*Article* **Evidence for Unknown** *Sarcocystis***-Like Infection in Stranded Striped Dolphins (***Stenella coeruleoalba***) from the Ligurian Sea, Italy**

**Federica Giorda 1,2,†, Umberto Romani-Cremaschi 3,†, Antoinette E. Marsh 4, Carla Grattarola 1, Barbara Iulini 1, Alessandra Pautasso 5, Katia Varello 1, Enrica Berio 1, Paola Gazzuola 1, Letizia Marsili 6, Cristina E. Di Francesco 7, Maria Goria 1, Federica Verna 8, Tania Audino 1, Simone Peletto 1, Maria Caramelli 1, Mercedes Fernández-Escobar 9, Eva Sierra 2, Antonio Fernández 2, Rafael Calero-Bernal <sup>9</sup> and Cristina Casalone 1,\***


**Simple Summary:** Two stranded striped dolphins presented meningoenchepalitic lesions associated with the presence of unknown protozoan tissue cysts. The present study aimed at fully characterizing these previously undescribed parasites. Light microscopy re-examination of affected CNS areas showed high numbers of tissue cysts with morphological features resembling those of *Sarcocystis* species. Tissue cyst bradyzoites positively stained when labeled with polyclonal antisera but crossreactivity could not be precluded. *Sarcocystis* sp. sequences with high homology to species infecting livestock were amplified by means of PCR from myocardial and muscle tissues. This is the first report of *Sarcocystis*-like tissue cysts in the cerebral tissue of stranded cetaceans with muscular sarcocystosis in Mediterranean dolphins. The obtained results may suggest a land-to-sea cycling of Apicomplexan parasites in this region and the need for further investigations in order to foster marine mammal conservation.

**Abstract:** Two striped dolphins (SD1, SD2), stranded along the Ligurian coast of Italy, were diagnosed with a nonsuppurative meningoencephalitis associated with previously undescribed protozoan tissue cysts. As tissue cysts were morphologically different from those of *Toxoplasma gondii*, additional histopathological, immunohistochemical, ultrastructural, and biomolecular investigations were performed, aiming to fully characterize the organism. Histopathology revealed the presence of large *Sarcocystis*-like tissue cysts, associated with limited inflammatory lesions in all CNS areas studied. IHC was inconclusive, as positive staining with polyclonal antisera did not preclude crossreaction with other Sarcocystidae coccidia. Applied to each animal, 11 different PCR protocols

#### **Citation:** Giorda, F.;

Romani-Cremaschi, U.; Marsh, A.E.; Grattarola, C.; Iulini, B.; Pautasso, A.; Varello, K.; Berio, E.; Gazzuola, P.; Marsili, L.; et al. Evidence for Unknown *Sarcocystis*-Like Infection in Stranded Striped Dolphins (*Stenella coeruleoalba*) from the Ligurian Sea, Italy. *Animals* **2021**, *11*, 1201. https://doi.org/10.3390/ ani11051201

Academic Editor: Pablo Diaz

Received: 20 March 2021 Accepted: 20 April 2021 Published: 22 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

precluded a neural infection by *Sarcocystis neurona, Sarcocystis falcatula, Hammondia hammondi*, and *Neospora caninum*. *T. gondii* coinfection was confirmed only in dolphin SD2. *Sarcocystis* sp. sequences, showing the highest homology to species infecting the Bovidae family, were amplified from SD1 myocardium and SD2 skeletal muscle. The present study represents the first report of *Sarcocystis*like tissue cysts in the brain of stranded cetaceans along with the first description of *Sarcocystis* sp. infection in muscle tissue of dolphins from the Mediterranean basin.

**Keywords:** striped dolphin; tissue cysts; neuropathology; *Toxoplasma gondii*; *Sarcocystis*-like; genotype

#### **1. Introduction**

Tissue cyst-forming coccidia from the genera *Toxoplasma*, *Sarcocystis*, and *Neospora* (Apicomplexa) are capable of infecting several species of marine mammals and are responsible for either chronic diseases or acute mortality [1]. While *Neospora caninum* does not seem to pose a major threat to marine wildlife, *Toxoplasma gondii* and *Sarcocystis neurona* are the two coccidian parasites most widely reported in North American marine mammals [1], especially in coastal species that are more likely to be overexposed to immunosuppressant chemical pollutants and to high concentrations of land-derived oocysts [2–4]. The most probable exposure route in these animals is through ingestion of environmentally resistant oocysts or sporocysts shed on land by the definitive hosts and passed into the sea through freshwater runoffs or the release of contaminated ship waters. Eventually, protozoal infectious stages may then accumulate in marine invertebrates, bivalve mollusks, or fish on which intermediate hosts prey [5]. In the Mediterranean basin, *T. gondii* is a frequent finding in stranded odontocetes, and it is often associated with protozoal meningoencephalitis [6,7].

On the other hand, neither muscular nor neural sarcocystosis has ever been officially reported in this geographical area, to the authors' best knowledge. However, a fatal case of hepatic sarcocystosis [8], caused by an unknown species, is the only account of a *Sarcocystis* sp. infection in a Mediterranean cetacean.

In this study, we provide evidence of an infection sustained by a *Sarcocystis*-like organism in two striped dolphins (*Stenella coeruleoalba*) stranded along the Ligurian coast of Italy, in the marine protected area of the Pelagos Sanctuary, in 2011 and 2017.

#### **2. Materials and Methods**

#### *2.1. Naturally Infected Dolphins*

The cases, SD1 and SD2, included in the present study were diagnosed during routine pathological and cause-of-death assessment in stranded cetaceans at the Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta. The two striped dolphins (*Stenella coeruleoalba*), stranded along the Ligurian Sea coast in 2011 (SD1) and 2017 (SD2) (Figure 1), were submitted for a complete postmortem examination, according to standard protocols [9]. Dolphin SD1 was a 206 cm (total length, TL) adult male, in a poor nutritional status and in a postmortem condition code 3 (moderately decomposed). Dolphin SD2 was a 177 cm (TL) juvenile male, in a moderate nutritional status and in postmortem condition code 2 (fresh). Neither animals displayed any evidence of interaction with fishing activities, and the stomach chambers were devoid of intake. Initially, postmortem findings from SD1 and SD2 were previously published [10,11] and, for the present study, these two cases were re-examined with the focus on neurotrophic causes of inflammation, CNS, and tissue protozoal cysts, parasite identification, and distribution.

**Figure 1.** Map of the study area (Ligurian coastline), displaying the stranding locations (red and green dots) of the two striped dolphins infected by Sarcocystidae organisms. The map was created by A.P. with QGIS (QGIS Development Team (2018). QGIS Geographic Information System. Open Source Geospatial Foundation Project. http://qgis.osgeo.org, accessed on 17 August 20).

During necropsy, the tissue samples of all the major organs and lesions were collected and split into aliquots for subsequent analyses: one was kept frozen at −20 ◦C for microbiological and toxicological investigations, one was kept frozen at −80 ◦C for biomolecular analyses, and the other was preserved in 10% buffered formalin for histological and immunohistochemical (IHC) investigations. Blood serum, aqueous humor, and cerebrospinal fluid (CSF) were collected, when available, and kept frozen at −20 ◦C for serological investigations.

#### *2.2. Histology and Immunohistochemistry*

Representative tissues from SD1 (brain, lung, heart, liver, spleen, kidney, prescapular lymph node, urinary bladder, and reproductive system) and from SD2 (brain, tonsils, lung, prescapular and tracheobronchial lymph nodes, heart, liver, spleen, pancreas, intestine, skeletal muscle, skin, kidney, urinary bladder, adrenal gland, tongue lesion, and reproductive system) were collected and fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4 ± 2 μm, stained with hematoxylin and eosin (H&E), and examined through a light microscope.

Nine different areas from the brain were sampled and examined, including basal nuclei, thalamus, mesencephalon, pons, obex, and frontal, parietal, occipital, and cerebellar cortex. Immunohistochemistry (IHC) for *Morbillivirus* was performed on tissue sections from SD1 (brain) and from SD2 (brain, tonsils, lung, prescapular and pulmonary lymph nodes, spleen, kidney, urinary bladder, liver, skin, and muscle), using a monoclonal anti-*Canine distemper virus* (CDV) antibody (VMRD, Pullman, WA, USA) [6]. *Toxoplasma gondii* IHC was carried out on the nine aforementioned brain tissues of each case, using a polyclonal serum of caprine origin (VMRD, Pullman, WA, USA) [6].

#### *2.3. PCR and Sequence Analysis*

Molecular detection of *Dolphin morbillivirus*(DMV) [12], *Herpesvirus*(HV) [13], *T. gondii* [14], and *Brucella* spp. [15] was routinely achieved on target tissues available from each case, consisting of brain, lung, tonsils, lymph nodes, liver, spleen, kidney, bladder, and blood for DMV, brain, lung, lymph nodes, spleen, and kidney for HV, brain, lymph nodes, liver, spleen, heart, and muscle for *T. gondii*, and brain, lung, tonsils, lymph nodes, liver, spleen, kidney, and blood for *Brucella* spp.

For DMV assays, amplicons were directly sequenced using PCR primers on a 3130XL Genetic Analyzer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Sequences were aligned using the SeqMan software (Lasergene package. DNASTAR Inc., Madison, WI, USA) to obtain a consensus sequence and compared with available sequences retrieved from the National Center for Biotechnology Information (NCBI) database through the BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 20 February 2021).

#### *2.4. Serological, Toxicological, and Microbiological Analyses*

Serological investigations to screen for the presence of specific antibodies against DMV and *T. gondii* were performed [6] on serum, CSF, and aqueous humor, when these samples were available from SD1 and SD2. These same samples were also tested by rapid serum agglutination (Rose Bengal plate test, RBT) using RBT antigen produced from *B. abortus* strain S99 [6,16] to detect anti-smooth *Brucella* spp. antibodies.

Toxicological investigations were carried out only in SD2. The toxicological analysis did not include tissues from SD1 as they were collected before implementing a sampling protocol that included contaminant analyses. Polychlorinated biphenyls (PCBs), hexachlorobenzene (HCB), and dichlorodiphenyltrichloroethanes (DDTs) were measured in blubber. Measurements were made according to Environmental Protection Agency method 8081/8082, with modifications [17], and toxicological stress was evaluated using a theoretical model [18].

Tissue samples including brain, lung, lymph nodes, liver, spleen, and kidney (SD1 and SD2) were processed for standard aerobic, anaerobic, and microaerobic (5% CO2) bacterial culture and identification, using biochemical and/or molecular analyses. Following international recommendations [19], samples from target tissues underwent specific bacteriological procedures to screen for *Salmonella* spp., *Listeria* spp., and *Brucella* spp.

#### *2.5. Light Microscopy Re-Examination for Parasite Characterization: Histology and Immunohistochemistry*

CNS and heart sections from both SD1 and SD2 and skeletal muscle tissue only from SD2 were hematoxylin and eosin (H&E)-stained for light microscopy re-examination. For each organ and the aforementioned CNS areas studied, nine additional 5 μm thick sections were cut in series from stored paraffin blocks. Sections n◦ 1, 4, and 7 were stained with H&E for light microscopy examination. For IHC, only sections adjacent to the H&E slides presenting tissue cysts which were clearly distinct from *T. gondii* tissue cysts were used (well-defined cyst wall and >8 μm long bradyzoites). The IHC protocols included *S. neurona* polyclonal antibodies (PoAb Rabbit 1 R81, [20]), *S. falcatula* polyclonal antibodies (PoAb Rabbit 2 R-anti SF, [20]), and *S. neurona* monoclonal antibody (MAb 2G5, [21]). The IHC analyses were performed as previously described [21,22]. *Sarcocystis neurona*-infected and noninfected murine brains from interferon gamma (IFN-γ) knockout B6.129S7-Ifngtm1Ts (*Mus musculus*) (Jackson Laboratories, Bar Harbor, ME, USA) mice [23] were used, respectively, as positive and negative controls for the polyclonal antibodies, whereas *S. neurona*-infected opossum (*Didelphis virginiana*) intestine tissues and the brain of a bottlenose dolphin (*Tursiops truncatus*) foetus born under human care were used, respectively, as positive and negative control for the monoclonal antibody.

#### *2.6. Electron Microscopy Examination*

Transmission electron microscopy (TEM) was performed on CNS samples of both specimens at the Spanish National Center for Electron Microscopy (Complutense University of Madrid). For each animal, two cysts were excised from the paraffin block and samples were prepared for TEM as previously described [24]. Ultra-thin sections were cut on a Leica UC6 ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany), mounted onto TEM grids, and stained with 6% saturated uranyl acetate and 3% lead citrate. Sections were examined with a JEOL JEM 1400 Plus (JEOL USA Inc., Peabody, MA, USA) transmission electron microscope operated at 80 kV.

#### *2.7. Molecular Analyses: Parasite Detection, Identification, and Characterization*

Genomic DNA was extracted from CNS, myocardium, and skeletal muscle tissue samples from SD1 and SD2 and screened for the presence of tissue-cyst forming coccidia (*Sarcocystis* spp., *T. gondii* and *N. caninum*) DNA using 11 different PCR protocols, detailed in Supplementary material Table S1. DNA extraction from frozen samples was achieved using a "four-step" method, namely, ReliaPrep™ gDNA Tissue Miniprep System (Promega Italia S.r.l. Milan, Italy), whereas DNA extraction from paraffin-embedded samples, previously purified using the QIAamp DNA FFPE Tissue Kit, was achieved using QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany).

For each positive PCR result, amplicons of the expected size were sequenced using the BigDye® Terminator kit v 3.1 (Applied Biosystems, Foster City, CA, USA) and analyzed on an ABI 3130 Genetic Analyzer (Applied Biosystems). The obtained sequences were curated manually if necessary and analyzed using BioEdit software, version 7.0.5.3 [25]. Generated DNA consensus sequences were aligned to appropriate reference sequences using MEGA X software (http://www.megasoftware.net/ accessed on 20 April 2021) [26], and compared with available sequences retrieved from the NCBI database through the BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 20 February 2021).

*Toxoplasma gondii* strain genotyping analyses were carried out at the Complutense University of Madrid. DNA extracts from SD2 heart, muscle, and brain tissues were subjected to the widely used Mn-PCR restriction fragment length polymorphism (RFLP) method, with the markers *SAG1, SAG2 (5 –3' SAG2,* and alt. *SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico*, and *CS3* [27,28]. ToxoDB RFLP genotype was identified according to http://toxodb.org/toxo/ accessed on 20 April 2021.

#### **3. Results**

#### *3.1. Naturally Infected Dolphins*

Results of *postmortem*, routine investigations, along with anamnestic data, are summarized in Table 1.

#### *3.2. Histology and Immunohistochemistry*

Significant histopathological lesions detected for SD1 and SD2 are detailed in Table 1. Both SD1 and SD2 were diagnosed with severe and diffuse NS meningoencephalitis in association with the detection of protozoan tissue cysts (a single cyst for SD1 and two cysts for SD2). The cysts were morphologically distinct from *T. gondii*. In addition, SD2 was also diagnosed with *T. gondii*, whereas SD1 samples demonstrated no detectable antibodies for *T. gondii* nor detectable *T. gondii* DNA by PCR. No cysts were observed in the other target tissues investigated: muscle of SD2 and heart of both SD1 and SD2. *Morbillivirus*-specific antigens were detected in brain, urinary bladder, and muscle of SD2 by IHC, while SD1 did not show any specific staining.

All brain cysts observed in both animals stained positive with the polyclonal Ab raised for *T. gondii*. Since these data were not supported by molecular and serological investigations performed in SD1, an antigenic cross-reactivity among genetically related protozoa was hypothesized.


YS = year of stranding; DC = decomposition code (2, fresh; 3, moderate autolysis); NuS = nutritional status; M = male; NS = nonsuppurative; DMV = *Dolphin morbillivirus*; VN = virus neutralization; CNS = central nervous system; PCR = polymerase chain reaction; IHC = immunohistochemistry; CFS = cerebrospinal fluid; IFAT = indirect fluorescent antibody technique; CAN = canonical variable.

#### *3.3. PCR and Sequence Analysis*

No biomolecular evidence of DMV, HV, *T. gondii* or *Brucella* spp. was found in SD1. A systemic DMV infection was demonstrated in SD2, through PCR, in brain, lung, laryngeal tonsils, tracheobronchial lymph node, spleen, kidney, and bladder, and subsequently confirmed through amplicon sequencing and BLAST analysis.

However, in SD2, molecular data supported a coinfection with both *T. gondii*, in brain, liver, muscle, spleen, and tracheobronchial and pulmonary lymph nodes, and *Brucella* sp., by PCR detection, in brain, liver, lung, spleen, and tracheobronchial, pulmonary, and prescapular lymph nodes. The HV analysis was negative for SD2.

#### *3.4. Serological, Toxicological, and Microbiological Analyses*

Anti-*Morbillivirus* antibodies (1:16) were detected in serum of SD1, while anti-*Morbillivirus* (1:8 serum) and anti-*T. gondii* antibodies (>1:640 serum; 1:160 CSF; 1:80 aqueous humor) were detected in SD2. No evidence of anti-*Brucella* spp. antibodies was demonstrated in sera, cerebrospinal fluid, or aqueous humor samples from either of the dolphins.

For SD2, the levels of PCBs, HCB, and DDTs, expressed in ng·g−<sup>1</sup> on a lipid weight basis (PCBs: 136810.3; DDTs: 69866.92; HCB: 153.6; canonical variable value (CAN) = 0.688), confirmed the presence of immunotoxic levels of OC pollutants (CAN > 0.47).

*Photobacterium damselae* subsp. *damselae* was isolated by microaerobic bacterial culture from blowhole and lungs of SD1; no other significant bacteria, including *Salmonella* spp., *Listeria* spp., and *Brucella* spp., were isolated. *Brucella ceti* was isolated from CNS, spleen, and lung of SD2; no other significant bacteria, including *Listeria* spp. and *Salmonella* spp., were isolated.

#### *3.5. Light Microscopy Re-Examination: Histology and Immunohistochemistry*

For both SD1 and SD2, several non-*Toxoplasma* protozoal tissue cysts were observed in all the CNS areas studied, with major involvement of the brain cortical areas. Overall, neural tissue cysts were round to oval in shape, from 27 to 119.3 μm in diameter, presented with a distinguishable, apparently smooth outer wall, and they were filled with mild basophilic bradyzoites (Figure 2). Within the range of the available magnifications, neither internal septations nor villous protrusions were visible by light microscopy. Moreover, it was not possible to detect the presence of free schizonts or merozoites in any of the brain sections examined. The histomorphological appearance was consistent with that of other apicomplexan coccidia, with most features resembling the genus *Sarcocystis.* No other cysts were observed in the heart of either case, while, in the skeletal muscle from SD2, only one tissue cyst morphologically resembling *T. gondii* was observed.

*Sarcocystis*-like tissue cysts in the brains of both SD1 and SD2 stained immunopositive with the anti-*S. neurona* polyclonal antiserum. Similar results were obtained with the anti-*S. falcatula* polyclonal antiserum whereas MAb 2G5 failed in labeling protozoal antigens. Overall, the application of the polyclonal antisera resulted in a negative staining of the cyst wall and in a sparse labeling of the enclosed bradyzoites (Figure 2).

#### *3.6. Ultrastructural Description of Tissue Cysts*

After processing of samples, no tissue cysts could be observed from the SD1 specimenderived blocks, but a mature thin-walled (400 nm) cyst resembling *T. gondii* (Figure 3) was examined in dolphin SD2. Typical morphology with simple wall structure presenting vesicles, absence of septae, and small bradyzoites of 5.3 × 1.4 μm in size (*n* = 10) were observed.

**Figure 2.** *Sarcocystis*-like tissue cysts in the brain of striped dolphins (*Stenella coeruleoalba*) SD1 and SD2 from Liguria, Italy. (**A**) Parietal cortex (SD1). Protozoan tissue cyst measuring 70 × 50 μm. H&E. (**B**) Occipital cortex (SD1). Protozoan tissue cyst measuring 44.6 × 58.1 μm. H&E. (**C**) Frontal cortex (SD2). Protozoan tissue cyst measuring 72.83 × 116.34 μm. H&E. (**D**) Basal ganglia (SD2). Protozoan tissue cysts measuring (left-right reading) 110 × 119.3 μm, 40 × 19.8 μm and 50 × 99.1 μm. H&E. (**E**) Mesencephalon (SD1). Negative immunostaining of a protozoan tissue cyst (arrow). Monoclonal Ab anti-*S. neurona*. (**F**) Cerebellum (SD1). Positive labeling of *Sarcocystis*-like tissue cyst. Polyclonal Ab anti-*S. falcatula.* (**G**) Mesencephalon (SD1). Positive labeling of a protozoan tissue cyst bradyzoites. Polyclonal Ab anti-*S. neurona.* (**H**) Parietal cortex (SD2). Positive immunostaining of a protozoan tissue cyst bradyzoites. Polyclonal Ab anti-*S. neurona.*

**Figure 3.** Transmission electron microscopy micrographs of the *Toxoplasma gondii* tissue cyst studied from central nervous system of striped dolphin (*Stenella coeruleoalba*) SD2 in Liguria, Italy. (**A**) Section of the thin-walled tissue cyst; note the cyst wall (arrowheads) and densely packaged bradyzoites (br). (**B**,**C**) Details of the simple and thin cyst wall (arrowheads) and granular layer (gl) presenting vesicles (arrows). (**D**,**E**) Ultrastructural details of bradyzoites, note: nucleus (nu), micronemes (mi), dense granules (dg), amylopectin granules (am), conoid (co), and rhoptries (rh).

#### *3.7. Molecular Detection and Parasite Identification*

The findings on PCR screening for tissue-cyst forming coccidia DNA are summarized in Supplementary material Table S2. PCR n.1 (18S region) and n.9 protocols showed the presence of cyst-forming coccidia DNA in at least one tissue of each animal, in agreement with histological findings. Sequencing of PCR products confirmed the absence of *T. gondii* DNA in SD1 and the presence of *T. gondii* in SD2. PCR n.5 and n.6 protocols showed the presence of *Sarcocystis* sp. DNA in the tissues from both SD1 and SD2. The *Sarcocystis* sp. DNA detection was confirmed by sequencing of the amplicons produced from DNA extracted from SD1 myocardial tissue and SD2 skeletal muscle tissue. BLAST® analysis retrieved different homology to *Sarcocystis* species infecting members of the family Bovidae, *S. hirsuta* (99.4%), and *S. buffalonis* (97.8%), respectively. These sequences were deposited in GenBank® with the following accession numbers: MW151248 (*Sarcocystis* sp.) and MW151249 (*Sarcocystis* sp.).

PCR n.7 and n.8 protocols resulted in the detection of Sarcocystidae-unspecific products in heart and brain tissues from SD2 followed by PCR n.8, and DNA sequencing which was used to confirm the presence of *T. gondii* DNA in SD2 heart and brain tissues. This finding was further documented by PCR n.10 (specific for *T. gondii* amplification). Molecular methods confirmed the presence of *T.gondii* in addition to the detection of *Sarcocystis* DNA. The molecular results suggest a potentially greater distribution of *T. gondii* parasites or higher concentration of DNA from *T. gondii* as compared to the *Sarcocystis* sp.

Phylogenetic analysis of the 18S rRNA *Sarcocystis* sp. sequence from SD2 indicated a *Sarcocystis buffalonis*-like organism (MW151249) when the sequence was compared to genetically similar species and the *Sarcocystis* spp. infecting major livestock species in Mediterranean Europe (Figure 4). The *S. hirsuta*-like sequence (MW151248) was not included in the tree because of its short length (122 bp). PCR n.11 screening for *N. caninum*

DNA resulted in none detected in both cases. All SD2 target organs tested for the presence of *T. gondii* DNA were strongly positive (100% homology with other *T. gondii* sequences deposited such as MH793505). Furthermore, all three DNA samples from SD2 were genotyped by PCR-RFLP method as ToxoDB genotype #3 showing type II alleles for all the markers except *Apico* (type I allele) (Table 2).

**Figure 4.** Phylogenetic positioning of the *Sarcocystis*-like organism found in muscle of striped dolphin (*Stenella coeruleoalba*) SD2 in Liguria, Italy. The evolutionary history was inferred using the maximum parsimony (MP) method. Tree n.1 out of three most parsimonious trees (length = 1532) is shown. The consistency index is (0.750000), the retention index is (0.820830), and the composite index is 0.689026 (0.615623) for all sites and parsimony-informative sites (in parentheses). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches [29].The MP tree was obtained using the subtree–pruning–regrafting (SPR) algorithm [30] with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates). This analysis involved 21 nucleotide sequences from *Sarcocystis* species infecting domestic hosts that are raised in Europe (Bt, *Bos taurus*; Bb, *Bubalus bubalis*, Ch, *Capra hircus*; Oa, *Ovis aries*; Ss, *Sus scrofa*). In cluster B and C, a high or moderate bootstrap (BP) value at each node supported each group containing closely related *Sarcocystis* species with canids as the definitive host, respectively, whereas, in cluster A, some low BP values indicated that the phylogenetic position of *Sarcocystis* species with felids as definitive hosts is not conclusive (BP = 34–38%). These results are probably due to the fact that the 18S rRNA locus is not the most appropriate to infer phylogenetic relationships; moreover, the short length of the SD2 sequence obtained is a limitation identified here. Nonetheless, it should be noted that neighbor-joining and maximum-likelihood methods also resulted in phylogenetic trees in which *S. hirsuta*, *S. buffalonis*, and SD2 *Sarcocystis* sp. grouped together (data not shown). There were a total of 1974 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [26].


**Table 2.** Results of genotyping analysis carried out on *Toxoplasma gondii* strain identified in dolphin SD2.

#### **4. Discussion**

NS meningoencephalitis in dolphins is usually related to *B. ceti* infections, to viruses such as DMV and HV, and to protozoa, especially *T. gondii* [7,31]. In this study, involving two striped dolphins (*Stenella coeruleoalba*) stranded along the Ligurian coast of Italy, samples from SD2 demonstrated the presence of *T. gondii*, DMV, and *B. ceti*. This coinfection, alongside with the toxicological stress detected (CAN > 0.47, [18]), has been considered responsible for the cerebral impairment and the consequential animal's stranding [10]. In contrast, samples from SD1 lacked direct detection of commonly recognized neurotropic agents to explain the observed neuroinflammatory pattern present. SD1 demonstrated serological evidence of DMV infection, evident by a very low titer of antibodies, and suggestive of contact with the virus, rather than the disease, i.e., subclinical infection [32]. Therefore, a closer examination of the tissues for other potential neurotropic agents was undertaken for SD1 and SD2 for comparison.

From the histopathological examination, the morphological appearance of the unusual cysts observed in both animals was highly suggestive of a *Sarcocystis*-like coccidium. Although neither villous protrusions nor internal septations were observed, the protozoan tissue cysts were large (up to 116 μm in diameter), presented with a discernible thin outer wall, and the enclosed bradyzoites stained more basophilic than *T. gondii* ones.

As it is routinely done in suspected cases of toxoplasmosis, an immunohistochemical characterization of the unknown organism was attempted. The obtained results were inconclusive and consistent with the reviewed literature [20–22]. The reactivity of the two polyclonal *Sarcocystis* spp. antisera was mild and failed to label the tissue cyst wall. The absent reactivity of the cyst wall may be related to its maturity, as previously proposed [22]. This similar reactivity of both the anti-*S. neurona* and anti-*S. falcatula* antisera to the cysts is in agreement with what has been previously reported [20,21]. A possible broader spectrum of antigenic cross-reactivity between closely related *Sarcocystis* species and other Apicomplexa has been suggested [20,33].

It should be also noted that, during previous standard investigations, large *Sarcocystis*like tissue cysts in the CNS of both animals stained positively to the anti-*T. gondii* PoAb, even in the absence of a molecular and serological confirmation of *T. gondii* in SD1. This finding suggests that common epitopes may be shared between these two protozoa and that the commercial anti-*T. gondii* PoAb is not specific in discriminating between these two protozoa, as previously documented by other authors [34], in cases of closely related cyst-forming apicomplexan parasites. The anti-*S. neurona* 2G5 monoclonal antibody did not stain the bradyzoites or the cyst wall of the protozoan tissue cyst in SD1 brain. Although this monoclonal antiserum is directed against a more conserved epitope among *S. neurona* strains and it is suitable to stain FFPE tissues [21], low reactivity was also observed by other authors [35] in the IHC investigations performed on the brains from California Sea otters (*Enhydra lutris nereis*) with PCR-confirmed *S. neurona* infection.

Therefore, the diagnostic value of the IHC staining is questionable and, in this study, could be limited in that both the monoclonal and polyclonal antibodies were raised against merozoite epitopes and may not be suitable to label the cyst wall or the enclosed bradyzoites [35].

As the IHC evaluations were insufficient for parasite identification, in order to have a final confirmation of the parasites detected, 11 different PCR protocols were employed in an attempt to molecularly characterize the tissue-cyst forming protozoa. *Sarcocystis* sp. infections, previously unreported in muscle tissue of Mediterranean cetaceans, were confirmed by PCR means in the myocardium and in the skeletal muscle.

PCR n.1 excluded parasite identification as *S. neurona* or *S. falcatula* in either SD1 or SD2. Moreover, the sequencing of the 18S rDNA amplicon to discern the presence of mixed protozoal infections [35] was nonspecific in SD1, resulting in exclusion of other known tissue cyst-forming coccidia, such as *T. gondii*, *N. caninum*, and *Hammondia hammondi*. *Neospora caninum* infection was also excluded by specific PCR n.11, and the thickness of the observed tissue cyst walls did not correspond to what is expected in such an organism.

The results obtained from SD2 tissues should be carefully interpreted. A majority of the DNA sequencing showed homology to *T. gondii* sequences deposited in GenBank®. SD2 was already proven coinfected with *T. gondii* by means of PCR [14] and IHC [6] and the massive infection by *T. gondii* could have masked the detection of other pathogens, including *Sarcocystis* sp. Perhaps, in SD2, DNA extraction from purified tissue cyst would be more effective as compared to genomic DNA extracted without isolating tissue cyst from the surrounding tissue [36].

PCR protocol n.6 succeeded in amplifying *Sarcocystis* sp. DNA sequences from target organs. A short sequence (122 bp), showing high BLAST homology with *S. hirsuta*, was obtained from SD1 myocardium, whereas a longer (188 bp), high-quality sequence was obtained from SD2 skeletal muscle showing 97.8% homology with *S. buffalonis* isolates.

To date, *S. hirsuta* and *S. buffalonis* have been reported only in cattle and water buffaloes [33] and seem to be strictly intermediate host-specific, like other livestock *Sarcocystis* species, with no reported infection in non-ruminant intermediate hosts. Furthermore, in the present study, parasite identification was achieved targeting the 18S rRNA gene but other authors [37,38] recommend the analysis of *cox1* gene to molecularly discriminate between *Sarcocystis* species-infecting hosts from the Bovidae family.

Although lacking additional natural cases or experimental infections, the possibility for marine wildlife to share *Sarcocystis* species with domestic animals cannot be ruled out completely, especially when intermediate hosts are phylogenetically related. Specifically, the order Cetacea is a sister-group to the family Hippopotamidae and to the Ruminantia taxa [39] which includes also the Bovidae family.

However, considering the results of the BLAST analysis in relationship to sequence quality and the host specificity of this protozoal family, it is most likely that a previouslyundescribed *Sarcocystis*-like protozoa, within the family Sarcocystidae and phylogenetically related to species cycling in livestock, is infecting Mediterranean marine mammals. The lack of reports and of prevalence data on muscular sarcocystosis in the Mediterranean makes it difficult to determine the origin of this pathogen.

Since the highest degree of similarity was observed with bovine and bubaline *Sarcocystis* species, a land-to-sea transfer can be hypothesized for this protozoon even though, in the absence of an exhaustive characterization, a marine cycle cannot be discarded. Being that most *Sarcocystis* species are highly host-specific, the hypothesis of a two-host heteroxenous marine cycle, like the one proposed for *Sarcocystis balaenopteralis* [40], seems unlikely as dolphins are apex predators rather than prey in the Mediterranean food web.

To date, *S. neurona* is commonly reported in cases of muscular and neural sarcocystosis diagnosed in marine mammals in North America [1], but its presence has not yet been reported in Europe. However, a *S. neurona* infection in Mediterranean dolphins is highly improbable due to the lack of the definitive hosts (*D. virginiana* and *D. albiventris*) which are geographically confined to the New World.

In the Mediterranean basin, protozoal meningoencephalitis has been limited to reports associated with *T. gondii* subacute to chronic infections [6,7]. A *T. gondii* infection was also confirmed and fully characterized by means of TEM, IHC, and PCR in the present study. Multilocus RFLP PCR genotyping of the *T. gondii* strain infecting SD2 retrieved a type II PRU variant genotype (ToxoDB#3), which is common in felids, livestock, and wildlife around Europe [41,42]. To date, type II genotypes account for the totality of toxoplasmosis reports in Mediterranean cetaceans [43–45] (reviewed in Table 3). Although a marine cycle for this parasite cannot be precluded, the results from SD1 and SD2 support the role of protozoa as a land-base "pollutant" that have expanded the range of their intermediate hosts to the marine environment.

**Table 3.** Summary of the available literature reporting genotyping data on *Toxoplasma gondii* strains infecting dolphins.


Previously, *Sarcocystis* spp. have not been observed in histopathological brain sections of stranded cetaceans. The only documented *Sarcocystis* sp. infection in a wild Mediterranean marine mammal is a hepatic sarcocystosis due to a *S. canis*-like protozoan infecting a striped dolphin stranded along the Spanish coast [8]. *Sarcocystis* spp. have already been reported in other marine mammals [50,51]. In aquatic species, *S. canis*-like infection causes a fatal and acute hepatitis with microscopic lesions confined to the liver [8,24,50]. Mature and immature schizonts are the protozoal stages observed during histopathological and TEM investigations, whereas tissue cysts have not been observed [8,50].

However, sarcocysts in muscle tissue are chronic lesions that have been incidentally observed in mysticetes and odontocetes from other geographical areas without associated pathology [52–56]. In previous reports, protozoal tissue cysts were attributed to *Sarcocystis* spp. only on the basis of light microscopy and TEM findings. In our study, muscular sarcocystosis was evidenced by means of PCR in each stranded dolphin. To the

authors' best knowledge, no other *Sarcocystis* species have been previously observed or isolated in Mediterranean cetaceans. Moreover, the prevalence of muscular sarcocystosis in Mediterranean marine mammals is unknown as such infections are likely overlooked during routine investigations. Therefore, it is not possible to state whether or not tissue cysts are a common finding in muscle tissues and which species of *Sarcocystis* are prevalent in Mediterranean marine mammals.

The most likely hypothesis would be to consider the same protozoa in the muscle tissues as the etiologic agents of the cysts observed in the CNS. Nevertheless, we cannot discard the possibility of two different species infecting the same host. The morphology of the observed neural cysts is highly suggestive of a *Sarcocystis*-like coccidium. It was not feasible to perform a morphological comparison with the muscle tissue cysts because SD1 FFPE skeletal muscle was not available and only *T. gondii* muscular cysts were observed in SD2 skeletal muscle sections (*data not shown*), while no cysts were identified in the heart sections of either SD1 or SD2.

As the biomolecular investigations failed in amplifying specific *Sarcocystis* sequences in the CNS, further investigations are needed to confirm our putative diagnosis.

#### **5. Conclusions**

The present study represents the first description of a *Sarcocystis*-like infection in muscle tissue of dolphins from the Mediterranean basin along with the first report of *Sarcocystis*-like tissue cysts in the brain of stranded cetaceans.

The *T. gondii* strain detected belongs to a common genotype circulating in Europe, while the unknown organisms were genetically similar to *Sarcocystis* species infecting the Bovidae family. Such results might suggest a land-to-sea cycling of these Apicomplexan parasites and the need for further investigations.

Because of the novelty of these findings, special attention should be reserved for the differential diagnosis of protozoal infections when performing sanitary surveillance on stranded Mediterranean cetaceans, including collection and preservation of tissues to enable a panel of characterization studies.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ani11051201/s1, Table S1: Selected PCR protocols used in the present study to detect DNA from tissue-cyst forming coccidian, Table S2: Results of the PCR protocols screening for tissue cyst-forming coccidian DNA in target organs.

**Author Contributions:** Conceptualization, F.G., E.S., A.F. and C.C.; formal analysis, F.G., U.R.-C., A.E.M., C.G. and R.C.-B.; funding acquisition, R.C.-B. and C.C.; investigation, B.I., A.P., K.V., E.B., P.G., L.M., C.E.D.F., M.G., F.V., T.A., S.P. and M.F.-E.; methodology, F.G., U.R.-C., A.E.M., C.G. and R.C.-B.; supervision, M.C., R.C.-B. and C.C.; writing—original draft, F.G. and U.R.-C.; writing—review and editing, A.E.M., M.C., R.C.-B. and C.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Italian Ministry of Health [Ricerca Corrente 2016 IZS PLV 05/16 RC].

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available within the article and the Supplementary Materials. The sequences generated in the present study were submitted to the GenBank database with the following accession numbers: MW151248-MW151249.

**Acknowledgments:** The authors are grateful to Milena Monnier and Laura Serracca for their support in biomolecular analyses, to Walter Mignone for necropsy investigations and to Agustín Rebollada-Merino (VISAVET Health Surveillance Centre, UCM) for his assistance with microscope images. Rafael Calero-Bernal is part of the TOXOSOURCES consortium supported by the funding from the European Union's Horizon 2020 Research and Innovation Program under the grant agreement No 773830: One Health European Joint Program.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


## *Article* **The Role of Mustelids in the Transmission of** *Sarcocystis* **spp. Using Cattle as Intermediate Hosts**

**Petras Prakas \*, Linas Balˇciauskas, Evelina Juozaityte-Ngugu and Dalius Butkauskas ˙**

Nature Research Centre, Akademijos Str. 2, LT-08412 Vilnius, Lithuania; linas.balciauskas@gamtc.lt (L.B.); evelina.ngugu@gamtc.lt (E.J.-N.); dalius.butkauskas@gamtc.lt (D.B.)

**\*** Correspondence: petras.prakas@gamtc.lt

**Simple Summary:** Members of the genus *Sarcocystis* are worldwide distributed protozoan parasites. *Sarcocystis* infections cause great losses in economically important animals. There is a lack of studies on *Sarcocystis* in naturally infected wild predators, especially of the family Mustelidae which represent a presumably important group of definitive hosts of these parasites. The objective of the present study was to examine the small intestine samples of various mustelid species from Lithuania serving as a possible source of *Sarcocystis* spp. using cattle as intermediate hosts. Overall, 84 samples collected from five mustelid species were analyzed. Oocysts/sporocysts of *Sarcocystis* spp. were detected in 75 animals (89.3%). Using molecular methods four *Sarcocystis* spp., *S*. *bovifelis*, *S*. *cruzi*, *S*. *hirsuta* and *S*. *hominis* were identified, with the first two being the most prevalent. These results indicate that mustelids are involved in the transmission of *Sarcocystis* spp. using cattle as intermediate hosts. The determined high prevalence of *Sarcocystis* spp. rates cause concerns about food safety issues. To control the spread of infection, further studies on the way carcasses of cattle or beef waste become accessible to mustelids are needed.

**Abstract:** There is a lack of research on the role of mustelids in the transmission of various *Sarcocystis* spp. In the present study we tested the hypothesis that widespread mustelids in Lithuania could be involved in the transmission of *Sarcocystis* spp. using cattle as intermediate hosts. In 2016–2020, intestinal samples of 84 mustelids were examined. *Sarcocystis* spp. were identified by species-specific PCR targeting the *cox1* gene and subsequent sequencing. Under a light microscope, oocysts/sporocysts of *Sarcocystis* spp. were observed in 40 samples (47.6%), while using molecular methods, they were detected in 75 animals (89.3%). Four *Sarcocystis* spp. were identified in the intestinal samples of American mink (*Neovison vison*), Beech marten (*Martes foina*), European pine marten (*Martes martes*), European badger (*Meles meles*) and European polecat (*Mustela putorius*). The prevalence of predominant *Sarcocystis* spp., *S*. *bovifelis* (89.3%) and *S*. *cruzi* (73.8%) was significantly higher than that of *S*. *hirsuta* (3.6%) and *S*. *hominis* (1.2%). In an individual sample, most frequently two *Sarcocystis* spp. were identified (69.0%), then a single species (15.5%) and three species (4.8%). The present study provides strong evidence that mustelids serve as definitive hosts for *Sarcocystis* spp. using cattle as intermediate hosts.

**Keywords:** *Sarcocystis*; cattle; mustelidae; life cycle; *cox1*; molecular identification

#### **1. Introduction**

Representatives of the genus *Sarcocystis* (Apicomplexa: Sarcocystidae) are cyst forming coccidians with an obligatory prey-predator two-host life cycle. Asexual multiplication with the formation of sarcocysts takes place in the extra-intestinal tissues of the intermediate host (IH), while sexual stages (oocysts-sporocysts) develop in the small intestine of the definitive host (DH) [1]. Predators and scavengers serve as DH for *Sarcocystis* spp., whereas prey animals become IH [2].

Members of the family Mustelidae may act as IH or DH for several *Sarcocystis* spp. The agent of equine protozoal myeloencephalitis, *S*. *neurona* was also detected in the muscles

**Citation:** Prakas, P.; Balˇciauskas, L.; Juozaityte-Ngugu, E.; Butkauskas, D. ˙ The Role of Mustelids in the Transmission of *Sarcocystis* spp. Using Cattle as Intermediate Hosts. *Animals* **2021**, *11*, 822. https://doi.org/ 10.3390/ani11030822

Academic Editors: Rafael Calero-Bernal and Ignacio Garcia-Bocanegra

Received: 15 February 2021 Accepted: 12 March 2021 Published: 15 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of a fisher (*Martes pennanti*), ferret (*Mustela putorius furo*) and American mink (*Neovison vison*) [3]. Additionally, eight species of *Sarcocystis* have been observed in the muscles of various mustelids [4]. Recently described *S*. *lutrae* [5] was identified in the muscles of several Carnivora families, Canidae, Mustelidae and Procyonidae [5–7]. The role of mustelids as DH of *Sarcocystis* spp. has not been investigated [8].

Mustelidae is the largest and most diverse family in the order of Carnivora in Lithuania, with nine species present [9]. Representatives of mustelids occur in all habitats, including the urban ones [10,11]. The broad habitat niches of the American mink, the Beech marten (*Martes foina*), European badger (*Meles meles*), European pine marten (*Martes martes*) and European polecat (*Mustela putorius*) are reflected in their diverse diets [10,11]. In general, members of the family Mustelidae are opportunistic predators and their diet consists of birds, various mammals, fish, amphibians, invertebrates, fruits, ungulate carcasses, plants and mushrooms [12–16]. In Lithuania, the food chains of mustelids, including cattle carrion, were not investigated in detail, with exception of the European pine marten [17]. Diet of this species in the cold period included 5.3% of carcasses of domestic animals according to the biomass consumed. Thus, far no studies on the role of mustelids in the transmission of *Sarcocystis* in Lithuania have been undertaken.

Recently, a high prevalence of *Sarcocystis* spp. in cattle from Lithuania has been recorded [18]. By performing trypsinization of the diaphragm muscles and species-specific PCR targeting the *cox1* (mitochondrial gene encoding subunit 1 of cytochrome c oxidase), *S*. *cruzi* was identified in 96.1% of the samples, *S*. *bovifelis* was detected in 71.6% of the samples, *S*. *hirsuta* was confirmed in 30.4% of the samples and *S*. *hominis* was observed in 13.7% of the samples [19]. Canids are DH for *S*. *cruzi*, humans are DH for *S*. *hominis*, whereas *S*. *hirsuta* and *S*. *bovifelis* are transmitted via felids [19]. The Eurasian lynx (*Lynx lynx*) is the only wild member of the felids in Lithuania [9]. However, this species is not abundant and there were approximately 160 lynx individuals in Lithuania in 2018 [20]. Thus, the high prevalence of *S*. *bovifelis* implies that it is not solely felids that contribute to the spread of this species. Therefore, we put forward the hypothesis that mustelids can act as DH of *S*. *bovifelis*. In order to test the assumption, the aim of the present study was to examine the small intestines of various mustelids from Lithuania for the presence of *Sarcocystis* spp. employing cattle as IH.

#### **2. Materials and Methods**

#### *2.1. Sample Collection*

Between 2016 and 2020, intestine samples of 84 mustelids (40 American mink, 4 Beech marten, 5 European badger, 20 European pine marten and 15 European polecat) were studied for the presence of *Sarcocystis* spp. The animals were collected from hunters, taxidermists, or biologists who found dead animals on the roadways in different regions of Lithuania (Figure 1).

**Figure 1.** *Sarcocystis* spp. in the species of Mustelidae in Lithuania. Red color means positive individuals, green color represents negative individuals.

#### *2.2. Examination of Intestines*

Oocysts/sporocysts of *Sarcocystis* spp. were excreted from the entire intestine of each mustelids using a slightly modified Verma et al. [21] technique. At first, faeces of each intestine were squeezed and the entire intestine was cut lengthwise. The intestinal epithelium was lightly scraped with the help of a scalpel blade and suspended in 50 mL of water. The homogenate was centrifuged for 10 min at 1000 rpm, 25 ◦C in 50 mL centrifuge tubes. The supernatant was discarded and sediments were re-suspended in 50 mL water. Subsequently, the homogenate was centrifuged for 10 min at 1000 rpm, 25 ◦C and the supernatant was discarded. The examination of the sediments for oocysts/sporocysts under a light microscope was repeated. The 200 μL of re-suspended sediments were taken from each sample and used for DNA extraction. DNA was isolated from all mustelid samples.

#### *2.3. Molecular Analysis*

DNA extraction from mucosal suspension was performed using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific Baltics, Vilnius, Lithuania). *Sarcocystis* spp. were identified by nested PCR of partial *cox1* sequences. Primers used in the present study are listed in Table 1. PCRs were conducted in the final volume of 25 μL made of 12.5 μL of DreamTaq PCR Master Mix (Thermo Fisher Scientific, Vilnius, Lithuania), 0.5 μM of each primer, 0.04 μg template DNA and nuclease-free water. The first run of nested PCR began with one cycle at 95 ◦C for 5 min followed by 35 cycles of 94 ◦C for 45 s, 58–60 ◦C, depending on primer pair for 60 s and 72 ◦C for 80 s and ending with one cycle at 72 ◦C for 7 min. For the second PCR assay, 1 μL from the first PCR assay was used. Visualization, purification and sequencing of PCR products were carried out using a previously described protocol [22]. The obtained *cox1* sequences were compared with the Nucleotide BLAST program (megablast option) [23]. The *cox1* sequences generated in the present study are available in GenBank with Acc. No. MW595468–MW595608.


#### **Table 1.** The primers used for the nested PCR.

<sup>1</sup> [24], <sup>2</sup> [19].

#### *2.4. Statistical Tests*

The prevalence and 95% CI for prevalence were calculated using OpenEpi epidemiological software [25], following the Wilson method for calculating score interval [26]. Differences in the prevalence of the identified *Sarcocystis* spp. were evaluated using the Chi-squared test, calculated in WinPepi, ver. 11.39 and using Upton's approximation for small and medium sample sizes [27]. Comparing the prevalence of *Sarcocystis* spp., the effect size was expressed according to adjusted Cohen's w [28].

#### **3. Results**

#### *3.1. Differences in Prevalence of Sarcocystis spp. Using Microscopic and Molecular Methods*

Based on microscopic examination, the prevalence of *Sarcocystis* spp. in mucosal scrapings was 47.6% (Table 2). Under a light microscope usually free sporocysts measuring 11.8 × 8.3 μm (7.1–14.5 × 6.5–10.9 μm; *n* = 219) were seen. Sporulated oocysts of *Sarcocystis* 17.7 × 13.1 μm (12.5–23.7 × 10.5–18.3 μm; *n* = 100) were also noticed. With the help of nested PCR and subsequent sequencing *Sarcocystis* spp. were confirmed in 75 animals (89.3%). In general, as compared with morphological examination, the detection rate of *Sarcocystis* spp. was significantly higher (χ<sup>2</sup> = 33.56, *p* < 0.0001; adjusted Cohen's w = 0.709, large effect size) when a molecular method was employed. The molecular method yielded significantly more detections in the American mink, European polecat and European badger (Cohen's w = 1.083, 0.606 and 1.061, respectively, large effect size). Differences between the two methods in the Beech marten and European pine marten were not significant (Table 2). In one American mink and three Beech marten samples, oocysts/sporocysts were detected microscopically, however, these samples were negative for the examined *Sarcocystis* spp. using a molecular analysis.

Based on molecular analysis, the highest prevalence of *Sarcocystis* spp. was observed in the Beech marten, followed by the American mink and European polecat; however, even the lowest prevalence of *Sarcocystis* spp. detected in the European badger and European pine marten were 75% and higher (Table 2). The prevalence of *Sarcocystis* spp. observed in the Beech marten, American mink and European polecat did not differ statistically (species cluster with the highest prevalence). The prevalence of *Sarcocystis* spp. observed in the American mink was significantly higher (χ<sup>2</sup> = 5.09, *p* < 0.025; Cohen's w = 0.435, medium effect size) than that detected in the European pine marten. Other differences were not significant and the effect size was either small or absent.


**Table 2.** Identification of *Sarcocystis* spp. oocysts/sporocysts in mustelids using microscopic and molecular examination.

Significance of differences between methods is shown in superscript: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.0001, NS not significant.

#### *3.2. Molecular Identification of Sarcocystis spp.*

The comparison of sequences generated in the present study showed the presence of four *Sarcocystis* spp. (*S*. *bovifelis*, *S*. *cruzi*, *S*. *hirsuta* and *S*. *hominis*) in the analyzed samples of Mustelidae (Table 3).

**Table 3.** Intra- and inter-specific genetic variability of identified *Sarcocystis* spp.


#### *3.3. Distribution of Sarcocystis spp. in the Intestine Samples of Mustelids*

Irrespective of the host species, *S*. *bovifelis* in the examined samples was identified most often (Figure 2A). The prevalence of *S*. *bovifelis* (89.3%) was significantly higher than that of *S*. *cruzi* (73.8%, a small effect size), *S*. *hirsuta* (3.6%, a large effect size) and *S*. *hominis* (1.2%, a large effect size). The prevalence of *S*. *cruzi* was significantly higher than that of *S*. *hirsuta* (3.6%) and *S*. *hominis* (a large effect size both).

**Figure 2.** Prevalence of *Sarcocystis* spp. in the examined samples of mustelids. (**A**)—in the pooled sample of all host species, (**B**)—in American mink, (**C**)—in Beech marten, (**D**)—in European pine marten, (**E**)—in European badger, (**F**)—in European polecat. Differences of prevalence in A: *S*. *bovifelis* > *S*. *cruzi* (χ<sup>2</sup> = 6.65, *p* < 0.01; Cohen's w = 0.288), >*S*. *hirsuta* (χ<sup>2</sup> = 123.32, *p* < 0.001; w = 2.376) and >*S*. *hominis* (χ<sup>2</sup> = 130.79, *p* < 0.001; w = 2.688); *S*. *cruzi* > *S*. *hirsuta* (3.6%, χ<sup>2</sup> = 86.83, *p* < 0.001; w = 1.472) and >*S*. *hominis* (χ<sup>2</sup> = 93.94, *p* < 0.001; w = 1.604); in B: *S*. *bovifelis* >*S*. *cruzi* (χ<sup>2</sup> = 5.10, *p* < 0.025; w = 0.372); in E: *S*. *bovifelis* > *S*. *cruzi* (χ<sup>2</sup> = 3.24, *p* < 0.075; w = 1.064).

The prevalence of *S*. *bovifelis* was the highest, exceeding that of *S*. *cruzi* in the examined samples of the American mink (a medium effect size, Figure 2B) and European badger (a large effect size, Figure 2E). The prevalence of *S*. *bovifelis* and *S*. *cruzi* did not differ significantly in European polecat (Figure 2F) and Beech marten (Figure 2C); in European pine marten they were equal (Figure 2D). The prevalence of predominant *Sarcocystis* spp., *S*. *bovifelis* and *S*. *cruzi*, was significantly higher than that of *S*. *hirsuta* and *S*. *hominis*, in all host species (Figure 2B–F). Both predominant species were observed in all five examined host species. *Sarcocystis hirsuta* was identified in two American mink individuals and one European polecat individual; whereas *S*. *hominis* was confirmed in one European pine marten individual.

Up to three *Sarcocystis* spp. were identified in one host individual (Figure 3). No examined *Sarcocystis* spp. were found in approximately one tenth of the investigated animals (10.7%). The prevalence of single species infections was 15.5%; in all cases when a single species was detected in individual samples, it was *S*. *bovifelis*. Two *Sarcocystis* spp. (69.0%) were most frequently identified in one host individual and in all such cases it was *S*. *cruzi*/*S*. *bovifelis* co-infection. Three *Sarcocystis* spp. were confirmed in four animals (4.8%), one European polecat individual, one European pine marten individual and two American minks. In three of these cases, it was *S*. *bovifelis*/*S*. *cruzi*/*S*. *hirsuta* co-infection, in one case—*S*. *bovifelis*/*S*. *cruzi*/*S*. *hominis* co-infection.

**Figure 3.** Distribution of the number of *Sarcocystis* spp. identified in the examined samples of mustelids.

#### **4. Discussion**

In the present study, high rates (89.3%) of *Sarcocystis* spp. employing cattle as IH were observed in mustelids from Lithuania. Under a light microscope oocysts/sporocysts were detected in 40 out of 84 samples (47.6%). In comparison, the presence of *Sarcocystis*

spp. in 75 (89.3%) mucosal scrapings of mustelids were confirmed by molecular methods. Usually, molecular analysis is performed when oocysts/sporocysts of *Sarcocystis* spp. are microscopically detected in intestine mucosal or faecal samples [2,29–31]. However, the results of the present study reveal that molecular methods should be applied in testing all examined samples rather than only microscopically positive ones. No *Sarcocystis* spp. were identified in the mucosal scrapings of a single American mink and three European pine martens using species-specific PCR; however, oocysts/sporocysts were detected in these samples under a light microscope. Thus, these animals were most likely infected with oocysts/sporocysts of *Sarcocystis* spp., which employ other than cattle IH. There are a few reports on mustelids as DH for *Sarcocystis* spp. Transmission experiments have shown that mustelids are DH of several *Sarcocystis* spp., *S*. *campestris*, *S*. *muris*, *S*. *putorii*, *S*. *undulati* and *S*. *citellivulpes* (invalid species by Dubey [1]) using members of the order Rodentia as IH [8]. Further studies are needed to reveal the role of mustelids in the transmission of *Sarcocystis* spp. using various mammals and birds as IH.

*Sarcocystis* spp. identified in the present study, namely, *S. bovifelis, S. cruzi, S. hirsuta* and *S. hominis*, are specific to their IH [32]. Molecular data suggest that *S. cruzi* might occasionally infect water buffaloes (*Bubalus bubalis*) [33]. However, sheep, goats, pigs, horses and other domestic animals raised in Lithuania cannot serve as IH of the abovementioned *Sarcocystis* spp. [1]. Of the Lithuanian wild fauna, only the European bison (*Bison bonasus*) can possibly act as an IH of some *Sarcocystis* spp. detected in this study [34–36]. However, the *B. bonasus* population in Lithuania is not large, it stands at less than 300 individuals and their distribution range does not intersect with the sites of our material on mustelids [9–11]. Therefore, it is impossible for *B. bonasus* to be responsible for the high rates of *S. bovifelis* and *S. cruzi* in the intestinal samples of mustelids.

The forest is considered a primary habitat of two mustelid species, European pine marten and European badger, though they are frequent visitors to the surrounding woodlots, meadows and riversides [9]. The habitat of the American mink is related to water they inhabit banks of rivers, lakes and ponds. These mustelid species are not closely related to human settlements. Two other investigated mustelids, American mink and European polecat, are more often related to settlements than to other habitats, such as forests and shrubby areas [9]. Habitats preferred by mustelids in Lithuania are similar to those in other countries [37]. Diet peculiarities of the investigated mustelids are not directly related to the involvement of these species in the transmission of *Sarcocystis* spp. using cattle as IH. All the investigated mustelid species are opportunistic feeders. Among such diet sources as fruits, berries and other plant materials, invertebrates, fish, amphibians, birds and various mammals [12–17], only one source, namely, cattle carrion, or other sources of cattle meat may be related to *Sarcocystis* spp. we have identified. Mustelid species that we have investigated [12–17], with the exception of the American mink [38], use carrion of wild ungulates.

Cattle are too large prey for mustelids to hunt; therefore, mustelids become infected with *S*. *bovifelis*, *S*. *cruzi*, *S*. *hirsuta* and *S*. *hominis* species by scavenging carcasses of cattle. However, habitat distribution of the five investigated mustelid species in Lithuania (see above) should exclude contact with carrion of at least two species, American mink and European pine marten. Therefore, the first assumption about high rates of *Sarcocystis* spp. employing cattle as IH is related to food safety issues. In further studies we are going to examine in what way cattle carcasses or beef waste become accessible to mustelids in Lithuania. It is important to understand whether there are gaps in the management of anthropogenic carrion [39] and if this has already become a source of predictable resources accessible to mustelids. Improper carrion management may be related to (i) dumping sites, (ii) treatment of the waste from meat processing factories, especially small ones and located in the countryside and (iii) raw meat waste from homesteads and farms. The two last sources may be neighboring forests and water bodies, therefore becoming sources of possible infection and available even to the American mink and European pine marten, otherwise having no contact with cattle carrion.

Historically, the disclosure of DH of *Sarcocystis* spp. was performed by transmission experiments [40]. Among carnivorous mammals, transmission experiments of *Sarcocystis* spp. have mainly been carried out with dogs, foxes and cats [41,42]. Recently, molecular methods have been applied for the identification of *Sarcocystis* spp. from fecal or mucosal scraping samples of various wild predators or scavengers infected under natural conditions [2,29–31]. The present work is the first study of the molecular identification of *Sarcocystis* spp. in mustelids. Further molecular examination of oocysts/sporocysts detected in the intestine or fecal samples of mustelids can help to clarify the role of these carnivorous mammals in the transmission of *Sarcocystis* parasites.

It is well known that *Sarcocystis* spp. transmitted via canids cannot be spread via felids and vice versa [1]. However, there is a lack of data on whether *Sarcocystis* spp. transmitted via canids and/or felids can be spread via mustelids. It was demonstrated that mustelids and canids could serve as DH of *S*. *undulati* and *S*. *citellivulpes* [8,43], whereas mustelids and felids could act as DH for *S*. *muris* [8]. Two species, *S*. *bovifelis* (89.3%) and *S*. *cruzi* (73.8%), were most common in the analyzed intestinal samples of mustelids (Figure 2), whereas *S*. *hirusta* and *S*. *hominis* were confirmed in three and single samples, respectively. Canids serve as DH for *S*. *cruzi*, felids act as DH for *S*. *hirsuta* and *S*. *bovifelis* and humans are DH for *S*. *hominis* [19]. Thus, our results indicate that mustelids might be involved in the transmission of *Sarcocystis* spp. which were confirmed to be transmitted via canids and felids. Nevertheless, further detailed studies on this subject are required. Considering a low abundance of wild felids in Lithuania, we speculate that *S*. *hirsuta* is mainly transmitted via felids and *S*. *bovifelis* is mainly transmitted via mustelids. To test the hypothesis, the prevalence of *S*. *hirsuta* and *S*. *bovifelis* in muscles of cattle can be examined in European countries where wild felids are more prevalent. Estonia and Finland are the nearest countries with similar environments and with similar abundances of mustelids but with the high abundances of Eurasian lynx, while Germany or Belgium may be the reference countries with the European wildcat (*Felis silvestris*) populations [44].

#### **5. Conclusions**

Using a molecular analysis four *Sarcocystis* spp. employing cattle as IH (S. *bovifelis*, *S*. *cruzi*, *S*. *hirsuta* and *S*. *hominis*) were identified in the intestine mucosal scrapings of five Mustelidae species for the first time. Thus, the results of the present study indicate that a wide range of mustelids serve as DH of these *Sarcocystis* spp. Therefore, it is necessary to identify gaps in the management of cattle carrion and beef waste.

**Author Contributions:** Conceptualization, P.P. and D.B.; methodology, P.P.; software, L.B.; validation, D.B. and P.P.; formal analysis, L.B. and P.P.; investigation, E.J.-N.; resources D.B.; data curation, P.P.; writing—original draft preparation, P.P., L.B., E.J.-N. and D.B.; writing—review and editing, P.P., L.B., E.J.-N. and D.B.; visualization L.B. and E.J.-N.; supervision, P.P.; project administration, P.P. and D.B.; funding acquisition, D.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the Research Council of Lithuania (grant number S-MIP-20-24).

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Data supporting the conclusions of this article are included in the article. The sequences generated in the present study were submitted to the GenBank database under accession numbers MW595468–MW595608.

**Acknowledgments:** This study was supported by the Open Access research infrastructure of the Nature Research Centre under the Lithuanian open access network initiative. The authors are grateful to Valentinas Pabrinkis (Nature Research Centre, Vilnius, Lithuania) who provided samples for the study.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

#### **References**

