Diversity of Sarcocystis parasites in southeastern Baltic Sea catchment ecosystems.

Currently, research on apicomplexan Sarcocystis parasites is mainly carried out by analyzing animal carcasses. However, environmental studies would not only allow faster detection of possible sources of infection but also avoid the use of animals for investigations. Therefore, in the current study, we aimed to identify tested Sarcocystis species in sediment collected from water bodies located in the southeastern Baltic countries. A total of 99 sediment samples were collected during the summer from different types of water bodies in Estonia, Latvia, Lithuania, and Poland. Species-specific nested PCR targeting cox1 gene was used for the detection of selected Sarcocystis species (S. cruzi, S. bovifelis, S. hirsuta, S. arieticanis, S. tenella, S. capracanis, S. miescheriana, and S. bertrami) infecting livestock. The results showed a statistically lower (p < 0.05) occurrence of Sarcocystis parasites in Estonia (50%) compared to three countries, where the detection rate of Sarcocystis spp. DNA was remarkably higher, ranging from 88 to 100%. Among Sarcocystis species tested, S. cruzi (83.8%) and S. arieticanis (55.6%) using cattle and sheep as their intermediate hosts were most commonly identified. The detection rates of some of the analyzed Sarcocystis species were significantly different in southeastern Baltic countries. It is discussed that the detection rates of certain Sarcocystis species depend not only on the number of animals per 1 km2 but also on various ecological factors and farming practices that differ in the amount of contact domestic animals have with predators and the potential for animals to become infected through natural water or food sources.

First Observations of Buzzards (Buteo) as Definitive Hosts of Sarcocystis Parasites Forming Cysts in the Brain Tissues of Rodents in Lithuania.

Representatives of the genus Sarcocystis are worldwide distributed apicomplexan parasites characterised by two-host prey-predator relationships. Sarcocystis spp. produce sarcocysts in the muscles and brains of intermediate hosts and develop sporocysts in the intestines of definitive hosts. Two species, Sarcocystis glareoli and Sarcocystis microti, previously assigned to the genus Frenkelia, form cysts in the brains of rodents and are transmitted through the common buzzard (Buteo buteo). In our study, brain samples of 694 small mammals caught in different regions of Lithuania were examined for Sarcocystis spp. Additionally, 10 B. buteo and two rough-legged buzzards (Buteo lagopus) were tested for sporocysts of the analysed parasites. Sarcocystis species were identified based on 28S rRNA sequence comparison. Of the eleven species of small mammals tested, Sarcocystis parasites were observed only in the bank vole (Clethrionomys glareolus). Cysts of S. glareoli were detected in 34 out of 374 C. glareolus (9.1%, 95% CI = 6.4-12.5%). Molecular investigation showed the presence of only S. glareoli in the intestines of 50% of B. buteo. Furthermore, two species, Sarcocystis sp. Rod3 and Sarcocystis sp. Rod4, were confirmed in B. lagopus. Our results demonstrate the need for further studies on Sarcocystis cycling between rodents and birds.

Detection of Three Sarcocystis Species (Apicomplexa) in Blood Samples of the Bank Vole and Yellow-Necked Mouse from Lithuania.

The genus Sarcocystis is an abundant group of Apicomplexa parasites found in mammals, birds, and reptiles. These parasites are characterised by the formation of sarcocysts in the muscles of intermediate hosts and the development of sporocysts in the intestines of definitive hosts. The identification of Sarcocystis spp. is usually carried out in carcasses of animals, while there is a lack of studies on the detection of Sarcocystis species in blood samples. In the current study, blood samples of 214 yellow-necked mice (Apodemus flavicollis) and 143 bank voles (Clethrionomys glareolus) from Lithuania were examined for Sarcocystis. The molecular identification of Sarcocystis was carried out using nested PCR of cox1 and 28S rRNA and subsequent sequencing. Sarcocystis spp. were statistically (p < 0.01) more frequently detected in the bank vole (6.3%) than in yellow-necked mice (0.9%). The analysed parasites were observed in four different habitats, such as mature deciduous forest, bog, natural meadow, and arable land. Three species, Sarcocystis funereus, Sarcocystis myodes, and Sarcocystis cf. glareoli were confirmed in the bank vole, whereas only Sarcocystis myodes were found in yellow-necked mice. The obtained results are important in the development of molecular identification of Sarcocystis parasites in live animals.

The Distribution of Sarcocsytis Species Described by Ungulates-Canids Life Cycle in Intestines of Small Predators of the Family Mustelidae.

PURPOSE: Using molecular techniques, we have previously shown that carnivorous mammals of the family Mustelidae might be common definitive hosts for various protozoan Sarcocystis species. In the present study we aimed to unravel whether Sarcocystis species using ungulates as intermediate hosts and canids or felids as definitive hosts can be found in intestine of mustelids. METHODS: Small intestine samples of 93 individual mustelids of five different species from Lithuania were examined. Sarcocystis species were identified based on species-specific PCR and subsequent cox1 sequencing. RESULTS: Six Sarcocystis species (S. arieticanis, S. bertrami, S. capracanis, S. capreolicanis, S. linearis and S. morae) defined by ungulate-canid life cycle were detected for the first time in small intestines of mustelids. By contrast, the prevalence of Sarcocystis characterised by ungulate-felid life cycle was low (3.2%). Overall, 76% of the examined animals were positive for at least one of the studied Sarcocystis species. Four species, S. arieticanis, S. bertrami, S. capracanis and S. morae were most commonly found, with the detection rate of about 40%. CONCLUSIONS: The current finding, in addition to our previous studies, suggests that mustelids play an important role in the spread of various Sarcocystis species.

Identification of Sarcocystis and Trichinella Species in Muscles of Gray Wolf (Canis lupus) from Lithuania.

Apicomplexan Sarcocystis and Trichinella nematodes are food-borne parasites whose life cycle is carried-out in various wildlife and domestic animals. The gray wolf (Canis lupus) is an apex predator acting as an ecosystem engineer. This study aimed to identify the species of Sarcocystis and Trichinella found in the muscles of gray wolves in Lithuania. During the 2017-2022 period, diaphragm, heart, and hind leg samples of 15 animals were examined. Microscopical analysis showed the presence of two types of Sarcocystis parasites in 26.7% of the analyzed muscle samples. Based on the sequencing of five loci, nuclear 18S rDNA, 28S rDNA, ITS1, mitochondrial cox1, and apicoplast rpoB, S. arctica, and S. svanai were identified. The current work presents the first report of S. svanai in gray wolf. Phylogenetically, S. svanai clustered together with S. lutrae, infecting various carnivorans, and S. arctica was most closely related to S. felis from domestic cats. Trichinella spp. were found in 12 gray wolves (80%). For the first time, Trichinella species were molecularly identified in gray wolves from Lithuania. Trichinella britovi was confirmed in all of the isolated Trichinella larvae using a multiplex PCR. Gray wolves in Lithuania may serve as a major source of zoonotic pathogens due to the presence of these parasites.

Low Genetic Variability of the Tundra Vole in Lithuania.

The distribution and spread of the tundra vole (Alexandromys oeconomus) in Lithuania have been documented over the last 70 years, but the genetic diversity of the species has not been studied. In this study, we examined A. oeconomus trapped in three sites in northern and western Lithuania using mtDNA sequence analysis of the cytb and control region. The western and northern sites are separated by anthropogenic landscape barriers. The western site is subject to regular spring flooding. Phylogenetic analyses of the studied individuals placed them in the Central European phylogroup, suggesting that Lithuanian A. oeconomus originated from northeastern Poland. In Lithuania, the genetic diversity of A. oeconomus at both mtDNA loci was relatively low (Hd < 0.6, π < 0.002) compared to that found in other European samples (Hd = 0.833-0.958; π = 0.00402-0.01552). Individuals analyzed in Lithuania were genetically different from samples collected in Poland and Northern Europe (ΦST > 0.15, p < 0.05). The genetic divergence between the western and northern samples of A. oeconomus in Lithuania, together with the low genetic variability among the voles studied, provides new insights into the phylogeography of the species and the influence of barriers on the colonization of the country.

First report of Sarcocystis halieti (Apicomplexa) in bearded vulture (Gypaetus barbatus).

At least three Sarcocystis species (S. falcatula, S. halieti and S. wobeseri-like) have been detected infecting raptorial birds. By histopathology and PCR-sequencing of the ITS1 marker, S. halieti was detected in a bearded vulture (Gypaetus barbatus) and a black kite (Milvus migrans) from the Catalonia region in North Spain. The 241 bp-long sequences obtained from the Sarcocystis organisms detected in both raptors showed 97.5-99.6% and 97.9-100% similarity with those of previously identified S. halieti; also, the phylogenetic trees generated placed the identified sequences together with other sequences of S. halieti available in GenBank. In sum, the description of the bearded vulture as a new intermediate host for S. halieti adds new insights on the complex epidemiology of the genus involving avian hosts.

Trends of Eurasian Perch (Perca fluviatilis) mtDNA ATP6 Region Genetic Diversity within the Hydro-Systems of the Eastern Part of the Baltic Sea in the Anthropocene.

The intraspecific genetic diversity of freshwater fish inhabiting hydro-systems of the macrogeographic area spreading from the Black to Baltic Seas requires comprehensive investigation from fundamental and practical perspectives. The current study focused on the involvement of the mtDNA ATP6 region in the adaptability and microevolution of Perca fluviatilis within phylogeographic and anthropogenic contexts. We sequenced a 627 bp fragment encompassing the ATP6 region and used it for genetic analysis of 193 perch caught in Latvia, Lithuania, Belarus, and Ukraine, representing natural and anthropogenically impacted populations. We evaluated patterns of intraspecific genetic diversity in the ATP6 region and phylogeographic trends within the studied area compared with previously established D-loop trends. Evaluation of ATP6 coding sequence variability revealed that among 13 newly detected haplotypes, only two were caused by non-synonymous substitutions of amino acids of the protein. PCoA revealed three genetic groups (I-III) based on the ATP6 region that encompassed four previously described genetic groups established based on the mtDNA D-loop. The two mtDNA regions (D-loop and ATP6) have microevolved at least partially independently. Prolonged anthropogenic impacts may generate new point mutations at the ATP6 locus, but this phenomenon could be mainly concealed by natural selection and reparation processes.

Molecular differentiation of Sarcocystis miescheriana and Sarcocystis suihominis using a new multiplex PCR targeting the mtDNA cox1 gene in wild boars in southern Italy.

The increase of wild boar populations density and their meat consumption across Europe could expose humans to a plethora of foodborne diseases as sarcocystosis, caused by the zoonotic protozoan Sarcocystis suihominis. Humans become infected by eating raw or undercooked pig (Sus scrofa domesticus) containing S. suihominis sarcocysts. Despite this, to date very few data are available on the risk of infection by this parasite to wild boar (Sus scrofa) meat consumers. Thus, the present study aimed to assess the occurrence of Sarcocystis spp. in wild boars from southern Italy, applying both histology and a new multiplex PCR assay targeting the cox1 gene. Between 2019 and 2020, 997 muscle tissues (i.e., n = 269 oesophagus, n = 277 diaphragms, n = 298 hearts, n = 153 tongues) from 311 wild boars were collected and screened by a combined histological and molecular approach. Overall, 251 (80.7%) animals tested were positive for Sarcocystis spp., and S. miescheriana whose definitive hosts are canids, was the only molecularly identified species. A statistically significant difference (p < 0.05) in the prevalence of Sarcocystis infection was found according to the wild boar age and muscle tissue. Findings outlined the low zoonotic potential of infection to humans via wild boar meat consumption in Italy and the importance of the application of new molecular methods in distinguishing different Sarcocystis species.

Sarcocystis spp. Macrocysts Infection in Wildfowl Species in Eastern Baltic Region: Trends in Prevalence in 2011-2022.

Wildfowl meat infected with S. rileyi macrocysts is not suitable for human consumption. Ducks are among the main game birds in Europe, and S. rileyi infections cause significant economic losses. In 2011-2022, a total of 2649 anseriforms collected in Lithuania and 619 Mallards (Anas platyrhynchos) hunted in the Kaliningrad region of Russia, Belarus, and Latvia were tested for macrocysts. In Lithuania, macrocysts were detected in 206 of 2362 Mallards (8.7%) and in two of 88 (2.3%) Eurasian Teals (Anas crecca). The prevalence of macrocysts in the other three countries, Belarus (5.9%), Russia (5.0%), and Latvia (3.1%), was similar. For species identification, macrocysts isolated from 37 Mallards (21 from Lithuania, 8 from Russia, 6 from Belarus, and 2 from Latvia) were subjected to sequencing of the ITS1 region. Based on DNA analysis, S. rileyi was confirmed in all tested birds. By comparing the infection rates of macrocysts in Mallards in Lithuania, significant differences were observed in different years (p = 0.036), and a significantly higher prevalence of infection was established in November-December than in September-October (p = 0.028). Given the amount of data per decade on the prevalence of S. rileyi, awareness of infection needs to be increased.

Sarcocystis Species Richness in Sheep and Goats from Lithuania.

Contradictory data is available on the intermediate host specificity of Sarcocystis spp. in farm animals. Therefore, the current work aimed at molecularly testing samples of sheep and goats reared in Lithuania to identify Sarcocystis species described in other intermediate hosts but suspected to be non-canonical parasites to these small ruminants. For this purpose, muscle samples from 47 domestic sheep and nine goats were examined. Sarcocystis species were identified using direct and nested PCR targeting cox1 and sequencing of positive amplified products. Along with the detection of the canonical Sarcocystis spp. in their respective intermediate hosts, the DNA of S. capracanis and S. morae was detected in sheep, although these species were previously thought to be specific to goats and deer, respectively. In addition, DNA from S. arieticanis and S. tenella was found in goats, even though these two species were believed to be sheep-specific. Notably, under light microscopy, only sarcocysts of S. capracanis specific to goats were observed. Thus, future research on the life cycle and host-specificity of Sarcocystis spp. examined is warranted.

Dynamics of Polyamines, Proline, and Ethylene Metabolism under Increasing Cold in Winter Oilseed Rape.

Cold stress is among the most important environmental factors reducing the yield of crops. The present study aimed to investigate the impact of increasing cold stress conditions on winter oilseed rape polyamines, proline, and ethylene metabolism in acclimated and non-acclimated winter oilseed rape. This study was carried out under controlled conditions in the laboratory. The winter oilseed rape hybrid 'Visby' was used in the experiment. Acclimated and non-acclimated plants were subjected to a two-day-long increasing cold (from -1 °C to -3 °C) treatment. HPTLC, RT-qPCR, spectral analysis, and gas chromatography methods were used to analyse the levels of polyamines, gene expression, proline, and ethylene, respectively. This study showed a decrease in putrescine, spermidine, and spermine content during cold acclimation and a decrease in putrescine and spermidine levels at sub-zero temperatures. There were intensive changes in ADC2 gene expression, proline, and ethylene levels in non-acclimated plants: a substantial increase after exposure to -1 °C temperature and a sharp decrease after exposure to -3 °C temperature. The changes in these parameters were lower or absent in acclimated plants. The phenomena observed in this study add new insights to the knowledge about the plant stress response and suggest questions to be answered in the future.

Protozoan Parasites of Sarcocystis spp. in Rodents from Commercial Orchards.

Small mammals are an important group of wildlife that can transmit pathogens to humans and animals. There is a lack of comprehensive studies on the protozoan parasites of the genus Sarcocystis in agricultural areas. The aim of the current research was to evaluate the prevalence of Sarcocystis spp., and to identify the parasite species found in the skeletal muscles of rodents and insectivores from commercial orchards. A total of 679 muscle samples from small mammals, mainly rodents (n = 674), belonging to eight species were examined. Muscle samples were pooled into groups, then digested, and the presence of the Sarcocystis species was confirmed by molecular methods. The examined parasites were determined in five rodent species, Apodemus agrarius, A. flavicollis, Clethrionomys glareolus, Microtus arvalis, and M. oeconomus. The prevalence of Sarcocystis spp. was low: 2.23% in voles and 0.79% in mice. Based on a sequence comparison of cox1 and 28S rDNA, four species were identified: S. myodes, Sarcocystis cf. strixi, Sarcocystis sp. Rod1, and Sarcocystis sp. Rod2. This is the first report of S. myodes in A. agrarius, A. flavicollis, and M. arvalis. The identified species were most closely related to Sarcocystis spp., and were transmitted by predatory mammals and birds. Future studies are needed to describe the species morphologically, as well as to define the host spectrum and to evaluate their possible pathogenicity.

Molecular Confirmation of Accipiter Birds of Prey as Definitive Hosts of Numerous Sarcocystis Species, including Sarcocystis sp., Closely Related to Pathogenic S. calchasi.

The present study aimed to test intestinal scrapings of the Northern Goshawk (Accipiter gentilis) and the Eurasian Sparrowhawk (Accipiter nisus) from Lithuania for S. calchasi and other Sarcocystis species characterised by bird-bird life cycles. The protozoan parasite Sarcocystis calchasi can cause respiratory and neurological diseases in a variety of birds; however, the distribution of this parasite is not well-examined. Sarcocystis species were identified with nested PCR and sequencing of the partial ITS1 region. Sporocysts and/or sporulated oocysts of Sarcocystis spp. were observed in 16 (100%) Northern Goshawks and 9 (56.3%) Eurasian Sparrowhawks. Four species, S. columbae, S. halieti, S. turdusi, and S. wobeseri, were confirmed in the Eurasian Sparrowhawk. Apart from the latter four species, S. calchasi, S. cornixi, S. kutkienae, and S. lari were established in the Northern Goshawk. A higher prevalence of Sarcocystis spp. and species richness in Northern Goshawks is associated with the differences in the diet of two examined Accipiter species. This study is the first report of S. calchasi in Lithuania. Furthermore, the genetically distinct species Sarcocystis spp. 23LTAcc, which is most closely related to S. calchasi, was found in three Northern Goshawks.

Molecular Identification of Sarcocystis rileyi and Sarcocystis sp. (Closely Related to Sarcocystis wenzeli) in Intestines of Mustelids from Lithuania.

The genus Sarcocystis is a group of numerous protozoan parasites having a two-host life cycle. Based on laboratory experiments and/or phylogenetic analysis results it was shown that seven Sarcocystis spp. producing sarcocsyts in bird tissues are transmitted via predatory placental mammals. To date the role of small mammals of the family Mustelidae in the distribution of avian Sarcocystis spp. have not been studied. During the current investigation, intestinal mucosa scrapings of 115 mustelids belonging to five species were tested for S. albifronsi, S. anasi, S. rileyi, and S. wenzeli infecting anseriforms and chickens. Microscopically, free sporocysts, sporulating oocysts, and loose oocysts were found in 61 samples (53.0%). Using nested PCR targeting the ITS1 region and sequencing, S. rileyi was confirmed in eight American minks, two European polecats and single European badger. Sarcocystis sp. was identified in one American mink and one European pine marten. Based on the partial ITS1 region this parasite showed that 100% identity to pathogenic Sarcocystis sp. caused a fatal infection in backyard chickens from Brazil. Phylogenetically, the Sarcocystis sp. identified in our study was most closely related to S. wenzeli parasitising domestic fowl (Gallus domesticus).

Molecular identification of Sarcocystis species in sika deer (Cervus nippon) of free-ranging populations in Germany and Austria.

In this study, for the first time, Sarcocystis species were identified molecularly in sika deer (Cervus nippon) that form free-ranging populations in several countries of Europe. Diaphragm muscle samples from 151 deer from 10 populations in Germany and Austria were examined for sarcocysts. By one-gram methylene-blue staining, sarcocysts were recorded in samples of 114 animals (75.5%) which originated from all populations. Sarcocysts were more often (p < 0.0001) recorded in yearling and adult deer than in calves. Infection intensity was generally low with ~ 70% of the sarcocyst positive deer harbouring ≤ 10 sarcocysts per 1-gram diaphragm muscle. Based on cox1 sequence comparison, 10 species of Sarcocystis, all previously reported parasitizing cervids, were identified: S. elongata, S. entzerothi, S. hjorti, S. iberica, S. japonica, S. linearis, S. morae, S. pilosa, S. silva and S. truncata. The prevailing S. hjorti was detected in sika deer of all 10 populations. The identification in sika deer of S. hjorti, S. iberica, S. elongata, S. linearis, S. morae and S. silva constitutes new host records. With the additional species records of this study, the highest number of Sarcocystis species, at least 16, was identified in this host.

Molecular Identification of Sarcocystis Species in Sheep from Lithuania.

Data on the distribution of different Sarcocystis species in various muscles of sheep are scarce. In the present study, 190 diaphragm, oesophagus, and heart muscle samples of 69 sheep raised in Lithuania were examined for the presence of Sarcocystis spp. Under a light microscope, two morphological types of microcysts corresponding to S. arieticanis and S. tenella were detected. Eight and 12 sarcocysts of S. arieticanis and S. tenella, respectively, were isolated and characterised by the sequencing of a portion of cox1. The sequence comparisons revealed the highest similarity between European and Asian isolates of S. arieticanis and S. tenella obtained from domestic sheep and other wild Caprinae hosts. Based on peptic digestion, nested PCR targeting cox1, and sequencing, a 100% infection prevalence of S. arieticanis and S. tenella was observed in the 69 studied animals. The occurrence of S. tenella was significantly higher in the diaphragm than in the oesophagus (χ2 = 13.14, p < 0.001), whereas differences in the prevalence of S. arieticanis in the studied muscle types were insignificant (χ2 = 1.28, p > 0.05). Further molecularly based epidemiological studies are needed to compare the prevalence of Sarcocystis species in various muscles of sheep raised in different geographic regions.

Molecular Identification of Parasitic Protozoa Sarcocystis in Water Samples.

Sarcocystis parasites are among the most common parasitic protozoa in farm animals. So far, the diversity of these parasites has been mainly studied in animal carcasses by morphological or molecular methods. Research on parasitic protozoa in environmental samples is scarce due to the lack of an appropriate methodology and low concentrations of parasites. For these reasons, there is a paucity of validated methods for Sarcocystis identification from environmental samples. Therefore, the present study aims to investigate various molecular methods for Sarcocystis parasite identification in water samples. In the present study, the sample volume, sporocysts isolation, and various conventional PCR were evaluated, and species-specific primers for the identification of different Sarcocystis species have been developed. Of the methods studied, based on data the most appropriate method for the identification of analyzed Sarcocystis spp. in water bodies is nested PCR, using species-specific primers targeting the cox1 gene. Sarcocystis DNA was detected in 111 out of 114 (97.4%) samples. This paper represents the first identification of S. bovifelis, S. cruzi, S. hirsuta, S. arieticanis, S. tenella, S. capracanis, S. bertrami, and S. miescheriana by PCR and sequencing in environmental water samples. Our pilot study is useful in developing techniques for the identification of Sarcocystis species from water samples.

Molecular characterization of Sarcocystis spp. in seabirds from southern Brazil.

Sarcocystis spp. are cyst forming apicomplexan parasites that infect many vertebrates including birds. Sarcocystis spp. infection was investigated in tissue samples (pectoral muscles, heart, and brain) of 47 dead seabirds collected from the coastline of Santa Catarina State SC - Brazil, between August 2019 and March 2020. A portion of each tissue was fixed in 10% buffered formalin for histopathologic analysis while DNA was extracted from another portion and screened using nested-PCR targeting ITS1. Based on molecular analysis, Sarcocystis spp. were identified in 15/47 (31.9%) seabirds of five species, kelp gull (Larus dominicanus), manx shearwater (Puffinus puffinus), neotropic cormorant (Phalacrocorax brasilianus), brown booby (Sula leucogaster) and great skua (Stercorarius skua). Microscopically visible sarcocysts were observed only in the pectoral muscle of four seabirds 8.5% (4/47), while in one brown booby, sarcocysts were seen in both pectoral and cardiac muscles. Two types of sarcocysts, thin walled (≤1 µm) and thick-walled (≥ 2 µm) were identified. Based on ITS1 sequence comparison, S. halieti, S. falcatula and three not yet described Sarcocystis spp. were detected. Phylogenetically, S. falcatula isolates were classified as two distinct clusters. This is the first confirmation of S. halieti in seabird's species in South America and S. falcatula in birds of the order Charadriiformes. Further molecular studies are needed to understand the epidemiology of the Sarcocystis spp. infection and its impact on the health of seabirds.

A novel RFLP method for identification of morphologically similar avian Sarcocystis species.

Protozoans of genus Sarcocystis are widespread parasites infecting mammals, birds, and reptiles. Morphology of their sarcocysts is an important criterion for species identification. However, as more and more morphologically similar Sarcocystis species are being found and described, additional methods for their routine diagnostics are needed. We investigated restriction fragment length polymorphism (RFLP) as potential alternative to sequencing data analysis for the identification of Sarcocystis species using birds as an intermediate host. The internal transcribed spacer 1 (ITS1) region sequences of seventeen Sarcocystis species (S. albifronsi, S. anasi, S. calchasi, S. columbae, S. cornixi, S. corvusi, S. cristata, S. halieti, S. falcatula, S. fulicae, S. kutkienae, S. lari, S. lindsayi, S. rileyi, S. turdusi, S.wenzeli, and S. wobeseri) of interest were analysed and five best-fitting endonucleases generating most informative restriction fragments were selected for routine testing. In general, RFLP analyses are always inconclusive as they target very short DNA sequences. However, it can be an irreplaceable technique when fast and cheap identification and discrimination of known species are required, which was our main goal and preliminary results indicate that RFLP could be successfully used when identifying closely related avian Sarcocystis species with just two nucleases.