Morishitium polonicum as a Cause of Severe Respiratory Disease in Eurasian Blackbirds (Turdus merula) in Central Italy

Air sac trematodosis (AST) is a parasitic disease of birds caused by digenean trematodes belonging to the family Cyclocoelidae. The life cycle includes terrestrial snails (e.g., Helicella spp.) as intermediate hosts (Hoffman 2008). In Europe, the most common AST is associated with Morishitium polonicum species and occurrs in Song Thrushes (Turdus philomelos) and Eurasian Blackbirds (Turdus merula; Machalska 1980). Severe M. polonicum infections have been described in T. merula by Galosi et al. (2019), but to date insufficient morphologic, pathologic, and molecular data are available on Morishitium spp.

The objective of our report was to support the role of M. polonicum as a potentially life-threatening pathogen in Eurasian Blackbirds.

During spring 2019, two free-range juvenile (hatch-year) Eurasian Blackbirds with severe respiratory distress were recovered in Perugia and Arezzo provinces (belonging to Umbria and Tuscany Regions, respectively) and hospitalized at the University Veterinary Teaching Hospital of Perugia and in the “Centro Recupero Uccelli Marini ed Acquatici,” respectively. The birds showed similar clinical signs: reduced responsiveness, hypothermia, ruffled feathers, tremors, emaciation, mucous congestion, and rapid and open-beak breathing. One also showed paresis of the legs and inability to fly; this bird spontaneously died a few hours after hospitalization. The second bird was euthanized on ethical grounds due to concurrent severe external traumatic injuries.

Both birds underwent complete necropsy. The second bird had cutaneous wounds on the left wing in association with a complete fracture of the radius, perhaps due to predator aggression. Otherwise, the main lesions in both birds were observed in the coelomic cavity. In addition to hepatomegaly, several yellowish, elongated, dorso-ventrally flattened parasites, resembling trematodes, were free in the coelomatic cavity and on the surface of liver and all the other organs, including the air sacs (Fig. 1A). Diffuse cloudiness of the air sacs, with a scant amount of yellowish material (fibrin strands) and mild diffuse pulmonary hyperemia were also observed (Fig. 1B).

Organ sections were fixed in 4% buffered formalin, paraffin-wax embedded, sectioned at 4 mm, and routinely stained with H&E. At histologic examination in both birds, aggregations of lymphocytes, macrophages, epithelioid, and multinucleated giant cells around degenerated trematodes (granuloma) were identified in the air sacs (Fig. 2A). Intact trematodes were also evident on the surface of the air sacs. They were characterized by a thick eosinophilic tegument (up to 25 µm), an outer and inner muscular layer, and a spongy parenchyma containing loosely arranged parenchymal cells (Fig. 2B). Severe thickening of the air sacs was associated with diffuse abundant amorphous and fibrillar eosinophilic material (edema and fibrin), with a high number of heterophils and mononucleated cells, including histiocytes and lymphocytes. Lymphocytes occasionally formed follicular-like patterns (Fig. 2C). Moderate multifocal lung inflammation was also observed. Lumens of parabronchi contained eosinophilic and fibrillar material (edema and fibrin; Fig. 2D), while a number of mononucleated cells and heterophils expanded the subpleural and air capillary interstitium (Fig. 2E). In the spleen, multifocal lymphoid hyperplasia was seen. In the liver of one bird, multifocal lymphoid follicles were associated with a focal granuloma (Fig. 2F).

Flukes were collected from the coelomic cavities and air sacs: 76 from one bird, 54 from the other (Fig. 3A), and fixed in 70% ethanol; 20 specimens (10 for each bird), after clearing in Amann lactophenol, were mounted in Canada balsam and then subjected to morphometric analysis by light microscopy The flukes had a tongue-shaped elongate body, 10.08–11×2.11–2.30 mm, tapered anteriorly and rounded at the posterior end (Fig. 3B). The mouth was slightly oval and subterminal with a weakly developed oral sucker (Fig. 3C). The uterus was broad, between the ceca, and filled with brown-yellow eggs (Fig. 3D). The two testes were large and globular, placed obliquely in the posterior part of the body, with the globular ovary placed in a longitudinal straight line with the testes (Fig. 3E). The two ceca were joined posteriorly, while the vitelline glands were arranged symmetrically and were not confluent posteriorly (Fig. 3E). Morphologically, the flukes resembled M. polonicum (Machalska 1980; Visconti et al. 1988; Galosi et al. 2019). Because most cyclocoelid species of Degenea are morphologically similar, and measurements of specific structures often overlap extensively, biomolecular techniques were applied to support the morphologic identification and define the taxonomic position of our specimens.

Five specimens from each bird were subjected to genomic DNA extraction using a commercial kit (Exgene Clinic SV, Geneall Biotechnology, Seoul, South Korea). We carried out PCR of DNA of the specimens following a previously described protocol targeting the ITS2 region of the mitochondrial genome (Urabe et al. 2020). The obtained amplicons were purified (QIAquick Gel extraction kit, Qiagen, Hilden, Germany) and commercially sequenced (Bio-Fab Research, Rome, Italy) in both directions using PCR primers. A total of 563 base pairs of the obtained sequences were compared with each other and with those of other cyclocoelid species available in GenBank using the nucleotide BLAST (National Center for Biotechnology Information [NCBI 2020]). The sequences of the obtained amplicons were identical to each other and showed a similarity score of 99.38% with the M. polonicum (GenBank accession no. KU877887) sequence from NCBI. The sequence obtained was deposited in the NCBI database under number MT738315.1 and phylogenetically analyzed in comparison with those available in GenBank. Selected isolates previously sequenced for M. polonicum were included in the analysis. The evolutionary history was carried out using the neighbor-joining method and evolutionary distances were computed using MEGA7 (Kumar et al. 2006). The bootstrap consensus trees inferred from over 1,000 replicates were considered to represent the evolutionary history of the analyzed taxa. The taxon identification tree indicates very high similarity of the present specimens with M. polonicum isolated from Turdus philomenus (no. KU877908.1) and Turdus merula (no. KU877909.1) in the Czech Republic (see Supplementary Material).

Morishitium polonicum is considered a specific cyclocoelic trematode of Turdidae (order Passeriformes), although it may be found occasionally in birds belonging to the Sturnidae, Muscicapidae, Prunellidae, and Fringillidae families across Europe (Kirillova et al. 2019; Sitko and Heneberg 2020).

Morishitium polonicum has been documented in several countries in Central Europe (e.g., Poland, Czech Republic) and the associated AST has been described in southern countries (e.g., Italy, Spain; Galosi et al. 2019). The wide distribution of this parasitosis might be due to the migratory habits of thrushes, which are considered the natural reservoir of M. polonicum. For this reason the parasitic infection may diffuse within Central European regions and in Mediterranean regions where thrushes commonly winter (Milwright 2006). Most reports describe single individuals or small case series without taking into account the migratory patterns of the birds; therefore, insufficient data are available to effectively understand the distribution of M. polonicum.

The presence of M. polonicum in Central Italy is not surprising; indeed, two previous reports described AST, including fatal infection, in T. merula from the neighboring regions of Umbria and Tuscany (Galosi et al. 2019).

The two birds described here were heavily infected. A high environmental and intermediate host parasite pressure could result in the morbidity found in these juveniles. In both the cases, the high environmental parasite pressure should be considered in the pathogenesis. Moreover, the autochthonous life-cycle of the parasite may be maintained thanks to the presence of the intermediate hosts in these areas, namely the snails which, together with other invertebrates (e.g., earthworms and insects), may present an important component of the Eurasian Blackbird's diet.

Gastropods of the genera Helicella and Cernutella, known as hosts of the trematoes causing dicrocoeliosis in ruminants (Otranto and Traversa 2002), are found in this area and may also potentially act as intermediate hosts for M. polonicum. Unfortunately, no studies have been carried out on the competence of gastropod populations of Central Italy as hosts of M. polonicum.

Diffusion of M. polonicum in these geographic areas could be linked to similar drivers at the basis of the epidemiologic changes occurring for other parasites, including some emergent metastrongyloid nematodes of pets and wildlife animals (Di Cesare et al. 2013).

It is interesting to note that the Eurasian Blackbird does not represent the typical definitive avian host for M. polonicum. However, compared to other passerine birds, such as Blue-crowned Laughingthrush (Garrulax courtoisi), Blackbirds seem to show a greater intensity of infestation and much-more severe effects, suggesting a possible higher susceptibility (Machalska 1980; Delaski et al. 2015; Galosi et al. 2019; Sitko and Heneberg 2020). The small body size of Blackbirds and the associated high risk of tracheal and bronchiolar obstruction by massive infections of cestode, nematode, and acanthocephalan species (Galosi et al. 2019), as well as local inflammatory reaction, might increase the pathogenic effect of M. polonicum in this species.

Regarding local prevalence of this condition, 16 Eurasian Blackbirds were examined in our laboratories from November 2018 to November 2020, with only these two cases of AST being detected (12.5%).

Lesions related to AST caused by M. polonicum have been occasionally reported in the literature (Delaski et al. 2015; Galosi et al. 2019). The pathologic findings we report confirm granulomatous airsacculitis as the primary expression of the severe respiratory clinical form associated with M. polonicum (Galosi et al. 2019). Additionally, for the first time, airsacculitis was observed in association with heterophilic interstitial pneumonia and multiple granulomas around degenerated trematodes in air sacs and other organs. In contrast to previous reports (e.g., Delaski et al. 2015), no signs of airway obstruction were identified in our cases. The severe thickening of the air sacs and the pulmonary interstitium extension due to edema, fibrin deposition, and inflammatory cells infiltration, as well as the presence of numerous trematodes in the lumen of the air sacs, might be considered, in our cases, the most relevant cause of the respiratory failure and consequent death, as seen in one Blackbird, or making the animal less responsive and more prone to predation, as seen in the second case.

Based on our results and on previously published findings (Galosi et al. 2019), severe health implications for Eurasian Blackbirds caused by M. polonicum infection should be considered in Italy and in other European or Mediterranean Countries.

Supplementary material for this article is online at http://dx.doi.org/10.7589/JWD-D-20-00163.