a macroscopically visible lymphoid structure located in the anal canal, found in some cetacean species [6,11]. The anal tonsil may function in antigen presentation, reacting to waterborne antigens entering during diving [8]. It consists of lymphoid cell clusters with epithelial crypts and occasional mucous glands. In advanced involution stages, glandular structures suggest a shift from immune to mechanical functions, such as lubrication [13].

In contrast to the GIT, the histological structure of the lymph nodes (LNs) has been described in several studies conducted on cetaceans [8,14], and a morphology similar to that of terrestrial mammals’ LNs has been observed. However, in the odontocetes harbour porpoise (Phocoena Phocoena) and common dolphin (Delphinus delphis), it has been reported that a pig-like structure may occasionally be present [15], in which the cortical and medullary regions exhibit an inverted pattern.

Taking into account that lack of information, the aims of this study were to (i) histologically characterize the GIT and associated lymphoid tissue of a harbour porpoise which died due to bycatch, (ii) quantify and describe the distribution of macrophages, T lymphocytes (TL) and B lymphocytes (BL) in the lymphoid tissue present in the GIT and regional LNs and (iii) to establish a reliable method for further histological structure studies of GIT and LNs in cetaceans.

2. Materials and Methods

2.1. Sampling

One subadult male harbour porpoise, which died because of bycatch in the Bay of Biscay, was used in this study. Necropsy was performed in 48 hours and samples from the GIT and associated lymphoid tissue were collected and fixed in 10% buffer formalin for further histological and immunohistochemical (IHC) studies. GIT samples included all three chambers from the gastric complex (i.e., forestomach, main stomach and pyloric stomach), the transition between gastric compartment and intestine, duodenal ampulla, intestine and rectum. The intestine was divided into three regions: proximal, medial and distal, with a length of six meters each. Three samples were taken from equidistant points from each region, making a total of nine samples from the intestine (Figure 1). Intestinal samples were collected in a closed state to maintain the tubular shape, and formalin was carefully injected into the lumen to attempt to preserve the mucosal integrity. LNs were taken from the gastro-splenic and pancreatic regions. Additionally, the mesenteric LN was sampled and subdivided into proximal, middle, and distal portions. The rectal LN and the anal canal were also collected (with its associated anal tonsil). Pathological evaluation confirmed the absence of any disease.

2.2. Histology and Immunohistochemistry

For each sample (n=16), serial paraffin-embedded sections (3 µm) were used for histological and IHC studies. The staining techniques included the hematoxylin and eosin standard stain and Masson’s trichrome stain, for specific visualization of the connective tissue.

IHC consisted of the detection of three cell populations using different primary antibodies: ionized calcium-binding adaptor molecule 1 (IBA1) for macrophages, CD3 for TL and CD20 for BL. After being deparaffinized, antigen retrieval was carried out with sodium citrate buffer (10 mmol/L, pH 6.0) with heat induction by microwave for 20 min. Endogenous peroxidase activity was subsequently blocked by incubation in hydrogen peroxide (0.5%) solution in distilled water for 30 min at room temperature. The tissue sections were then incubated overnight at 4°C in a humidified chamber with commercial monoclonal or polyclonal antibodies diluted in TBS+BSA 0.1% (Table 1), washed with TBS 1x, and incubated with a secondary antibody (Vector Laboratories, California, USA), diluted 1:200 in TBS+BSA 0.1% (Table 1), followed by incubation with the Avidin-biotin-peroxidase complex reagent-method (ABC Standard, Vector Laboratories, California, USA) in TBS 1x for 30 min. Labeling was visualized using the Vector® NovaRed™ peroxidase substrate kit (Vector Laboratories, California, USA) as chromogen substrate. Slides were counterstained with Mayer´s haematoxylin, dehydrated and mounted with DPX (Fluka, Sigma, St. Louis, MO, USA). The negative control consisted of an additional slide without the primary antibody. Lymph node tissue from badger was used as a positive control for the three antibodies.

2.3. Measurement of Histological Layer Thickness in the GIT

Samples were examined using a Nikon E600 microscope and a Nikon DS-FI1 camera. Image analysis software (Nikon NIS-Elements Br, Nikon, Japan) was used to measure the thickness of the different layers of the GIT. Five measurements were taken for each sample: mucosa or mucosa-submucosa layers (when muscularis mucosae was absent), submucosa, and internal and external muscularis layers. A total of 350 measurements were taken.

2.4. Evaluation and Quantification of Cell Types in the Gastrointestinal Tract (GIT) and Lymph Nodes (LNs)

Immunostained tissue sections were scanned at the Microscopy Service of the University of León. An Olympus BX51 microscope (Olympus, Tokyo, Japan) and an Olympus XC10 camera (Olympus, Tokyo, Japan) were used. The digitally scanned samples were then analyzed using the QuPath v. 0.5.1 image analysis software [16] (QuPath, University of Edinburgh, Scotland).

For quantification of cells in the GIT, five fields were randomly sampled from mucosa, submucosa, and muscularis layers (a total of 15 counts/sample). Regarding LNs, five fields were randomly sampled from the diffuse lymphoreticular tissue of the cortex, lymphoid follicles, and medulla, obtaining 15 cell counts/LN.

For cell counting in each field, an area of ​​62,000 μm2 was delimited, yielding the following results: total number of cells detected, number of positive and negative cells, percentage of positive cells detected in the area, and number of positive cells per mm2. A total of 745 positive cell count fields were obtained.

2.5. Statistical Analysis

Statistical analysis was performed using IBM SPSS Statistics v. 22.0 software (IBM SPSS Statistics, IBM, USA). Since the values did not meet the assumptions of normality (Kolmogorov–Smirnov and Shapiro–Wilk tests), nonparametric tests were used. Statistical significance between categories was assessed using the Mann–Whitney U test and the Kruskal–Wallis test to compare two or more independent groups, respectively. When statistical differences were found, a pairwise comparisons were conducted. The level of statistical significance was set at p = 0.05.

3. Results and Discussion

3.1. Histological Structure of the Gastrointestinal Tract (GIT)

Main histological findings in the GIT are shown in Table 2. In the histological study of the gastric complex, the adaptation of forestomach to mechanical digestion [1], was reflected in the robust lining epithelium (Figure 2), the absence of glands, and the marked development of the muscularis (Table 2). In turn, the pronounced development of the mucosa (Table 2) and the high number of secretory cells in the main stomach highlighted the relevance of this chamber in chemical and enzymatic digestion. Although the literature considers that the duodenal ampulla and the remaining segments of the duodenum share the same structure [1], in this study the ampulla exhibited a greater development of the muscularis mucosae and lacked folds (Figure 2), which were present in the subsequent intestinal sections. Furthermore, the thickness of all histological layers was notably greater in the duodenal ampulla compared to the other intestinal regions (see Table 2). Consistent with previous descriptions, folds were observed in the intestinal mucosa [1,3,17], being more abundant in the proximal sections and gradually decreasing towards the more distal regions of the intestine.

The presence of glands in the submucosa has been reported to vary among different cetacean species [3]. In this study, no glands were observed in any of the gastrointestinal segments examined. Regarding villi, while some authors reported the absence of well-defined villi [7], others described short and thin villi [1,17], or even their complete absence in the most distal regions of the intestine [18]. In this porpoise, no villi were observed. Given these discrepancies, it is important to consider the challenges associated with cetacean sample collection and the rapid degeneration of intestinal mucosa after death. In the same line, no muscularis mucosae was observed in the rectum, which contrasts with its presence in other terrestrial mammalian species [19,20]. Also, numerous lymphoid follicles have been identified in the mucosa of this segment in other studies [4], although they were not observed in the harbour porpoise studied.

Finally, the statistical analysis of the thickness of the nine intestinal samples, taken from the duodenal ampulla to the rectum, showed no significant differences, making impossible to distinguish between the small and large intestine based solely on wall thickness.

3.2. Distribution of Immune Cells Within Gastrointestinal Tract (GIT) and Associated Lymphoid Tissue

In the GIT, the most frequently detected cells were macrophages, followed by BL and TL. Likewise, the submucosa represented the layer with the highest density of immunopositive cells, followed by the mucosa and muscularis.

Within the gastric chambers, the pyloric stomach exhibited the highest percentage of immunopositive cells, with an average of 6.73% of IBA1-stained cells in the mucosa, 8.83% in the submucosa, and 3.44% in the muscularis layer. However, the higher cellular positivity in this chamber, at its transition with the intestine and in the duodenal ampulla itself, is likely associated with the presence of lymphoid cell clusters (Figure 3).

In the intestine, significant differences were observed in the distribution of the three cell populations across the nine collected segments (p = 0.005, p = 0.000, and p = 0.034 for macrophages, TL, and BL, respectively) (Figure 4B). Macrophages were mainly located in the apical region of the mucosa, forming an almost continuous layer of cells (Figure 3). TL and BL were diffusely distributed near the lamina propria of the mucosa layer and predominated in the lymphoid aggregates observed in the submucosa, with BL being the most abundant cell (Figure 3). While immune cells in the proximal and medial intestine samples appeared only occasionally as aggregates, in the distal segment, aggregates were more frequently observed, forming well-defined lymphoid follicles in the submucosa and infiltrating the mucosa layer (Figure 3). This was reflected in the marked increase in immune cell detection in this last segment (Figure 4). Furthermore, the follicles found in the distal portions were structures similar to the Peyer’s patches of terrestrial mammals [20]. These structures appeared as well-defined, circular lymphoid follicles in the submucosa layer, either isolated or in contact with nearby follicles, sometimes infiltrating the entire thickness of the mucosa (Figure 3). They were frequently detected in mucosal folds, and no well-defined germinal centers were observed. Also, macrophages were diffusely distributed within these Peyer’s patches, BL were the most abundant cells predominating in all follicles and mucosal infiltrations, and TL were diffusely distributed, mainly in the interfollicular areas. Although these lymphoid structures are consistent with those described by Silva et al. (2016) [12], the authors reported their presence in the medial region and final third of the intestine, whereas in this study they were exclusively observed in the final third. In this line, Cowan and Smith (1999) [8] described the presence of a continuous layer of lymphoid structures in the lamina propria of the distal intestinal segments beyond the splenic flexure, without providing a detailed description of the tissue morphology. Moreover, authors suggested that this segment was analogous to the vermiform appendix. In comparison to other terrestrial mammals, where Peyer’s patches are most frequently found in the ileum [19,21], our findings suggest that, in the harbour porpoise, these lymphoid structures are located further distally in the intestine, where the colon would typically be expected. However, it should be considered that the porpoise studied in this work was a subadult, in which Peyer’s patches were likely undergoing involution [13].

In addition, immune cells were identified in the anal canal, diffusely distributed within the keratinized stratified epithelium and the mucosa-submucosa (Figure 3 and Figure 4). However, most immune cells were concentrated in large aggregates of lymphoid tissue surrounding the epithelial crypts, corresponding to the anal tonsil. Although the histological structure observed was consistent with previous descriptions [8,12], the use of IHC techniques in the present study provided deeper insights into the cellular composition of this organ. In this regard, while macrophages were the most frequently detected immune cells throughout the GIT, in the anal tonsil they were less abundant than TL and BL, with the latter being the most prevalent (average percentage of positive cells in the mucosa-submucosa: 10.7%, 17.8%, and 24.3%, respectively) (Figure 4). The higher proportion of BL may be related to a stronger local humoral immune response at this level [9]. Regarding their distribution, BL and macrophages were observed diffusely in the epithelium and lamina propria-submucosa, as well as within the lymphoid aggregates, whereas TL were detected exclusively between these clusters. Those aggregates showed well-defined follicles, with macrophages diffusely distributed, TL predominating in the interfollicular regions, and BL distributed both diffusely or associated with the mantle and germinal center of the lymphoid follicles. Interestingly, the cellular detection levels of macrophages, TL, and BL were comparable to those observed in the LN analyzed in this study, as no significant differences were found. Therefore, based on its cellular composition, the anal tonsil might be considered a lymphoid organ analogous to the LNs.

Although some publications have described the LNs of the harbour porpoise as having an inverted structure similar to that of pigs [15], in the present study they exhibited a typical structure, with a well-differentiated cortex containing lymphoid follicles and a medullary region (Figure 5), consistent with descriptions in other cetacean species [17,22]. Overall, BL were the most frequently detected cells, followed by lower proportions of TL and macrophages (Figure 4). As expected, the cortical lymphoid follicles represented the region with the highest density of immunopositive cells, compared to the diffuse lymphoreticular tissue of both cortical and medullary regions (Figure 5). The percentage of macrophages did not show significant differences between the different LNs. In contrast, BL showed higher positivity in the cranial LNs, with the main detection in the pancreatic LN, followed by a gradual decrease in caudal LNs (Figure 4). In contrast, TL displayed an opposite pattern to that of BL, increasing in the caudal regions and reaching maximum positivity in the rectal LN (Figure 4).

A limitation of this work is the study of a single specimen; however, the inherent challenges in obtaining cetacean samples, particularly from healthy individuals, must be considered. In addition, IHC resulted an useful tool to detect immune cells in GALT, thus opening up the possibility of future immune response studies in cetaceans.

5. Conclusions

This study provides the first detailed histological and immunochemical characterization of the GIT and associated lymphoid tissue in harbour porpoise. The results highlight structural adaptations in the GIT for digestion and regional variation in immune cell distribution, which enables microscopic identification of the distal intestinal segment, even though distinguishing between the small and large intestines remains unfeasible. Lymphoid follicles resembling Peyer’s patches were restricted to the distal intestine, and the anal tonsil showed features comparable to LNs, supporting its relevant role as a major lymphoid organ. LNs showed typical mammalian organization, with B lymphocytes predominating and displaying cranio-caudal variation opposite to that of T cells. This study establishes a preliminary information for future research on cetacean health.