Introduction
Cormorants are very mobile, piscivorous birds that consume a wide variety of fish species. These birds are highly adaptable to changes in marine and riverine prey availability across coastal and inland habitats (Good et al., Reference Good, Weitkamp, Lyons, Roby, Andrews and Bentley2022). Cormorants received protective status in 1981 in the EU, and their global population has increased significantly in recent years, which has led to conflict with recreational and commercial fisheries and fish farms (Moravec and Scholz, Reference Moravec and Scholz2016; Dinesen et al., Reference Dinesen, Andersen, Worsaae and Støttrup2024). The subspecies Phalacrocorax carbo sinensis lives near large rivers, lakes, coasts, estuaries and lagoons in several regions of Europe (Bregnballe et al., Reference Bregnballe, Engström, Knief, Eerden, van Rijn, Kieckbusch and Eskildsen2003); indeed, most of their nesting colonies have been documented in the freshwater wetlands of Northern Italy and in the lagoons along the Adriatic coasts (e.g. Canneviè lagoon).
Parasitic nematode infection in wild animals contributes to changes in reproductive success, physiology and behaviour of the host (Dobson, Reference Dobson1988), and population genetic surveys can show complexes of morphologically indistinguishable, but genetically very different, cryptic species, which are common among the nematodes (Nadler and Perez-ponce de Leon, Reference Nadler and Perez-ponce de Leon2011). The factors determining the population genetics of nematodes in wild animals were detailed in a review by Cole and Viney (Reference Cole and Viney2018). Among the endoparasites infecting the alimentary canal of wild birds, the genus Contracaecum Raillet and Henry, 1912 is the most diverse and frequently observed group of anisakid reported. The genus includes over 50 species (D’Amelio et al., Reference D’Amelio, Barros, Ingrosso, Fauquier, Russo and Paggi2007), and Contracaecum rudolphii sensu lato (s.l.) Hartwich, 1964 usually infects cormorants of the genus Phalacrocorax, its definitive host. C. rudolphii (s.l.) is a complex of 6 sibling species with different genetic structure, geographical distribution, host preference and life cycle (Mattiucci and Nascetti, Reference Mattiucci and Nascetti2006; D’Amelio et al., Reference D’Amelio, Cavallero, Dronen, Barros and Paggi2012; Ángeles-Hernández et al., Reference Ángeles-Hernández, Gómez-de Anda, Reyes-rodríguez, Vega-sánchez, García-reyna, Campos-montiel, Calderón-apodaca, Salgado-miranda and Zepeda-velázquez2020). The use of nuclear and mitochondrial molecular markers confirmed the existence of cryptic species A and B (Zhu et al., Reference Zhu, D’Amelio, Paggi and Gasser2000; Li et al., Reference Li, D’Amelio, Paggi, He, Gasser, Lun, Abollo, Turchetto and Zhu2005) infecting the great cormorant, and the later identification of a third one, named C, in double-crested cormorants from west-central Florida (D’Amelio et al., Reference D’Amelio, Barros, Ingrosso, Fauquier, Russo and Paggi2007). In Australia, 2 additional subspecies were reported, named D and E, infecting P. carbo and Phalacrocorax varius (Shamsi et al., Reference Shamsi, Norman, Gasser and Beveridge2009a) and the last C. rudolphii F was recorded from the brown pelican Pelecanus occidentalis in the northern Gulf of Mexico (D’Amelio et al., Reference D’Amelio, Cavallero, Dronen, Barros and Paggi2012). In the Mediterranean areas, the occurrence of larvae of C. rudolphii A and B has been recorded in fish that inhabit both fresh and brackish waters, with C. rudolphii A more frequently reported in marine and brackish ecosystems (Amor et al., Reference Amor, Farjallah, Piras, Burreddu, Garippa and Merella2020; Roca-Geronès et al., Reference Roca-Geronès, Fisa, Montoliu, Casadevall, Tobella, Bas, Palomba and Mattiucci2023), while C. rudolphii B in freshwater environments (Mattiucci et al., Reference Mattiucci, Sbaraglia, Palomba, Filippi, Paoletti, Cipriani and Nascetti2020). C. rudolphii (s.l.) uses several fish species as paratenic hosts and cormorants as definitive hosts worldwide (Moravec, Reference Moravec2009; Mattiucci et al., Reference Mattiucci, Sbaraglia, Palomba, Filippi, Paoletti, Cipriani and Nascetti2020). Occurrence of the third-stage larvae (L3) of Contracaecum rudolphii A was encountered in Anguilla anguilla from Canneviè lagoon (Sayyaf Dezfuli et al., Reference Sayyaf Dezfuli, Pironi, Castaldelli, Giari, Lanzoni, Buchmann, Kania and Bosi2024), and this is the first account of the presence of the same nematode larvae in seabass Dicentrarchus labrax (Linnaeus, 1758) of the above lagoon.
P. carbo sinensis is the principal host of C. rudolphii (s.l.) in Europe, and in different regions the prevalence of infection was 100% (Rokicki et al., Reference Rokicki, Sołtysiak, Dziekońska-rynko and Borucińska2011; Amor et al., Reference Amor, Farjallah, Piras, Burreddu, Garippa and Merella2020; Caffara et al., Reference Caffara, Tedesco, Davidovich, Rubini, Luci, Cantori, Glogowski, Fioravanti and Gustinelli2023; Cammilleri et al., Reference Cammilleri, D’Amelio, Ferrantelli, Costa, Buscemi, Castello, Bacchi, Goffredo, Mancini and Cavallero2023; Roca-Geronès et al., Reference Roca-Geronès, Fisa, Montoliu, Casadevall, Tobella, Bas, Palomba and Mattiucci2023). The vast majority of the reports on the infection of great cormorant by Contracaecum rudolphii (s.l.) dealt with molecular identification of the nematodes and with ecological implications of species adaptation, and the studies on histopathology associated with the host tissue infection are limited (Kuiken et al., Reference Kuiken, Leighton, Wobeser and Wagner1999; Rokicki et al., Reference Rokicki, Sołtysiak, Dziekońska-rynko and Borucińska2011; Đurđević et al., Reference Đurđević, Vučićević, Bogunović, Pajić, Pelić, Todorović and Ćirković2024; Shamsi et al., Reference Shamsi, Nelson, Gordon, Markham, Francis, Suthar and Zhu2024).
In vertebrates, the alimentary canal is one of the primary routes of microbial and parasitic infections and affords a remarkable and rich environment for endoparasites (Leung et al., Reference Leung, Graham and Knowles2018). Most often, endoparasitic helminths induce inflammation and changes in the structure and function of the infected organ (Maizels and Gause, Reference Maizels and Gause2023), and in infected birds, the physical and immunological environment of the gut can be changed by both parasites and microbiota, creating a wide range of interactions (Nemathaga et al., Reference Nemathaga, Smith and Malatji2023).
The present investigation sought to answer the following questions: (1) Which organisms are involved in the life cycle of Contracaecum in the study area? (2) How many species of Contracaecum exist in cormorant and seabass? (3) What are the pathological effects of Contracaecum in the bird stomach, and what type of immune cells are involved and their quantitative estimate?
Materials and methods
Specimen collection and bird tissue preparation
Six freshly dead adult great cormorants and 41 seabass Dicentrarchus labrax (L.) were collected during the winter season 2025 from Canneviè lagoon (Northern Adriatic Sea, Italy, 44°48′07″N 12°13′49″E). Birds were subject to necropsy, and immediately afterwards, the whole alimentary tract of each bird was removed from the carcass and opened and searched for helminths. Furthermore, a random sample of 40 adult worms and 30 larvae from different birds was fixed in 70% ethanol for molecular investigation. From each bird, 25 pieces of proventriculus and gizzard and 10 pieces of oesophagus 15 × 15 mm in length with attached nematodes were excised and fixed in 10% neutral buffered formalin for 24–48 h. Then, the samples were dehydrated through an alcohol series and paraffin wax-embedded using a Shandon Citadel 2000 tissue processor. Multiple histological sections (5 µm thick) were taken from each tissue block, stained with Haematoxylin and Eosin, Alcian Blue PAS, Masson’s trichrome or Giemsa, and photographed under an optical microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan).
A morphological study was carried out on adult male nematodes (n = 115) and females (n = 38) following fixation and clearing methods suggested in Moravec (Reference Moravec2013). All the worms free in the lumen of the digestive tract of 6 great cormorants were morphologically attributed to the genus Contracaecum based on a comparison of apomorphic features for the genus provided by Li et al. (Reference Li, Xu and Zhang2013), Moravec and Scholz (Reference Moravec and Scholz2016) and Caffara et al. (Reference Caffara, Tedesco, Davidovich, Rubini, Luci, Cantori, Glogowski, Fioravanti and Gustinelli2023).
Counting and statistical analysis of host immune cells
The numbers of immune cells were counted on slides using a light microscope and image analysis software (Nis Elements AR 3.0). Gastric epithelial tissue was screened for mucous cells whilst connective tissues of stomach mucosa and submucosa were screened for mast cells (MCs) and macrophages. The cell numbers were determined through a blind count at 400× magnification in 120 areas (each area = 5.000 µm2): a total of 60 areas from 2 heavily infected cormorants (i.e. with more than 300 parasites) and 60 areas from 2 moderately infected cormorants (i.e. with less than 70 parasites). The cell density, expressed as the mean number of cells ± standard deviation in 5.000 μm2, was compared statistically between the cormorants with different intensity of infection applying a Student’s 2-tailed t-test. The level of significance was set at P = 0.05.
Parasite larvae and adult molecular identification
A subsample of nematodes of the Contracaecum genus collected from the cormorant digestive tract (adults = 40 and larvae = 30) and from the outer surface of the intestine of seabass (larvae = 40) were characterized at species level using the diagnostic key based on Restriction Fragment Length Polymorphism (RFLP) of 2 ribosomal regions, the nuclear ITS and the mitochondrial rrnS (12S), according to D’Amelio et al. (Reference D’Amelio, Barros, Ingrosso, Fauquier, Russo and Paggi2007). In particular, genomic DNA was isolated using Quick DNA Kit (Zymo Research) following the manufacturer’s instructions and the material was used to amplify the 2 regions of interest. For ITS, the forward primer NC5 (5′-GTAGGTGAACCTGCGGAAGGATCATT-3′) and the reverse primer NC2 (5′-TTAGTTTCTTCCTCCGCT-3′) were used (Zhu et al., Reference Zhu, D’Amelio, Paggi and Gasser2000). For the rrnS (12S), the forward primer MH3 (5′-TTGTTCCAGAATAATCGGCTAGACTT) and the reverse primer MH4.5 (5′-TCTACTTTACTACAACTTACTCC) were used. PCR conditions were as described by D’Amelio et al. (Reference D’Amelio, Barros, Ingrosso, Fauquier, Russo and Paggi2007). For each PCR, a negative control without genomic DNA was included, and individual PCR products were detected on agarose gels (1%), stained with SYBRSafe (Invitrogen, Waltham, MA, USA). Positive ITS and 12S PCRs were incubated with the endonuclease Tsp509I and RsaI, respectively, according to the best temperature for each enzyme and then resolved by electrophoresis in 2% agarose gels. The sizes of fragments were characterized by comparison to a 100 bp DNA ladder as size marker (Promega, Amsterdam, the Netherlands) and to reference patterns, according to D’Amelio et al. (Reference D’Amelio, Barros, Ingrosso, Fauquier, Russo and Paggi2007) and Cammilleri et al. (Reference Cammilleri, D’Amelio, Ferrantelli, Costa, Buscemi, Castello, Bacchi, Goffredo, Mancini and Cavallero2023).
Results
Parasitological analysis
All 6 P. carbo sinensis were infected with adult and larval specimens belonging to Contracaecum rudolphii (s.l.), and the intensity of infection varied from 39 to 550 worms per host (212.8 ± 80.7; mean ± s.e.; Figure 1A–C). A total of 1277 specimens of C. rudolphii (s.l.) were counted; larvae and adults were found to co-exist in the same host, but the vast majority of worms encountered in each single bird were mature nematodes (Figure 1A and B). During necropsy, many live adult nematodes were firmly attached, deep into the wall of the host proventriculus and gizzard; nevertheless, free adults and larvae were also observed within the lumen. The most parasitized regions of the cormorant digestive tract were the gizzard and proventriculus (Figure 1A and B); only in 2 hosts with the highest intensity of infection (310 and 550) were the nematodes also encountered in the distal oesophagus (Figure 1A). From the same lagoon, a subpopulation of 41 seabass, Dicentrarchus labrax (L.) were examined for helminths and in 27 fish specimens (66%, Confidence Interval: 50.4–79.6%) L3 of C. rudolphii (s.l.) were encountered on external surface of the intestine (Figure 1D and E) with the intensity of infection ranging from 5 to 50 nematode larvae per seabass (16.9 ± 2.2, mean ± s.e.).
(A–C) Digestive tract of the great cormorant, Phalacrocorax carbo sinensis infected with the nematode Contracaecum rudolphii (s.l.). (D–E) L3 of C. rudolphii A on outer surface of the intestine of seabass, Dicentrarchus labrax. (A) Photograph shows a heavily infected cormorant: oesophagus (curved arrow), proventriculus (arrow) and gizzard (thick arrow) harboured numerous C. rudolphii (s.l.); larval specimens (arrow heads) are whitish in colour. (B) Higher magnification of gizzard and proventriculus of (A): high dimensions of mature adult worms and deep penetration of C. rudolphii (s.l.) specimens (arrows) in the organ wall are evident. (C) Image of uninfected oesophagus (curved arrow) and presence of mature and larvae of C. rudolphii (s.l.) in proventriculus (arrow) and gizzard (white arrow) is visible, good condition of heart (asterisk) and liver (2 asterisks) is evident. Difference in colour of very heavily infected gizzard in (A) and less parasitized conspecific in (B) is appreciable. (D) Outer surface of D. labrax intestine, some C. rudolphii A L3 (arrows) are totally encircled by serosa (asterisks). (E) Photograph shows C. rudolphii A L3 (arrow) partially surrounded by serosa (asterisk).
A morphological study was performed on adult male nematodes (n = 115) and females (n = 38). Mature adult specimens of C. rudolphii (s.l.) were dark grey in colour and L4 larvae were whitish in colour and smaller in size (female larvae, 23.20 ± 6.38 mm, mean length ± s.e., 1.28 ± 0.02 mm, width, n = 10; male larvae, 18.20 ± 0.77 mm length, 0.98 ± 0.02 mm width, n = 10). Briefly, both sexes had a transversely striated cuticle, an evident cephalic collar, 3 well-developed lips and a conical tail. Females were larger than males in size (mature female, 33.63 ± 2.22 mm length, 1.59 ± 0.05 mm width, n = 10; mature male, 23.18 ± 0.82 mm length, 1.06 ± 0.03 mm width, n = 10).
Gross and histopathological findings
All 6 freshly dead great cormorants showed no visible sign of autolysis; the internal organs appeared intact and recognizable (Figure 1C). The gizzard and proventriculus mucosal layer of heavily infected birds was very dark in colour, while in birds with less parasites the organ was a clear colour (Figure 1C). The difference in colour of the infected versus uninfected oesophagus was also noteworthy (Figure 1A and C, respectively). Nematodes were firmly embedded in the gizzard and proventriculus. The depth of penetration by the nematodes was generally restricted to the level of the gastric glands of the proventriculus. Detachment-destruction of the epithelial cells of the organ was associated with the area of nematode attachment (Figure 2A). Damage to the epithelial cells in the infected gastric gland is present compared to adjacent uninfected areas (Figure 2B). In several sections, an inflammatory reaction was observed in the mucosa adjacent to the burrowing nematodes and consisted of focal accumulations of macrophages (Figure 2A and C). In some gastric glands, hyperplasia of the mucous cells and abundant mucus secretion was observed around the worm body (Figure 2D). In 2 gizzards, worms were present in the thick muscle layer and at the site of infection and in its proximity; this layer was necrotic and in a state of degeneration, forming a chasm (Figure 2E). In the muscle layer of the gizzard, the nematode induced intense inflammation with recruitment of collagen fibres (Figure 2E), MCs and macrophages (Figure 2F), which encircled the C. rudolphii (s.l.).
Histological sections of Phalacrocorax carbo sinensis infected digestive tract. (A) Deep penetration of nematode (arrow) within the gastric gland induced detachment and desquamation of the epithelial cells (thick arrow), note lack of desquamate epithelial cells in uninfected gastric gland (curved arrows). Inflammatory cells (arrow heads) in mucosal layer near nematode body, stain, Masson’s trichrome, scale bar = 200 μm. (B) Micrograph shows 3 gastric glands, in an infected one, near section of parasite body (thick arrow), the residue of desquamate epithelium is visible (arrow), in glands with no parasite intact aspect of the tissue can be seen (curved arrows), stain, Giemsa, scale bar = 200 μm. (C) Recruitment of inflammatory cells (arrows) near C. rudolphii (s.l.) body (asterisk), stain Masson’s trichrome, scale bar = 20 μm. (D) Inside the gastric gland, the body of nematode (arrow heads) is surrounded by abundant mucus (thick arrows), within the epithelium hyperplasia of mucous cells is visible (arrows), stain, Alcian Blue PAS, scale bar = 200 μm. (E) Muscularis layer is infected with a C. rudolphii (s.l.), inflammatory cells (arrow heads) encircled the nematode cuticle (arrows), numerous collagen fibres (curved arrows) and chasm (asterisk) are visible, stain Masson’s trichrome, scale bar = 200 μm. (F) High magnification on interface region between C. rudolphii (s.l.) body (asterisk) and mast cells (arrows) and macrophages (curved arrows) in muscularis layer of proventriculus, stain, Giemsa, scale bar = 10 μm.
The number of MCs, macrophages and mucous cells in birds with the highest intensity of infection was statistically significantly higher in comparison to hosts with less worms (Table 1).
Mean number (± standard deviation, s.d.) of mucous cells, mast cells and macrophages in 5.000 µm2 of gastric tissue from cormorants with high (more than 300 parasites) and low (below 70 parasites) intensity of infection. The cell densities were evaluated in 30 areas of tissue (each area 5.000 µm2) per cormorant specimen at 400× magnification
Table 1 Long description
Mean cell densities per five thousand square micrometers of cormorant gastric tissue are compared between stomachs with high versus low parasite infection. Mucous cells average 33.75 with a standard deviation of 11.32 in high infection, versus 27.59 with a standard deviation of 7.09 in low infection. Mast cells average 14.34 with a standard deviation of 5.71 in high infection, versus 11.32 with a standard deviation of 3.03 in low infection. Macrophages average 13.28 with a standard deviation of 4.64 in high infection, versus 10.36 with a standard deviation of 2.78 in low infection. For all three cell types, the high infection group shows higher mean counts than the low infection group. Superscript letters indicate that the high and low infection means differ significantly within each row based on a Student t test with a p value below 0.05.
Different superscript letters in the same line indicate significant differences (Student t-test, P < 0.05).
Molecular identification of parasite larvae and adults
Positive amplicons were obtained for more than 90% of the material regarding the adults (37/40) and for 63% of the larvae (19/30) from the cormorants and for 25% of larvae from the seabass (10/40) for both markers, with sizes of the target loci of ∼1000 bp for ITS and ∼600 for 12S. The 2 expected sibling species C. rudolphii A and B were identified, according to the banding patterns: C. rudolphii A showed bands of 300, 200, 150 and below 100 bp at ITS digested with Tsp509I and bands of 350, 130 and below 100 bp at rrnS cut with RsaI; C. rudolphii B showed bands of 500, 350 and below 100 bp at ITS and bands of 280 and below 100 bp at rrnS.
Regarding the adults and larvae infecting the cormorants, 83.9% (47/56) were identified as Contracaecum rudolphii B and the remaining 16.1% (9/56) were C. rudolphii A and all larval stages from the seabass were identified as C. rudolphii A.
Discussion
Cormorants – nematode system/parasitological survey
The role of parasitic nematodes in natural ecosystem regulation appears highly relevant; the population genetics of nematode parasites of wild animals may have consequences for host conservation, and host movement is essential for nematode population genetic structure (Cole and Viney, Reference Cole and Viney2018). There are about 40 species of shags and cormorants (Phalacrocoracidae) foraging in marine, fresh and brackish waters (Sibley, Reference Sibley2001), and all species are predominantly piscivorous. Conflict between conservation and fisheries stakeholders concerning the cormorant is one of the most relevant and current wildlife management issues (Ovegård et al., Reference Ovegård, Jepsen, Bergenius Nord and Petersson2021). Great cormorants are migratory birds; an insight into their role as carriers for the introduction and spread of infectious diseases in new areas was recently discussed by Lee et al. (Reference Lee, Alkathiri, Lee, Lee, Jeong, Kim, Choe and Lee2025). Among the great cormorant subspecies, Phalacrocorax carbo sinensis is a highly specialized fish-eating bird with a sub-cosmopolitan distribution and has been encountered near coasts, estuaries, lagoons, large rivers and lakes in different parts of Europe (Bregnballe et al., Reference Bregnballe, Engström, Knief, Eerden, van Rijn, Kieckbusch and Eskildsen2003). It migrates between October and April and winters in Italy; most of the nesting colonies are in the freshwater wetlands of Northern Italy and in the lagoons along the Adriatic coasts (Bregnballe et al., Reference Bregnballe, Engström, Knief, Eerden, van Rijn, Kieckbusch and Eskildsen2003). The largest number of breeders occurs in the Emilia-Romagna region, where the Canneviè lagoon is situated.
Species of cormorants have been reported as the main definitive hosts for C. rudolphii (s.l.) from several regions of the world (Moravec, Reference Moravec2009; Shamsi et al., Reference Shamsi, Norman, Gasser and Beveridge2009b). Multilocus allozyme electrophoresis analysis on specimens of C. rudolphii (s.l.) recovered from cormorant populations in Europe demonstrated the existence of 2 sibling species, namely C. rudolphii A and C. rudolphii B (D’Amelio et al., Reference D’Amelio, Nascetti, Mattiucci, Cianchi, Orecchia, Paggi, Berland and Bullini1990; Mattiucci et al., Reference Mattiucci, Turchetto, Brigantini and Nascetti2002). Subsequently, accounts on further sibling species of C. rudolphii parasites of piscivorous birds from different continents were published (Shamsi et al., Reference Shamsi, Norman, Gasser and Beveridge2009a; D’Amelio et al., Reference D’Amelio, Cavallero, Dronen, Barros and Paggi2012; Shamsi, Reference Shamsi2019) and papers on the species of Contracaecum in cormorants from Asia (Al-Moussawi, Reference Al-Moussawi2017), Africa (El-Dakhly et al., Reference El-Dakhly, Hussein and El-nahass2018) and in South America has been published recently in Garbin et al. (Reference Garbin, Servián, Fuentes, Morgenthaler, Millones, Fuchs, Diaz and Navone2024) and Villena et al. (Reference Villena, Ramos, Daga, Santillán, Cruces, Ñacari, Arias, Mondragón-martínez, Martínez-rojas, Luque and Chero2025).
Records on the presence of C. rudolphii A and C. rudolphii B in the great cormorant P. carbo sinensis from Europe are frequent and the 2 species may co-occur in a single individual host (Amor et al., Reference Amor, Farjallah, Piras, Burreddu, Garippa and Merella2020; Mattiucci et al., Reference Mattiucci, Sbaraglia, Palomba, Filippi, Paoletti, Cipriani and Nascetti2020; Carmeno et al., Reference Carmeno, Rusconi, Castelli, Prati, Bragoni, Santoro, Postiglione, Sassera and Olivieri2022; Cammilleri et al., Reference Cammilleri, D’Amelio, Ferrantelli, Costa, Buscemi, Castello, Bacchi, Goffredo, Mancini and Cavallero2023; Caffara et al., Reference Caffara, Tedesco, Davidovich, Rubini, Luci, Cantori, Glogowski, Fioravanti and Gustinelli2023; Roca-Geronès et al., Reference Roca-Geronès, Fisa, Montoliu, Casadevall, Tobella, Bas, Palomba and Mattiucci2023 and present study). Very limited sporadic observations in the wild and in a single study under experimental conditions have been performed by Moravec (Reference Moravec2009) to ascertain the life cycle of C. rudolphii (s.l.). It is interesting that the 2 species showed a peculiar ecological adaptation, with C. rudolphii A occurring in cormorants inhabiting marine and brackish ecosystems and C. rudolphii B infecting birds from freshwater sites (Mattiucci et al., Reference Mattiucci, Turchetto, Brigantini and Nascetti2002, Reference Mattiucci, Sbaraglia, Palomba, Filippi, Paoletti, Cipriani and Nascetti2020; Carmeno et al., Reference Carmeno, Rusconi, Castelli, Prati, Bragoni, Santoro, Postiglione, Sassera and Olivieri2022; Caffara et al., Reference Caffara, Tedesco, Davidovich, Rubini, Luci, Cantori, Glogowski, Fioravanti and Gustinelli2023; Palomba et al., Reference Palomba, Belli, Chiatante, Favero, Canestrelli, Nascetti and Mattiucci2025). Such differences have been recently investigated in vitro and explored the effect of temperature and salinity on egg hatching success in the 2 sibling species. The study reported species-specific dynamics, primarily driven by salinity, which supports the differential ecological adaptation of the 2 taxa in their respective aquatic habitats (Palomba et al., Reference Palomba, Belli, Chiatante, Favero, Canestrelli, Nascetti and Mattiucci2025).
Great cormorants are avian top predators, opportunistic feeders whose diet is almost exclusively based on a wide range of fish species. They are able to change their diet according to the seasonal availability of prey at a particular site (Leopold et al., Reference Leopold, Van Damme and Van Der Veer1998). The high adaptability of these piscivorous birds is very important for the establishment of the life cycle of their enteric worms such as Contracaecum (s.l.) (Mattiucci and Nascetti, Reference Mattiucci and Nascetti2006). The high prevalence of C. rudolphii B was reported in cormorants of different freshwater geographical areas (Rokicki et al., Reference Rokicki, Sołtysiak, Dziekońska-rynko and Borucińska2011; Moravec and Scholz, Reference Moravec and Scholz2016; Mattiucci et al., Reference Mattiucci, Sbaraglia, Palomba, Filippi, Paoletti, Cipriani and Nascetti2020; Carmeno et al., Reference Carmeno, Rusconi, Castelli, Prati, Bragoni, Santoro, Postiglione, Sassera and Olivieri2022). Our molecular investigation with PCR/RFLP on adults and larvae (47/56) recovered in the digestive tract of the great cormorant from Canneviè lagoon found that over 83% of the nematodes were C. rudolphii B and only 16% (9/56) were C. rudolphii A. Results of a similar molecular analysis on Contracaecum isolated from the alimentary canal of cormorants from Lake Como (North Italy) also found that C. rudolphii B was the dominant species (over 78%) and C. rudolphii A occurred less frequently (21%) (Carmeno et al., Reference Carmeno, Rusconi, Castelli, Prati, Bragoni, Santoro, Postiglione, Sassera and Olivieri2022). It is known that C. rudolphii B uses freshwater fish species as paratenic hosts while C. rudolphii A prefers brackish and marine fish (Mattiucci et al., Reference Mattiucci, Sbaraglia, Palomba, Filippi, Paoletti, Cipriani and Nascetti2020; Caffara et al., Reference Caffara, Tedesco, Davidovich, Rubini, Luci, Cantori, Glogowski, Fioravanti and Gustinelli2023; Roca-Geronès et al., Reference Roca-Geronès, Fisa, Montoliu, Casadevall, Tobella, Bas, Palomba and Mattiucci2023; Sayyaf Dezfuli et al., Reference Sayyaf Dezfuli, Pironi, Castaldelli, Giari, Lanzoni, Buchmann, Kania and Bosi2024), and the reason for the presence of C. rudolphii A in cormorants in a freshwater site was thought to be due to host individuals originating from brackish lagoons and marine costal sites which may transport the nematodes from breeding areas to migration stopovers (Li et al., Reference Li, D’Amelio, Paggi, He, Gasser, Lun, Abollo, Turchetto and Zhu2005; Szostakowska and Fagerholm, Reference Szostakowska and Fagerholm2007; Mattiucci et al., Reference Mattiucci, Sbaraglia, Palomba, Filippi, Paoletti, Cipriani and Nascetti2020). Lastly, data on the population genetics of C. rudolphii A suggest the probable role of the migration routes of wintering populations of cormorants in the Mediterranean Sea in influencing the parasite genetic structure (Roca-Geronès et al., Reference Roca-Geronès, Fisa, Montoliu, Casadevall, Tobella, Bas, Palomba and Mattiucci2023).
With reference to the co-existence of C. rudolphii A and B in bird hosts, this could be due to acquisition of the 2 sibling species at different times and in different areas (Mattiucci et al., Reference Mattiucci, Sbaraglia, Palomba, Filippi, Paoletti, Cipriani and Nascetti2020). Canneviè lagoon is situated near a tributary of the River Po (about 20 m) where freshwater fish are abundant and numerous cormorants actively prey upon such fish. Conversely, inside the Canneviè lagoon, the presence of seabass and eel of high commercial value results in a constant presence of staff workers, the use of bird-deterring devices (scaring laser) that reduce the access of the cormorants to the above lagoon. Therefore, a reasonable hypothesis for the presence of a high number of specimens of C. rudolphii B in the alimentary canal of great cormorants is that the birds came from very close to the river shore, where several colonies of resident cormorants live and feed on different wild fish species without human interference or limitation. With reference to the cormorant – C. rudolphii B system in Lake Como, it was stated that the resident cormorants have an essential role in the stability of nematode occurrence (Carmeno et al., Reference Carmeno, Rusconi, Castelli, Prati, Bragoni, Santoro, Postiglione, Sassera and Olivieri2022). However, until further evidence emerges, a reasonable supposition may be that the resident cormorants of the River Po might also provide stability to the life cycle of C. rudolphii B in Canneviè lagoon.
In Italy, molecular identification of larval stages of C. rudolphii (s.l.) encountered in Liza aurata, Dicentrarchus labrax and Sparus aurata from brackish lagoons in Sardinia Island confirmed the occurrence of C. rudolphii A (Merella and Garippa, Reference Merella and Garippa2001; Salati et al., Reference Salati, Meloni, Cau and Angelucci2013; Culurgioni et al., Reference Culurgioni, Sabatini, De Murtas, Mattiucci and Figus2014). Recently, the same species was identified with molecular analysis for the larvae recovered in Anguilla anguilla from Canneviè lagoon (Sayyaf Dezfuli et al., Reference Sayyaf Dezfuli, Pironi, Castaldelli, Giari, Lanzoni, Buchmann, Kania and Bosi2024). The present study indicates the occurrence of L3 nematodes isolated from the outer part of the intestine of seabass D. labrax from Canneviè lagoon, molecularly attributed to the species C. rudolphii A. Similar findings were reported in seabass from other parts of Italy (Culurgioni et al., Reference Culurgioni, Murtas, Cannella and Figus2010; Guardone et al., Reference Guardone, Susini, Castiglione, Ricci, Corradini, Guidi and Armani2020; Mattiucci et al., Reference Mattiucci, Sbaraglia, Palomba, Filippi, Paoletti, Cipriani and Nascetti2020). The reduced success of the amplification of larvae recovered from the outer surface of the seabass intestine was most probably due to the small size of the worms that were coiled and embedded in fish-derived tissue, which may have jeopardized the parasitic nematode-specific PCR efficiency. Similarly, the lack of desirable success in the amplification of larvae from the cormorant stomach might be related to dry and sometimes damaged worms, which were smaller in size and more delicate in comparison to adult nematodes.
Histopathology and bird immune response
It is known that the pathogenicity of most enteric helminths is attributed to 2 factors: intensity of infection and depth of worm penetration in host tissue (Sayyaf Dezfuli et al., Reference Sayyaf Dezfuli, Giari and Bosi2021). However, there is a dearth of detailed information on the pathology and cellular immune response of the great cormorant associated with Contracaecum infection. Within the very limited records available, only gross histology has been described, and no reliable evidence on the type of cells involved in the host bird reaction was provided (Kuiken et al., Reference Kuiken, Leighton, Wobeser and Wagner1999; Rokicki et al., Reference Rokicki, Sołtysiak, Dziekońska-rynko and Borucińska2011; El-Dakhly et al., Reference El-Dakhly, Hussein and El-nahass2018; Đurđević et al., Reference Đurđević, Vučićević, Bogunović, Pajić, Pelić, Todorović and Ćirković2024; Shamsi et al., Reference Shamsi, Nelson, Gordon, Markham, Francis, Suthar and Zhu2024). It was mentioned that the presence of a high number of penetrating nematodes had no observable effect upon the condition of the birds examined (Huizinga, Reference Huizinga1971), nor was a case of mortality among cormorants that harboured C. rudolphii encountered (Rokicki et al., Reference Rokicki, Sołtysiak, Dziekońska-rynko and Borucińska2011), and it was stated that Contracaecum spp. are an unlikely primary cause of bird mortality (Shamsi et al., Reference Shamsi, Nelson, Gordon, Markham, Francis, Suthar and Zhu2024). The results of our study match very closely with the above statements, as during necropsy, the internal organs were intact and recognizable.
Histopathological lesions documented in the current investigation were mainly on the mucosal layer of the gizzard and proventriculus of the cormorants. Cellular immune responses in birds against nematodes generally involve the following cells: MCs (Rehman et al., Reference Rehman, Meng, Umar, Mahrose, Ding and Munir2017; Nemathaga et al., Reference Nemathaga, Smith and Malatji2023), macrophages (Webb and Tait Wojno, Reference Webb and Tait Wojno2017) and mucous cells (Padilla-Aguilar et al., Reference Padilla-Aguilar, Romero-callejas, Ramírez-lezama, Osorio-sarabia, García-prieto, Manterola, García-márquez and Zarza2020; Shohana et al., Reference Shohana, Rony, Ali, Ali, Hossain, Labony, Dey, Farjana, Alam, Alim and Anisuzzaman2023). All the above cell types were observed in the present study. In particular, MCs are important components of the immune system, and they perform pro-inflammatory as well as anti-inflammatory roles in the complex process of immune regulation in health and disease (Cardamone et al., Reference Cardamone, Parente, De Feo and Triggiani2016). MCs are located at sites such as the skin, lung and digestive tract (Webb and Tait Wojno, Reference Webb and Tait Wojno2017). On the other hand, parasites are able to adopt strategies to ensure their survival by altering the activation of the immune response of the host (Rehman et al., Reference Rehman, Meng, Umar, Mahrose, Ding and Munir2017). Malatji et al. (Reference Malatji, van Marle-koster and Muchadeyi2019) reported the downregulation of MC protease 1a-like, cytochrome P450 and apolipoprotein B in chickens infected with the nematode Ascaridia galli parasites; data mentioned in Malatji et al. (Reference Malatji, van Marle-koster and Muchadeyi2019) tally with the results provided in Balqis et al. (Reference Balqis, Hambal and Priosoeryanto2013) that showed the essential role played by MCs in controlling A. galli infection.
Macrophages are the major population of tissue-resident mononuclear phagocytes and contribute significantly to the immune response during helminth infection (Coakley and Harris, Reference Coakley and Harris2020). Macrophages are activated quickly in the anti-helminth response following tissue insult and parasite recognition (Webb and Tait Wojno, Reference Webb and Tait Wojno2017; Coakley and Harris, Reference Coakley and Harris2020). Herein, the occurrence of macrophages was noticed in different layers of the gizzard and proventriculus; the same finding was reported in cormorants from Poland infected with C. rudolphii (Rokicki et al., Reference Rokicki, Sołtysiak, Dziekońska-rynko and Borucińska2011).
Mucous cells are frequently associated with gastrointestinal helminth infections, displaying hyperplasia in close vicinity to the worm’s body; these cells are able to produce high quantities of mucins and RELM-β with direct anti-helminth properties (Herbert et al., Reference Herbert, Yang, Hogan, Groschwitz, Khodoun, Munitz, Orekov, Perkins, Wang, Brombacher, Urban, Rothenberg and Finkelman2009). In mice infected with the nematode Nippostrongylus brasiliensis and the bacterium Citrobacter rodentium, an IL-13–independent route promoting mucous cell differentiation, driven by IL-22, has been discovered, which preferentially induces RELM-β (Lindholm et al., Reference Lindholm, Parmar, Drurey, Campillo Poveda, Vornewald, Ostrop and Oudhoff2022). Herein, we provided data on hyperplasia of mucous cells and excessive mucus secretion in the infected proventriculus and gizzard of the great cormorant; the same finding was mentioned in chickens infected with nematodes (Shohana et al., Reference Shohana, Rony, Ali, Ali, Hossain, Labony, Dey, Farjana, Alam, Alim and Anisuzzaman2023; Khatun et al., Reference Khatun, Al Mahmud, Akhi, Sadi, Das, Islam and Islam2025; Rus et al., Reference Rus, Matei-lațiu and Gal2025). The mucus layer acts as a barrier to prevent the attachment and colonization of harmful bacteria since it contains immunoglobulins such as IgA, which play a role in the immune defence by neutralizing pathogens and toxins before they can cause harm to the bird (Duangnumsawang et al., Reference Duangnumsawang, Zentek and Goodarzi Boroojeni2021).
We also provided the first quantitative assessment of MCs, macrophages and mucous cells in a bird-nematode system. Our results showed that the intensity of infection influenced the recruitment and density of the above immune cells in the gastric region. MCs and macrophages play critical roles in inducing and regulating both innate and adaptive immune responses to fight against pathogen infection; they enhance the host defences by triggering the cellular immune responses from blood and lymphoid organs at the site of infection (Rehman et al., Reference Rehman, Meng, Umar, Mahrose, Ding and Munir2017; Cui et al., Reference Cui, Franz, Fillon, Jannetti, Isambert, Fundel-Clemens, Huber, Viollet, Ghanem, Niwa, Weigle and Pflanz2021).
Conclusions
The present study was undertaken to find answers to some queries on the cormorant-Contracaecum (s.l.) system in a brackish environment; almost all the responses summarized below constitute the novelties of this research. In Canneviè lagoon, seabass acts as the paratenic host and cormorant as the definitive host for C. rudolphii (s.l.). No previous account exists on the occurrence of these nematodes in the stomach of cormorants from this study site. Molecular characterization of nematodes revealed that the majority of the worms found in cormorants belong to C. rudolphii B, typical of freshwater ecosystems and a lesser number to C. rudolphii A, infecting brackish and marine organisms, whilst the larvae from seabass were all C. rudolphii A. This mismatch might shed light on the diet of Canneviè cormorants, which apparently feed mainly on neighbouring freshwater prey. Lastly, for the very first time, photographic documentation of the immune cells in response to C. rudolphii (s.l.) and quantitative data on MCs, macrophages and mucous cells in cormorants with different intensity of infection were provided.
Acknowledgements
We thank E. Franchella from the University of Ferrara for technical assistance. We are indebted to C. Holland from Trinity College Dublin for the English correction of the manuscript.
Author contributions
B.S.D.: conceptualization, investigation, data curation, writing – original draft, funding acquisition; F.P., L.B. and L.M.: data gathering; S.C. and S.D.: molecular analyses; L.G.: data curation and writing – review & editing.
Financial support
This work was supported by grants from the University of Ferrara to B. Sayyaf Dezfuli (FAR 2025).
Competing interests
The authors declare no conflicts of interest exist.
Ethical standards
Not applicable.
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