Parasites, Bacteria, and Associated Pathological Changes in the Digestive System of Diurnal and Nocturnal Raptors in Central Italy

The knowledge of raptor pathogens and associated lesions may be extremely important to enhancing raptor conservation efforts and reducing pathogen spillover to humans and domesticated animals and vice versa. Parasite infections of the digestive system and associated bacteria and pathological changes were evaluated in deceased diurnal and nocturnal raptors in central Italy. Overall, the prevalence of parasites (nematodes, cestodes, trematodes, acanthocephalans, and protozoa) identified in the examined birds was 72.41%, and most of the positive raptors (71.42%) showed multiple parasite infections. Among bacteria, Salmonella typhimurium, Salmonella enterica subspecies diarizonae, Escherichia coli, Clostridium perfringens, Yersinia enterocolitica, and Pasteurella multocida were identified. The results obtained showed that both parasites and bacteria may cause severe lesions in the digestive system of diurnal and nocturnal raptors; parasites and bacteria may concur in causing these lesions; most severe lesions are caused by the interaction of multiple pathogens, both parasites and bacteria; and the same pathogen taxa are frequently associated with the same pathological changes. This study is the first report of S. typhimurium and S. enterica subspecies diarizonae in Buteo buteo, while Andracantha mergi, Spirocerca spp., Sarcocystis dispersa, Sarcocystis columbae, and Eumonospora spp. were recorded for the first time in Italy.


Introduction
Raptors play a fundamental role in ecosystems as apex predators and scavengers and are considered biological indicators of environmental pollution. Therefore, studies on pathogens of raptors may provide useful data for ecosystem health monitoring, the evaluation of the health status of raptors populations, and the role these birds may play in spreading some important pathogens, such as drug-resistant bacteria and potentially zoonotic bacteria and parasites [1][2][3][4]. These animals are protected species in many countries, such as in Italy [5]. Nonetheless, worldwide, many raptor species are highly threatened [6]. In Italy, some diurnal and nocturnal raptors are currently included among endangered species, such as the marsh harrier (Circus aeruginosus), the Eleonora's falcon (Falco eleonorae), the Egyptian vulture (Neophron percnopterus), and the Eurasian pygmy owl (Glaucidium passerinum) [6].
Environmental deterioration and modifications due to human activities, in combination with other factors, such as direct persecution, are the main causes that are currently contributing to the decline of raptor populations [4,[7][8][9][10][11]. The increase in urbanized areas and the conversion of natural environments to crop and livestock production with the destruction of wooded areas have greatly reduced habitats suitable for nesting, migratory stops, and hunting of raptors [9,11].

Results and Discussion
Most of the birds examined in this study were admitted to wildlife recovery centers and died from trauma, often caused by gunshot (Table 1). Nevertheless, catarrhal and/or hemorrhagic (gastro) enteritis and cachexia were frequently observed in raptors showing or not traumatic lesions (Table 1).
Overall, the prevalence (72.41%) of parasites found in the digestive system of examined birds was high, as 21 out of 29 examined raptors scored positive for at least a parasite species. In most positive birds (14/21, 66.67%), the positivity for nematodes and protozoa was evidenced also at coprological analysis. Moreover, most of the positive raptors (15/21, 71.42%) showed multiple parasite infections (Table 2), as frequently observed in previous European studies [24,30,[33][34][35][36][37][38]. Table 1. Examined diurnal (12) and nocturnal (17) raptors deceased in wildlife rescue centers in Tuscany (central Italy) and the results of post-mortem gross examination. Among identified parasites, helminths and especially nematodes (20/21, 95.23%) were more frequently recorded, as all positive raptors scored positive for these parasites, except for a barn owl found infected only by protozoa (Table 2). Capillariid nematodes were found in almost all the raptor species here examined and in most of the positive birds (15/21, 71.42%), followed by Procyrnea spp. (10/21, 47.61%), even if these latter nematodes were more frequently found among diurnal raptors (Table 2). Trematodes and, mainly, cestodes were more rarely found (Table 2).
Acanthocephalans were also frequently identified, especially in diurnal raptors (Table 2), while Sarcocystis spp. and Eumonospora spp. protozoa were identified in a few birds ( Table 2).
Lesions of different typologies and levels of severity were evidenced at histopathological analysis in the different digestive tracts of the examined raptors (Table 3).
Moreover, among selected bacteria, secondary bacterial infection and bacteria considered primary causes of infections and/or diseases in these birds were identified at bacteriological analysis (Table 4) [11,17,[26][27][28][29].    Table 3. Results of histopathological analysis of the different digestive system tracts of the 18 examined raptors. Score lesion is: 0 (lesion not observed); 1(+) = mild injury; 2(++) = moderate injury; 3(+++) = severe injury. NS = organ not sampled. Focus of chronic-active inflammation, characterized by the presence of heterophiles, lymphocytes, and macrophages infiltrating a segmental tract of the organ wall, with the presence of parasites in the endoluminal area but not encysted or with a granulomatous reaction; C: Diffuse chronic inflammation with the presence of transmural infiltrate of lymphocytes and plasma cells in the absence of visible parasites; C1: As above, but with an evident dysmicrobism, characterized by a strong presence of Gram-negative/Gram-positive bacteria and damage to the intestinal epithelium; C2: As above, but with evident clostridial overgrowth (presence at the level of the crypts and glandular lumens of the mucosa of large quantities of filamentous rod-cell bacteria with clear endosomatic endospore); C3: As above, but with the evident presence of endocellular protozoa in the epithelium (various phases of the cycle) or inside macrophages interspersed in the intestinal wall; C4: As above, but with the evident presence of sub-epithelial parasites encysted at the apical/superficial level of the intestinal mucosa. D: Micro-and macro-granulomatous foci, characterized by the presence of parasites (larvae/adults) in the muscle wall. Granulomas characterized by a necrotic center with the presence of dystrophic calcification, a cell wall consisting of numerous heterophilic granulocytes and numerous macrophages or sometimes of giant cells and mixed lymphocytes and/or plasma cells; D1: As above, but with the presence of ulcerations in the mucosa above the micro-granulomatous areas; D2: As above, but with lesions located mainly in the isthmus area (anatomical area between the glandular stomach and the muscular stomach); E: The presence of lesions of atypical micro-granulomatous type (type "typhoid nodules" = foci of caseous necrosis of sub-miliary size with around some epithelioid cells or 2-3 macrophages and lymphocytes, in the absence of an encapsulating reactive fibrous wall); F: The presence of parasites in the endoluminal area or attached to the mucosa, in the absence of specific lesions; G: The presence of parasites inside the muscle fibers of the wall, in the absence of pericystic reaction (with few calcified cysts). Among examined diurnal raptor species, all buzzards were found infected by Procyrnea mansioni in the gizzard and proventriculus, Porrocoecum angusticolle and Baruscapillaria falconis in the small intestine and Centrorhynchus spp. in the large intestine, although in some cases, the infection intensity was very different among the examined buzzards ( Table 2, Figures 1 and 2). Moreover, Eucoleus dispar in the esophagus of the common buzzards 3 and 4, Physaloptera alata in the gizzard of the common buzzard 1, Neodiplostomum attenuatum and Spirocerca spp. in the intestine of the common buzzard 2, and Cladotaenia globifera cestodes in the intestine of the common buzzard 4, were also identified ( Table 2, Figures 1 and 2).     Gross lesions were found mainly in the digestive tract of the common buzzards 2, 3, and 4 (Table 1).

Raptor Species
At the histopathological examination, areas of mucosal erosion, as well as hyperemia, foci of micro-and macro-granulomatosis, and chronic-active inflammation were evidenced in the gizzard and in the proventriculus of all these buzzards and especially in the isthmus in two of them (Table 3, Figure 3a,b). These lesions were found constantly observed in P. mansioni-infected birds, and the number and severity of these lesions were Regarding bacteriological analysis, Salmonella typhimurium and Salmonella enterica subspecies diarizonae were detected in the liver and intestine of the common buzzard 2 and 3, respectively (Table 4). To the best of our knowledge, this study is the first report of S. typhimurium and S. enterica subspecies diarizonae in the common buzzard (B. buteo).
Gross lesions were found mainly in the digestive tract of the common buzzards 2, 3, and 4 (Table 1).
At the histopathological examination, areas of mucosal erosion, as well as hyperemia, foci of micro-and macro-granulomatosis, and chronic-active inflammation were evidenced in the gizzard and in the proventriculus of all these buzzards and especially in the isthmus in two of them (Table 3, Figure 3a,b). These lesions were found constantly observed in P. mansioni-infected birds, and the number and severity of these lesions were also found related to P. mansioni intensity, which was higher in two buzzards ( Table 2). In previous studies [30,45], dark red nodular lesions, erosions, and ulcers of the gastric mucosa associated with Procyrnea sp. infection have been reported in diurnal raptors, including buzzards, while data on associated histological lesions are lacking.
Inflammation of the intestinal mucosa and infiltration by heterophils and macrophages and micro and macro-granulomatous lesions were also found constantly present in the large intestine of Centrorhynchus spp.-infected buzzards. These findings confirm previous observations in buzzards, and some authors consider these lesions a direct consequence of the mechanical damage caused by the proboscis these parasites use to anchor themselves to the intestinal wall [30,37].
As previously reported [30,37,46], esophageal inflammation, characterized by chronic and diffuse inflammation with transmural infiltrate consisting of lymphocytes and plasma cells, was highlighted in the esophagus of the two E. dispar-infected common buzzards.
In the small intestine of a buzzard found heavily infected by B. falconis, foci of acute inflammation characterized by microscopic agglomerates composed by heterophils and eosinophils and focal mucosal erosion were highlighted ( Figure 3C). Previous data on B. falconis-associated lesions are lacking. However, similar lesions have been observed in raptors infected by other intestinal capillariid species [47,48]. also found related to P. mansioni intensity, which was higher in two buzzards ( Table 2). In previous studies [30,45], dark red nodular lesions, erosions, and ulcers of the gastric mucosa associated with Procyrnea sp. infection have been reported in diurnal raptors, including buzzards, while data on associated histological lesions are lacking.  In a common buzzard, diffuse duodenal inflammation with the presence of transmural infiltrates of lymphocytes and plasma cells was found associated with a heavy infection caused by the trematode N. attenuatum, confirming previous findings [30,37], while mucosal erosion and severe hyperemia were evidenced in the small intestine of a buzzard found infected by a high number of C. globifera cestodes (Table 3).
Among the two positive honey buzzards, E. dispar, Physaloptera apivori, and Raillietina apivori were identified, respectively, in the esophagus, gizzard, and intestine ( Figure 3D) of honey buzzard 1 concurring with intestinal S. typhimurium infection, while honey buzzard 2 scored positive for E. dispar in the esophagus, Procyrnea leptoptera in the stomach, and B. falconis in the small intestine, along with E. coli infection in the liver (and brain) (Tables 2 and 4, Figure 4). However, macroscopic lesions of the digestive system associated with severe weight loss and dehydration were observed mainly in the honey buzzard 2. This was also evidenced at the histopathological examination, as the presence of helminths in the esophagus and gizzard of the honey buzzard 1 was found not associated with specific lesions (Table 3), while chronic-active inflammation was found associated with R. apivori ( Figure 3D). Conversely, granulomatous lesions were observed in the esophagus and proventriculus of the honey buzzard 2, associated with E. dispar and P. leptoptera infection, respectively. Interestingly, lesions found associated with P. leptoptera infection in this honey buzzard are very similar to those found in some of P. mansioni-infected European common buzzards examined here.
Pathogens 2021, 10, x FOR PEER REVIEW 12 of 25 pathogen often isolated from the intestinal content of diurnal birds of prey, both as a primary pathogen or a pathogen secondary to other infections [2,18], and may cause acute septicemic forms or sub-acute-chronic infections characterized by voluminous granulomas, aero-sacculitis, pneumonia, pericarditis, osteomyelitis, and nephritis [17,46]. Clinically, diarrhea, anorexia, and respiratory distress can be also observed [2,18,58]. Among positive sparrowhawks, sparrowhawk 1 showed catarrhal enteritis at necropsy, but no pathogens were detected at the bacteriological examination (Tables 1 and  4). However, at the parasitological analysis, P. leptoptera, Synhimantus (Dispharynx) falconis, and P. alata were identified in the proventriculus and the proximal tract of the small intestine (Table 2, Figure 5). At the histopathological examination, these parasites were found associated with foci of chronic-active inflammation, characterized by the presence of heterophiles, lymphocytes, and macrophages infiltrating the proventriculus and a segmental tract of the duodenal wall; parasites were also evidenced in the lumen of these Furthermore, the presence of atypical sub-miliaric micro-granulomatous lesions was observed at the histopathological examination in the S. typhimurium-infected honey buzzard 1, while chronic and diffuse inflammation with transmural infiltrates made of lymphocytes and plasma cells were highlighted throughout the intestinal tract of honey buzzard 2. These latter lesions, along with hepatomegaly and nephromegaly observed at necropsy, could be associated mainly with E. coli infection. In fact, E. coli is a bacterial pathogen often isolated from the intestinal content of diurnal birds of prey, both as a primary pathogen or a pathogen secondary to other infections [2,18], and may cause acute septicemic forms or sub-acute-chronic infections characterized by voluminous granulomas, aero-sacculitis, pneumonia, pericarditis, osteomyelitis, and nephritis [17,46]. Clinically, diarrhea, anorexia, and respiratory distress can be also observed [2,18,58].
Among positive sparrowhawks, sparrowhawk 1 showed catarrhal enteritis at necropsy, but no pathogens were detected at the bacteriological examination (Tables 1 and 4). However, at the parasitological analysis, P. leptoptera, Synhimantus (Dispharynx) falconis, and P. alata were identified in the proventriculus and the proximal tract of the small intestine (Table 2, Figure 5). At the histopathological examination, these parasites were found associated with foci of chronic-active inflammation, characterized by the presence of heterophiles, lymphocytes, and macrophages infiltrating the proventriculus and a segmental tract of the duodenal wall; parasites were also evidenced in the lumen of these organs (Table 3, Figure 6). As observed in the common buzzards, B. falconis found in the small intestine of this animal was instead associated with foci of acute inflammation of jejunum and ileum and with the presence of micro-agglomerates of heterophils and eosinophils, areas of mucosal erosion, and hyperemia (Table 3, Figure 7).  Parasitological analysis of sparrowhawk 2 ( Table 2) revealed P. leptoptera infection in the gizzard and the proventriculus associated with moderate histopathological lesions characterized by micro-and macro-granulomatous foci and ulcers on the mucosa overlying the micro-granulomatous areas (Table 3). Granulomas were characterized by a necrotic center with the presence of dystrophic calcification, with the cell wall consisting of numerous heterophilic granulocytes and macrophages and sometimes of giant cells and mixed lymphocytes and/or plasma cells. These findings agree with previous observations in sparrowhawks infected by P. leptoptera and with findings from this study in P. leptoptera and P. mansioni-infected honey and common buzzards, respectively [30,33].  Moreover, B. falconis and Spirocerca cysts (Table 2), each containing an encysted larva, were identified in the intestine of this animal. At the histopathological examination, capillariids were evidenced in the lumen and within the mucosa of the intestine associated with diffuse chronic interstitial inflammation with the presence of transmural infiltrate of lymphocytes and plasma cells (Table 3, Figure 7).
Microscopic examination of the intestinal content of this animal also revealed the presence of a high number of protozoan sporocysts, with a mean measurement of 12.35 × 8.72 µm (range 10.9-13.08 × 8.72 µm) ( Table 2), morphologically identifiable with Sarcocystis columbae [59]. In the whole intestinal tract, lesions and chronic diffuse inflammation were highlighted at the histological examination, with the presence of transmural infiltrates of lymphocytes and plasma cells associated with a high number of endocellular protozoa in the epithelium and the lamina propria of the small intestine (Table 3), concurrent with evident bacterial overgrowth in the crypts and glands of the intestinal mucosa.
Moreover, B. falconis and Spirocerca cysts (Table 2), each containing an encysted larva, were identified in the intestine of this animal. At the histopathological examination, capillariids were evidenced in the lumen and within the mucosa of the intestine associated with diffuse chronic interstitial inflammation with the presence of transmural infiltrate of lymphocytes and plasma cells (Table 3, Figure 7). C. perfringens is often a component of the normal intestinal microbiome of various birds, and it is particularly frequent in Falcon raptors [18,60]. However, in the presence of concurrent factors, such as diet changes, stressful conditions, and some intercurrent infections, it can cause necrotic enteritis with diarrhea, often bloody, progressive dehydration that may also lead diseased birds to death, and secondary hepatitis and splenitis [61,62]. In poultry, C. perfringens is the most important causative agent of necrotic enteritis [63,64], and this condition is especially observed when bacterial overgrowth concurs with a severely damaged intestinal epithelium, often due to heavy coccidian infections [64]. Therefore, the concurrent severe S. columbae infection and necrotic enteritis may suggest a possible role of this protozoan infection as a predisposing factor for the onset of clostridial necrotic enteritis in this bird.
Finally, sparrowhawk 3 showed hemorrhagic gastroenteritis at necropsy ( Table 1). The bacteriological analysis was negative, while B. falconis and P. angusticolle were evidenced in the intestinal tract at the parasitological examination (Table 2, Figure 5). The histopathological examination confirmed the presence of these nematodes in the endoluminal area or attached to the mucosa, and associated lesions were similar to those observed in the B. falconis-infected sparrowhawk 2 (Table 3).
At necropsy (Table 1), kestrel 1 showed serous and fibrin collection in the thoracoabdominal cavity and catarrhal-hemorrhagic enteritis, while the liver was dark reddish-brown in color. Three parasite species (Table 2), namely Diplotriaena falconis, Neodiplostomum spathoides, and Spirocerca spp., were identified in the intestine of this animal.
At bacteriological analysis (Table 4), Yersinia enterocolitica and P. multocida were identified in the intestine and in the brain. Y. enterocolitica has been reported in wild-living birds [65,66], including raptors [67]. Infected birds may die very rapidly after infection, or the disease may take weeks to manifest [68]. Intestinal mucinosis associate with Y. enterocolitica infection has been also reported [69]. On the other hand, P. multocida is the etiological agent of avian cholera and one of the bacterial species more frequently causing disease in birds [46,70]. Severe hyperacute and acute infections may occur, while hemorrhagic diarrhea, along with other signs, can be observed in sub-acute infections [46,70]. Chronic forms include serositis, necrotic foci, and small granulomatous lesions affecting all organs.
Several intestinal macro-granulomatous foci and intestinal mucinosis were observed at the histopathological examination in this bird. Interestingly, a weak inflammatory infiltrate, associated with the areas of colonic mucinosis, was typically associated to the sub-epithelial localization of some Spirocerca spp. larvae (Figure 8).  The common kestrel 2 did not show any significant lesions at necropsy, and no bacterial pathogens were identified (Tables 1 and 4). On the other hand, over 50 Synhimantus laticeps adults and a C. falconis acanthocephalan were found in the proventriculus and the intestine, respectively (Table 2, Figure 9). The histopathological examination revealed erosions of the stomach mucosa and the presence of parasites, some of which calcified, in the muscle fibers of the stomach wall that can be associated with the heavy S. laticeps infection observed in this bird, as previously reported [30,33]. However, serositis observed in the thoracoabdominal cavity, catarrhal-hemorrhagic enteritis, and intestinal macro-granulomatous lesions, as well as liver pathological changes observed in this bird, could be due mainly to P. multocida, while Y. enterocolitica infection should be considered co-responsible for enteritis and sepsis and co-responsible or the main cause for intestinal mucinosis observed in this bird.
The common kestrel 2 did not show any significant lesions at necropsy, and no bacterial pathogens were identified (Tables 1 and 4). On the other hand, over 50 Synhimantus laticeps adults and a C. falconis acanthocephalan were found in the proventriculus and the intestine, respectively (Table 2, Figure 9). The histopathological examination revealed erosions of the stomach mucosa and the presence of parasites, some of which calcified, in the muscle fibers of the stomach wall that can be associated with the heavy S. laticeps infection observed in this bird, as previously reported [30,33]. Regarding nocturnal raptors, the two scops owls scored negative at the paras cal and bacteriological examinations.
The barn owl 1 (Table 1) showed enteritis associated with Sarcocystis dispers tion, but no bacteria were identified, while parasitological and histopathological nations of the barn owl 2 revealed Spirocerca spp. cysts in the gastric and intestin along with enteritis and macro-granulomatous lesions (Tables 1-3; Figure 10). Finally, among diurnal raptors, the single osprey examined was found positive for Procyrnea leptoptera nematoda in the gizzard and proventriculus and for the acanthocephalan species Andracantha mergi in the intestine, but no bacteria were found.
Regarding nocturnal raptors, the two scops owls scored negative at the parasitological and bacteriological examinations.
The barn owl 1 (Table 1) showed enteritis associated with Sarcocystis dispersa infection, but no bacteria were identified, while parasitological and histopathological examinations of the barn owl 2 revealed Spirocerca spp. cysts in the gastric and intestinal wall along with enteritis and macro-granulomatous lesions (Tables 1-3; Figure 10).
Among positive little owls, the parasitological examination (Table 2, Figure 11) revealed Capillaria tenuissima infection in the small intestine of almost all of these birds, while little owl 2 was also found infected by P. leptoptera in the gizzard and proventriculus, and little owl 12 was infected by N. attenuatum in the intestine (Table 2). Bacteriological analysis revealed E. coli infection in the liver of little owl 6 and P. multocida infection in the liver, brain, and other organs of little owl 13, showing intestinal gross lesions, hepatomegaly, and obstruction of the cloaca, suggesting septicemia (Table 4). At the histopathological examination (Table 3), diffuse chronic inflammation with transmural infiltrates of lymphocytes and plasma cells was instead highlighted in the gizzard, proventriculus, and the entire intestinal tract of this bird, which was probably caused by P. multocida [46,70]. Severe intestinal dysmicrobism and damage to the intestinal epithelium, especially in the duodenum, were also evidenced.
Regarding nocturnal raptors, the two scops owls scored negative at the parasitological and bacteriological examinations.
The barn owl 1 (Table 1) showed enteritis associated with Sarcocystis dispersa infection, but no bacteria were identified, while parasitological and histopathological examinations of the barn owl 2 revealed Spirocerca spp. cysts in the gastric and intestinal wall along with enteritis and macro-granulomatous lesions (Tables 1-3; Figure 10). Among positive little owls, the parasitological examination (Table 2, Figure 11) revealed Capillaria tenuissima infection in the small intestine of almost all of these birds, while little owl 2 was also found infected by P. leptoptera in the gizzard and proventriculus, and little owl 12 was infected by N. attenuatum in the intestine (Table 2). Bacteriological analysis revealed E. coli infection in the liver of little owl 6 and P. multocida infection in the liver, brain, and other organs of little owl 13, showing intestinal gross lesions, hepatomegaly, and obstruction of the cloaca, suggesting septicemia (Table 4). At the histopathological examination (Table 3), diffuse chronic inflammation with transmural infiltrates of lymphocytes and plasma cells was instead highlighted in the gizzard, proventriculus, and the entire intestinal tract of this bird, which was probably caused by P. multocida [46,70].
Severe intestinal dysmicrobism and damage to the intestinal epithelium, especially in the duodenum, were also evidenced. C. tenuissima infection was instead found constantly associated with diffuse chronic inflammation of mild severity with transmural infiltrates of lymphocytes and plasma cells or with foci of acute inflammation, micro-aggregates of heterophils, and eosinophils. Areas of mucosal erosion and hyperemia were instead found associated with N. attenuatum infections. Moreover, mild atypical micro-granulomatous lesions were also found in the jejunal and ileal tract of little owl 6, probably due to E. coli infection [18]. In little owl 12, a heavy colonization of coccidian parasites, identifiable with Eumonospora mochogalegoi or Eumonospora henryae previously reported in the little owl [71], was also evidenced at histopathology ( Figure 12).  C. tenuissima infection was instead found constantly associated with diffuse chronic inflammation of mild severity with transmural infiltrates of lymphocytes and plasma cells or with foci of acute inflammation, micro-aggregates of heterophils, and eosinophils. Areas of mucosal erosion and hyperemia were instead found associated with N. attenuatum infections. Moreover, mild atypical micro-granulomatous lesions were also found in the jejunal and ileal tract of little owl 6, probably due to E. coli infection [18]. In little owl 12, a heavy colonization of coccidian parasites, identifiable with Eumonospora mochogalegoi or Eumonospora henryae previously reported in the little owl [71], was also evidenced at histopathology (Figure 12).

Animals
Twenty-nine deceased diurnal and nocturnal raptors were examined from 20 2005 for endoparasites and bacteria ( Table 1). The examined diurnal raptors (Acci formes and Falconiformes) included four common buzzards (B. buteo), two Euro honey buzzards (P. apivorus), three sparrowhawks (A. nisus), two common kestrels (F nunculus), and a western osprey (P. haliaetus) ( Table 1). Among nocturnal raptors (S formes), two barn owls (T. alba), thirteen little owls (A. noctua), and two scops owl scops) were examined ( Table 1). All the raptors examined in this study had died fr few hours to ten days after their arrival in wildlife rescue centers located in the distr Lucca (43°50′ N, 10°30′ E) (Tuscany, central Italy), with the exception of a barn owl (T. that was found dead in a wildlife rescue center located in the district of Livorno (43°3 10°18′ E, Tuscany, central Italy) and a common kestrel (F. tinnunculus) that was f dead in the district of Pisa (43°43′ N, 10°23′ E, Tuscany, central Italy). The examined had not been treated with antiparasitic drugs and were single-caged. In most cases ceased birds were placed in clean bags, refrigerated, and transported to the labor soon after their death. All examined birds were necropsied, and bacteriological and sitological analysis of the different tracts of the digestive system were performed. Or that scored positive for the presence of endoparasites and/or bacteria underwent fu histopathological examinations.

Animals
Twenty-nine deceased diurnal and nocturnal raptors were examined from 2003 to 2005 for endoparasites and bacteria ( Table 1). The examined diurnal raptors (Accipitriformes and Falconiformes) included four common buzzards (B. buteo), two European honey buzzards (P. apivorus), three sparrowhawks (A. nisus), two common kestrels (F. tinnunculus), and a western osprey (P. haliaetus) ( Table 1). Among nocturnal raptors (Strigiformes), two barn owls (T. alba), thirteen little owls (A. noctua), and two scops owls (O. scops) were examined ( Table 1). All the raptors examined in this study had died from a few hours to ten days after their arrival in wildlife rescue centers located in the district of Lucca (43 • 50 N, 10 • 30 E) (Tuscany, central Italy), with the exception of a barn owl (T. alba) that was found dead in a wildlife rescue center located in the district of Livorno (43 • 33 N, 10 • 18 E, Tuscany, central Italy) and a common kestrel (F. tinnunculus) that was found dead in the district of Pisa (43 • 43 N, 10 • 23 E, Tuscany, central Italy). The examined birds had not been treated with antiparasitic drugs and were single-caged. In most cases, deceased birds were placed in clean bags, refrigerated, and transported to the laboratory soon after their death. All examined birds were necropsied, and bacteriological and parasitological analysis of the different tracts of the digestive system were performed. Organs that scored positive for the presence of endoparasites and/or bacteria underwent further histopathological examinations.

Necropsy
An external examination was performed to evaluate eventual abnormalities of muscle masses, subcutaneous fat, and mucous membranes. All organs and systems were examined to assess the presence of macroscopic lesions.

Bacteriological Analysis
Brain, intestine, and liver samples taken from all birds were cultured on several culture media specific for the search of different selected bacteria, including Salmonella spp., Escherichia coli, Yersinia spp., Pasteurella spp., and Clostridium spp. [72].
For Salmonella spp., samples were inoculated in Buffered Peptone Water and incubated at 37 • C for 18 h. After incubation, the samples were inoculated into Rappaport-Vassiliadis soya broth (Oxoid, Milan, Italy) and incubated at 42 • C for 18 h. The cultures obtained were plated onto Xylose-lysinedeoxycholate agar (Oxoid, Milan, Italy) and Brilliant Green Agar (Oxoid, Milan, Italy), incubated at 37 • C, and examined after 24 h. Suspected colonies were then sub-cultured in nutrient agar, and the confirmation of Salmonella species was performed using oxidase, API 20E (BioMérieux, Florence, Italy), and polyvalent antisera. Salmonella spp. isolates were serotyped according to the Kauffmann-White scheme in collaboration with The Reference Center for Pathogenic Enterobacteria (IZSLT, Rome, Italy).
To isolate E. coli, samples were inoculated onto Colombia blood agar base (Oxoid, Milan, Italy) and MacConkey agar (Oxoid, Milan, Italy) and incubated at 37 • C for 18 h. The biochemical identification was performed using a miniaturized biochemical test galleries API 20E system (BioMérieux, Florence, Italy).
For Clostridium perfringens isolation, samples were inoculated onto Colombia blood agar base (Oxoid, Milan, Italy), incubated at 37 • C for 24-48 h anaerobically, and the glove box was used with the AnaeroGen (Oxoid, Milan, Italy). The identification of colonies with specific characteristics (β-haemolytic and lecithinase positive) was performed using API rapid 32A and API 20A (BioMérieux, Florence, Italy).
The isolation of Yersinia spp. was performed by direct inoculation in cold preenrichment broth phosphate-buffered saline and incubated at 25 • C for 48 h. After incubation, the samples were inoculated onto Colombia blood agar base (Oxoid, Milan, Italy), MacConkey agar (Oxoid, Milan, Italy) and Yersinia selective agar base cefsulodin-irgasannovobiocin (Oxoid, Milan, Italy) with incubation at 30 • C for 24-48 h. The confirmation of Yersinia enterocolitica was performed using oxidase, urease, and miniaturized biochemical test by using test galleries API 20E (BioMérieux, Florence, Italy).
For Pasteurella multocida, samples were inoculated onto Colombia blood agar base (Oxoid, Milan, Italy), MacConkey agar (Oxoid, Milan, Italy), and brain heart infusion agar broth and incubated at 24-48 h at 37 • C. The confirmation of P. multocida was obtained using oxidase and miniaturized biochemical test galleries API 20 NE (BioMérieux, Florence, Italy).

Parasitological Analysis
The whole digestive (liver, gall bladder, esophagus, stomach, duodenum, jejunumileum, ceca, and cloaca) system was opened. A batch of intestinal tract content samples was microscopically examined under an optical microscope, both as fresh smears and after the flotation test with saturated NaCl solution (specific gravity 1.2), to detect the presence of nematode eggs or protozoa cysts/oocysts/sporocysts. Then, each opened digestive tract was washed in saline and subjected to sedimentation in a refrigerator for approximately 24 h. After this time, the sediment was observed under a stereoscope to assess the presence of adult helminths that were collected, counted, washed in saline solution, and fixed in a glycerin-ethanol solution (medium of Looss). Helminths were cleared in lactophenol on a glass slide for identification under an optical microscope and then returned to the preservative. All measurements were taken with the aid of a micrometric eyepiece.
Fragments of at least 2.5 cm in length of the esophagus, proventriculus, ventriculus, duodenum, jejunum/ileum, and colon were collected. Samples were fixed in 10% neutral buffered formalin for a period of 24 h and routinely processed. Three-micrometer paraffin sections were placed on Superfrost Plus slides (Histoline, Milan, Italy). The slides were then dewaxed and stained with hematoxylin and eosin stain (H&E) for microscopic examination. The morphological evaluation of each digestive tract was carried out, with the determination of the location of the parasite and, in the case of an evident histological lesion, the characterization of the inflammation (acute, chronic-active, granulomatous inflammation). Moreover, the sections found infected by parasites were stained with Shiff's Periodic Acid (PAS).
The following parameters were used for the histological evaluation: for each lesion, a score evaluation was performed considering as: grade 0 (negative = lesion not observed); grade from 1 to 3 (indicated with the + sign) the progressive degree of the lesions. The lesion score was therefore 1 (+) = minor lesion; 2 (++) = moderate lesion; 3 (+++) = severe lesion. To characterize the lesions, a letter from A to G was used, as described in the caption of Table 3.

Conclusions
Worldwide, raptors are included among animals more frequently admitted to wildlife recovery centers. Moreover, many raptor populations are highly threatened, mainly due to human activities. Therefore, the collection of data on pathogens and associated lesions in raptors living in a specific area can provide useful information for enhancing raptor conservation efforts, reducing pathogen transmission between raptors, domesticated animals, and humans, and improving the knowledge on raptor pathogens.
To the best of the authors' knowledge, this study is the first organic investigation on endoparasite infections and associated bacteria and pathological lesions of the digestive tract of European raptors. Although most of the pathogens identified in this study are commonly found in European diurnal and nocturnal raptors, this study is the first report of S. typhimurium and S. enterica subspecies diarizonae in the common buzzard (B. buteo) and the first report in Italy of the parasite species Andracantha mergi, Spirocerca spp., Sarcocystis dispersa, Sarcocystis columbae, and Eumonospora spp.
The results obtained showed that both parasites, especially when in high number, and/or pathogenic bacteria may cause severe lesions in the digestive system of diurnal and nocturnal raptors; parasites and bacteria may concur in causing these lesions; and most severe lesions are caused by the interaction of multiple pathogens, both parasites and bacteria. Moreover, in most cases, the same parasite genus/species was frequently found associated with the same type of lesions in the histopathological analysis. The potential role of these birds for the dispersal of bacteria potentially pathogenic for humans, such as Salmonella spp. and Y. enterocolitica, and domesticated animals was also highlighted. Although the characterization of some bacteria, such as E. coli, was not performed in this study, the obtained data are indicative of associated septicemic forms. Finally, the severity of pathological changes found associated with parasitic and/or bacterial pathogens in some of these raptors could have greatly impaired the function of the digestive system and, in some cases, may have represented the main cause for the death of these birds.