Red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) as potential spreaders of Sarcocystis species.

Background: Sarcocystis includes a global group of apicomplexan parasites with two-host life cycle frequently circulating in wildlife and domestic hosts, including humans. Two of the most important wild terrestrial carnivores acting as definitive hosts are the red fox and raccoon dog, due to their wide distribution in Europe and usage of wild and farmed animals as prey. This study was conducted to determine the prevalence of Sarcocystis in hunted red foxes and raccoon dogs from nine regions of the Czech Republic and to identify isolated sporocysts by molecular techniques. Methods: Approximately 5 g of the contents of large intestine from 200 animals (197 red foxes and three raccoon dogs) were examined by flotation centrifugation coprological method. Only samples of 50 red foxes and one raccoon dog positive to Sarcocystis spp. were used for the nested PCR (nPCR) method to amplify a fragment or partial sequence on the cox1 gene. Ten species-specific primer pairs for detection of Sarcocystis spp. using farm animals as intermediate hosts were utilized. Results: In total, 38.1% of the red foxes and 66.7% of the raccoon dogs were positive to Sarcocystis by light microscopy. The molecular characterization resulted in the identification of five species in the red fox: S. arieticanis, S. capracanis, S. cruzi, S. miescheriana, and S. tenella, while the PCR was negative for the sole raccoon dog. The highest intraspecific variation was found for S. miescheriana, while S. tenella was the most prevalent. Co-infections occurred in the large intestine of the red fox. No zoonotic species were found in our samples. Conclusion: This is the first study where the potential role of the red fox and raccoon dogs as spreaders of Sarcocystis to farm animals in the Czech Republic is shown. The use of species-specific primers provides a fast and easy method for screening multiple samples for a particular Sarcocystis species.

The Tengmalm's owl Aegolius funereus (Aves, Strigidae) as the definitive host of Sarcocystis funereus sp. nov. (Apicomplexa).

Background: Owls have been reported as definitive hosts, whereas wild small mammals (naturally and experimentally) as intermediate hosts of several species of Sarcocystis. Recently, dead fledglings were found infected by an unnamed species of Sarcocystis since its intermediate host was unknown. After collecting additional samples of owls and wild small mammals, the present study focused on elucidating the identity, potential intermediate host, and complete life cycle of the found Sarcocystis through experimentally infected rodents. The developmental stages' morphological and molecular characterizations (28S rRNA gene, ITS1 region) are presented herein. Methods: In total, 21 Tengmalm's owl carcasses (15 nestlings, 5 fledglings, and 1 adult male) were collected in Kauhava (west-central Finland) and parasitologically examined by wet mounts. Intestinal mucosa scrapings were used to isolate oocysts/sporocysts and employed for experimental infections in dexamethasone-immunosuppressed BALB/cOlaHsd mice. Additionally, sarcocysts were searched in the skeletal muscle of 95 samples from seven wild small mammal species. All these developmental stages were molecularly characterized by the 28S rRNA gene and ITS1 region. Experimental infections were carried out by using immunosuppressed female 8-week-old BALB/cOlaHsd mice, divided into three groups: (1) water with 15 µg/mL of dexamethasone, (2) water with 30 µg/mL of dexamethasone, (3) no dexamethasone treatment. Each group consisted of four individuals. In each group, two mice were infected with 1,000 sporocysts each, and the remaining two with 10,000 sporocysts each. All mice were euthanized on specific days post-infection. Results: The intestinal mucosa of 11 nestlings and 5 fledglings of the Tengmalm's owl were positive for Sarcocystis funereus sp. nov. The adult male owl and all owls' breast and heart muscles were negative for Sarcocystis. Two dexamethasone-immunosuppressed BALB/cOlaHsd mice (group 2) were positive to S. funereus sp. nov. in diaphragm and leg muscles after 22- and 24-day post-infection. Some sarcocysts were found in the wild small mammals. Molecular identification at 28S rRNA revealed sequences from naturally infected Tengmalm's owls, as well as sarcocysts of dexamethasone-immunosuppressed BALB/cOlaHsd mice were 99.87-100% similar to Sarcocystis sp. isolate Af1 previously found in the Tengmalm's owl. At the ITS1 region, the S. funereus sp. nov. isolates Af2 haplotype B and Af3 haplotype A were 98.77-100% identical to Sarcocystis sp. isolate Af1. The sequences from sarcocysts of naturally infected wild small mammals were 75.23-90.30% similar at ITS1 region to those of S. funereus sp. nov. Conclusion: The morphological and molecular characterizations and phylogenetic placement of S. funereus sp. nov. are presented here for the first time and support the erection of the new species.

Sarcocystis moreliae sp. nov. in the imported green tree python Morelia cf. viridis (Reptilia, Pythonidae).

Species of Sarcocystis use various vertebrates as intermediate or definitive hosts in their life cycles. One of these is snakes, whose role as intermediate hosts for these protozoans is scarce; in fact, there are six records, but only three with molecular characterization. An imported green tree python was involved in the morphological and molecular characterization (four loci) of a new species of Sarcocystis localized in skeletal muscles. Sarcocystis moreliae sp. nov. has a type 1 sarcocyst with a smooth wall and is genetically similar (at the 18S rRNA gene) to two unnamed species of Sarcocystis found in Lytorhynchus diadema from Oman and Varanus salvator macromaculatus from Malaysia, but their detailed comparison is impossible. The new species showed lower similarity to its congeners in other loci (28S rRNA, ITS1, and cox1). This is the first morphological and genetic characterization of a Sarcocystis species in snakes of the genus Morelia, particularly M. viridis, using four loci, but more data are needed to fill the knowledge gap about snakes as intermediate hosts of Sarcocystis.

White-tailed eagle (Haliaeetus albicilla) as the definitive host of Sarcocystis lutrae in the Czech Republic.

The white-tailed eagle, Haliaeetus albicilla, has been involved in the life cycle of several Sarcocystis species as the intermediate and definitive host. To date, it has been supposed that the eagle might play the role as the definitive host for S. Lutrae, and, herein, we tried to elucidate it based on morphometric and molecular analyses. One out of two eagles harbored oocysts (17.0-17.4 × 11.3-11.9 µm) and sporocysts (11.3-12.3 × 8.3-9.3 µm) in the intestinal mucosa, whose sequences at 18S rRNA, 28S rRNA, ITS1, and cox1 showed similar identity (97.64-100%) to published sequences of S. lutrae from other hosts. The presence of sporulated oocysts in the lamina propria of villi confirms that S. lutrae truly infects the white-tailed eagle. The white-tailed eagle is confirmed as the definitive host of S. lutrae in the Czech Republic.

Role of three bird species in the life cycle of two Sarcocystis spp. (Apicomplexa, Sarcocystidae) in the Czech Republic.

Birds are one of the groups involved in the development of Sarcocystis Lankester (1882), serving either as intermediate or definitive hosts. The white-tailed sea eagle Haliaeetus albicilla (Linnaeus, 1758), red kite Milvus milvus (Linnaeus, 1758) (both Accipitriformes) and common starlings Sturnus vulgaris Linnaeus, 1758 (Passeriformes) were examined to elucidate their participation in the development of Sarcocystis, as well as to determine the specific identity of the parasites based on morphological and especially molecular analyses. In 2020-2021, one white-tailed eagle, one red kite and five common starlings were parasitologically examined for the presence of Sarcocystis using flotation centrifugation coprological method and by wet mounts of intestinal mucosa scrapings and/or muscle samples. Positive samples were processed by light microscopy, histologically and followed molecularly at four genetic markers (18S rRNA, 28S rRNA, ITS1 and cox1). The white-tailed eagle harboured oocysts/sporocysts of S. arctica Gjerde et Schulze, 2014 in the intestinal mucosa, while the intestinal mucosa of the red kite and breasts and leg muscles of one common starling were positive to S. halieti Gjerde, Vikøren et Hamnes, 2018. Sequences from eagle shared 99.6-100% identity with each other and S. arctica in the red fox (V. vulpes Linnaeus, 1758) from the Czech Republic. Sequences from the common starling and red kite shared 100% identity with each other and with S. halieti in the great cormorant (P. carbo [Linnaeus, 1758]) from Lithuania and H. albicilla from Norway. The white-tailed sea eagle might act as definitive host of S. arctica, whereas the common starling and red kite represent intermediate and potential definitive hosts, respectively, for S. halieti.

Sarcocystis cristata sp. nov. (Apicomplexa, Sarcocystidae) in the imported great blue turaco Corythaeola cristata (Aves, Musophagidae).

BACKGROUND: Species of Sarcocystis are parasitic protozoa in poikilothermic and homeothermic animals. Out of the 26 valid species in birds as intermediate hosts, none has been reported in those of the order Musophagiformes, such as the great blue turaco Corythaeola cristata (Vieillot, 1816), which is a bird endemic to Central and Western Africa. The examination of great blue turacos imported from the Central Africa Republic to Czech Republic allowed the morphological and molecular characterization of a new species of Sarcocystis. METHODS: Four turacos imported from the Central Africa Republic to a private breeder (Czech Republic) underwent parasitological examination for the presence of sarcocysts through wet mounts of breast, heart and leg muscles. Found parasites were molecularly and histologically studied by four loci (18S rRNA, 28S rRNA, ITS1 and cox1) and haematoxylin and eosin staining, respectively. RESULTS: Three out of four examined birds harboured numerous sarcocysts in the breast and leg muscles. No macroscopic lesions where observed. Sarcocysts were microscopic, elongate and ribbon-shaped with a wall characterised by the presence of finger-shaped villar protrusions and filled with numerous elongate, banana-shaped bradyzoites, 11.87-14.84 × 2.05-2.92 µm in size. The new species was most closely related to Sarcocystis albifronsi, Sarcocystis anasi, Sarcocystis atraii, Sarcocystis chloropusae, Sarcocystis rileyi, Sarcocystis wenzeli and Sarcocystis sp. isolate from chicken in the four loci. CONCLUSIONS: To our knowledge, this is the first species of Sarcocystis found in a musophagiform bird worldwide. Genetically, S. cristata sp. nov. represents a distinct species. Phylogenetic analyses are useful for predicting potential definitive hosts of the new Sarcocystis species.

Molecular Identification of Sarcocystis sp. (Apicomplexa, Sarcocystidae) in Offspring of Tengmalm's Owls, Aegolius funereus (Aves, Strigidae).

Background: Birds act as intermediate or definitive hosts of cyst-forming coccidia parasites of the genus Sarcocystis Lankester, 1882. However, the spectrum of species of Sarcocystis in birds and the role of the latter in the transmission of coccidia are still incomplete for many avian species, including the Tengmalm's owl Aegolius funereus (Linnaeus, 1758). During the research on Tengmalm's owls in Finland, some fledglings were found dead and subsequently parasitologically examined. Therefore, this study is focused on the morphological and molecular description of a Sarcocystis species found in the intestine of the Tengmalm's owl and its possible role as a definitive host. Methods: Eleven fledgling owls in the Kauhava region of west-central Finland were found dead and subsequently were submitted for necropsy and parasitologically examined through the flotation-centrifugation coprological technique for the presence of oocysts/sporocysts of the genus Sarcocystis by light microscopy. Wet mounts were used for the examination of muscle samples (breast, legs, and heart). Polymerase chain reaction (PCR) and nested-PCR were carried out using primers for 18S rRNA, 28S rRNA, ITS1 region, and CO1 genes. Results: All 11 examined owls were parasitized by numerous sporocysts and oocysts in the intestinal mucosa scrapings (prevalence, 100%). Sporulated oocysts and sporocysts measured 16.34-16.96 × 11.47-12.09 µm and 11.85-13.52 × 7.77-9.25 µm, respectively. The skeletal and heart muscles were negative for sarcocysts. Sarcocystis sp. ex Aegolius funereus (hereafter Sarcocystis sp. Af) is closely related to Sarcocystis strixi in the barred owl (Strix varia Barton, 1799) from the USA and Sarcocystis sp. isolate 5 in the European shrew (Sorex araneus Linnaeus, 1758) from the Czech Republic. Phylogenetic analysis allowed determining the relationship of the herein reported Sarcocystis sp. with its congeners. Conclusions: This work provided the first and most comprehensive record on Sarcocystis from owls obtained in Finland, thus highlighting the importance of molecular data in species identification.

Molecular identification of Sarcocystis lutrae (Apicomplexa: Sarcocystidae) from the raccoon dog, Nyctereutes procyonoides, and the common raccoon, Procyon lotor, in the Czech Republic.

BACKGROUND: Apicomplexan parasites of the genus Sarcocystis have an obligate two-host life-cycle and comprise about 200 species, which infect different cold- and warm-blooded hosts, including humans. Recently, morphological and molecular studies of sarcocysts in broadly spread carnivore hosts have been on the rise. The description of muscular tissues infection by Sarcocystis in the raccoon dog and the common raccoon from the Czech Republic is herein presented. METHODS: During January-August 2019, 15 raccoon dogs and 1 common raccoon were examined from 5 districts (Ceská Lípa, Liberec, Mladá Boleslav, Trutnov and Ústí nad Labem) of the Czech Republic. Muscle parts (diaphragm, forearm, hind-limb, tongue and heart) were examined in wet preparations under compression by light microscopy. After finding Sarcocystis sp., morphological characteristics and molecular analyses of 18S rRNA, 28S rRNA, ITS1 and cox1 loci were used to identify the species. RESULTS: Sarcocysts were detected and identified in 1 out of 15 raccoon dogs and in the single common raccoon. Preferential infection sites were diaphragm and tongue, followed by forearm and hind limb. To our knowledge, this is the first identification of microscopic sarcocysts by multi-locus genetic analysis from both host species. Molecular analyses revealed 100% similarity at 18S rRNA, 28S rRNA, and cox1 genes with S. lutrae for both hosts and 98-100% identity at the ITS1 region of the isolate from the common raccoon. CONCLUSIONS: Both widely distributed non-indigenous wild carnivores represent new intermediate host records for S. lutrae and the first report of this parasite in a member of the family Procyonidae, but still with an unknown natural definitive host. Molecular data revealed that this parasite species appears more closely related to the Sarcocystis spp. using raptorial birds as definitive hosts. Therefore, further studies aimed at its identification, including the complete life-cycle, remain necessary.

Molecular identification of Sarcocystis lutrae in the European otter (Lutra lutra) and the European badger (Meles meles) from the Czech Republic.

Muscular sarcosporidial infections by Sarcocystis lutrae (Apicomplexa: Sarcocystidae) from the otter (Lutra lutra) and badger (Meles meles) (Carnivora: Mustelidae) were found in the Czech Republic. As part of a diversity evaluation of Sarcocystis in wild carnivores during 2016-2017, samples of diaphragm, tongue and hind-limb muscles were collected from nine districts, examined by compression and characterized molecularly. Cyst walls were thin, with no visible protrusions, and histological sections of infected muscle tissue showed no host responses. Fourteen of 17 badgers (82% prevalence) and one otter (100% prevalence) were positive for sarcocysts. Sequence analyses at four loci (18S rRNA, 28S rRNA, ITS1 and cox1) confirmed the identity as S. lutrae. This is also the first report of a co-infection with muscular sarcocystosis and Trichinella in badger. The finding of Trichinella is important from the zoonotic point of view, since badgers are used for meat consumption. Similar and future monitoring of both parasitic taxa are needed.

Morphological and molecular identification of Sarcocystis arctica sarcocysts in three red foxes (Vulpes vulpes) from the Czech Republic.

Muscular sarcocystosis by Sarcocystis arctica was found for the first time in the Czech Republic, in different muscles of red fox (Vulpes vulpes). Cysts were slim, elongated, thread-like, whitish, 1-7mm long, and 206-270µm wide; bradyzoites were 7.9×2.7µm in unstained wet mounts and 9.2×2.9µm in cyst Giemsa-stained smears. The cyst wall was thin, with short villi-like protrusions, and no host response was observed in the histological sections. Examination of the distribution and intensity of sarcocysts in 17 different muscle groups revealed that the highest intensity was in the cranial tibial muscle (>15 cysts in compressoria), followed by the diaphragm, forearm, and other groups (with intensities of 3-15 cysts in compressoria). Sarcocysts were detected in 3 out of 86 foxes. Genetic characterization at 18S rRNA, 28S rRNA, ITS1 and cox1, consistently showed that the species was identical with S. arctica. Interestingly, this protozoan was also detected as a co-infection in 3 foxes with the nematode Trichinella spp. for the first time.

Cryptosporidium infections of ring-necked pheasants (Phasianus colchicus) from an intensive artificial breeding programme in the Czech Republic.

From July to November 2012, preliminary coprological examinations were carried out on 85 pooled faecal samples of different aged ring-necked pheasants (Phasianus colchicus) (hatches from May until July) from an intensive artificial breeding programme in the Czech Republic. Cryptosporidium oocysts were detected in 12 samples (14.1 %) of ages >12 weeks (August-September). These results were supported by findings of Cryptosporidium baileyi and Cryptosporidium meleagridis oocysts in intestinal, or cloacal contents, and/or the bursa of Fabricius in 9 from 36 examined dead pheasants (prevalence 25 %). We describe in detail the various age groups of pheasants after hatching and present graphically the overall results of coprological examinations, showing pathways of infection of C. baileyi and C. meleagridis during the full rearing seasons of 2013 and 2014. We found very similar mean proportions of Cryptosporidium-positive samples over the entire 2013 period in pheasantry (173 pooled samples tested, 25 positive, 14.5 %) and 2014 (238 samples tested, 43 positive, 18.1 %). All tests were verified as being Cryptosporidium positive in 9 from 219 (prevalence 4.1 %) and 4 from 168 (prevalence 2.4 %) post-mortem examinations. Significantly, C. baileyi was found more frequently in faeces, with positivities ranging from 11.1 to 100 % (4->16-week-old pheasants). Oocysts of C. meleagridis were detected at ages 6->15 weeks ranging from 7.1 to 100 % in faeces during the rearing seasons. The burdens of C. baileyi (7 of 14 and 10 of 16) and C. meleagridis (5 of 14 and 7 of 16) for each year, in monitored brooder houses, flight pens and spread across all open areas were recorded. Oocysts of C. baileyi and C. meleagridis obtained from this study, and Cryptosporidium galli (obtained in another aviary from 36-week-old pheasants), were sequenced, and we characterized the highly variable 60-kDa glycoprotein gene of C. meleagridis. These results highlight the real risk of transmission of Cryptosporidium to susceptible wild birds and other potential hosts after termination of rearing and release.

Stichorchis subtriquetrus (Digenea: Paramphistomatidae) from Eurasian beaver (Castor fiber) in the Czech Republic.

Between March 2012 and April 2014, we performed post-mortem parasitological examinations of 11 Eurasian beavers (Castor fiber Linnaeus, 1758) from the basins of four main rivers (Dyje, Labe, Morava, Vltava) in the Czech Republic. The cause of death of five adult animals was unknown, three adult animals died after being hit by cars, while one young and one adult as a result of serious injuries and one juvenile male drowned. The trematode Stichorchis subtriquetrus (Rudolphi, 1814) Lühe, 1909 was only found in the caecum body and caecum apex of nine beavers (82%), with no significant differences in parasite intensity among beavers. The highest number of trematodes (271) occurred in an adult female in July 2013; while a range of 1-57 individuals were found in other positive beavers. S. subtriquetrus size in both parts of the caecum was 11.0-17.0 × 5.5-8.0 mm (mean 14.3 × 6.9 mm). Results demonstrated that for the optimal detection of eggs, it was necessary to examine at least 10 g of faeces with a new modified method of sedimentation. The size range of 30 eggs was 157.1-182.5 × 99.3-109.8 µm (mean 168.0 × 104.4 µm). There were no differences in prevalence and seasonal occurrence of S. subtriquetrus between male and female beavers. We did not find any other intestinal endoparasites or tissue parasites (Sarcocystis spp., Trichinella spp.).

First finding of spontaneous infections with Cryptosporidium baileyi and C. meleagridis in the red-legged partridge Alectoris rufa from an aviary in the Czech Republic.

This paper represents the first report of spontaneous infection with Cryptosporidium baileyi and Cryptosporidium meleagridis in the red-legged partridge (Alectoris rufa), as well as the percentage of positive samples and age-associated dynamics of cryptosporidial infections in an aviary in the Czech Republic. The entire infection process was monitored over two semesters (July-December 2012 and 2013) until release of birds for hunting purposes. Coprological examination of 663 pooled fecal samples and 89 post-mortem examinations of red-legged partridges were carried out. Our results indicated that infections with C. baileyi only occurred in 5-7 week-old birds during 2013 (percentage of positivity, 1%) and those with C. meleagridis in 18-22 week (17%) and 17-21 week-old birds (24%) during 2012 and 2013, respectively. Molecular characterization of isolates of C. baileyi and C. meleagridis heat shock protein 70 and actin genes were analyzed in order to support our coprological results. DNA sequence analysis of the 60kDa glycoprotein gene was used to subtype C. meleagridis. Our findings extend the host range for C. baileyi.

Description of a new species of Eimeria Schneider, 1875 (Apicomplexa: Eimeriidae) from the western Derby eland Taurotragus derbianus derbianus Gray (Artiodactyla: Bovidae) in Senegal.

Examination of faecal samples from semi-captive western Derby elands Taurotragus derbianus derbianus Gray, in the Bandia and Fathala Reserves of Senegal, revealed the presence of oöcysts of the genus Eimeria Schneider, 1875 that we considered to represent a new species, Eimeria derbiani n. sp. The new species possesses nearly ellipsoidal oöcysts (length/width ratio 1.3) with a bi-layered wall and an average size of 27.6 × 21.5 µm. E. derbiani possesses a micropyle covered by a micropylar cap and ovoidal, single-layered sporocysts with an average size of 14.9 × 7.7 µm, each with a Stieda body. Sporozoites of E. derbiani possess a large refractile body and a nucleus. Sporulation lasted for 2 days at 23°C. The new species is differentiated from the two species parasitising Taurotragus oryx Pallas, E. canna Triffitt, 1924 and E. triffittae Yakimoff, 1934.