Molecular Identification of Parasitic Protozoa Sarcocystis in Water Samples
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Sample Collection
2.2. Isolation of Sporocysts and Genomic DNA Extraction
2.3. Identification of Sarcocystis Species Using PCR Procedures
2.4. Statistical Analysis
3. Results
3.1. Selection of PCR Conditions
3.2. Identification of Sarcocystis spp. from in Water Bodies
3.3. Summary of Molecular-Based Sarcocystis Identification from Water Samples
3.4. Sarcocystis spp. Occurrence Rates in the Analyzed Water Samples
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Species | Primers | Ta, °C | Extension Time, s | Product Size (bp) | Reference | ||
---|---|---|---|---|---|---|---|
Name | Orientation | Sequence (5′–3′) | |||||
Primers for Direct and Nested PCR | |||||||
Sarcocystis spp. | SF1 | Forward | ATGGCGTACAACAATCATAAAGAA | [15] | |||
SR8D | Reverse | CATTGCCCATDACTACGCC | 55 | 70 | 1072 | [15] | |
SR9 | Reverse | ATATCCATACCRCCATTGCCCAT | 60 | 70 | 1085 | [16] | |
SR12H | Reverse | AAATACCTTGGTGCCCGTAG | 56 | 60 | 952 | [17] | |
S. bovifelis | GaBfEF | Forward | ATCAACTTCCTAGGTACAGCGGTATT | 56 | 45 | 523 | [11] |
GaBfER | Reverse | CCACATCATTGGTGCTTAGTCTAGTA | [11] | ||||
S. cruzi | GaCrEF | Forward | GCTATGTATCTACTTACGGCAGGTATC | 56 | 45 | 608 | [11] |
GaCrER | Reverse | GAATATAATGGCCCAGGTAAATAATG | [11] | ||||
S. hirsuta | GaHiEF | Forward | GTTGTGCGGTATGAATTATCAACCT | 56 | 45 | 513 | [11] |
GaHiER | Reverse | GGTAAGAACTGGAATGGTTAATATCAG | [11] | ||||
S. arieticanis | GsSariF | Forward | TTCTTGGTATGGCTATTCTTGGACT | 65 | 45 | 586 | [18] |
GsSariR | Reverse | GATATGTCAATCCAGAGATCGGTAG | [18] | ||||
S. tenella | GsStenF | Forward | TACTCGGAGCGGTGAACTTCTTA | 63 | 35 | 451 | [18] |
GsStenR | Reverse | ATAGTCACGGCAGAGAAGTAGGAC | [18] | ||||
S. capracanis | GsScapF | Forward | AGCGGTAAACTTCCTGGGTACT | 63 | 35 | 467 | [18] |
GsScapR | Reverse | GCCTATCCAGTTGAATATCTTGGT | [18] | ||||
Primers for multiplex-nested PCR | |||||||
1st round | |||||||
Sarcocystis spp. | SF1 | Forward | ATGGCGTACAACAATCATAAAGAA | [15] | |||
S. arieticanis, S. cruzi | SsunR2 | Reverse | GTGCCTCCCAGGCTGAAYAG | 56 | 70 | 1055 | [19] |
S. bertrami, S. tenella, S. capracanis | SsunR1 | Reverse | GTACCGCCCAGGCTGAAYAG | 56 | 70 | 1055 | [18] |
S. bovifelis, S. hirsuta | SkatR | Reverse | CAGGCTGAACAGHABTACGA | 56 | 70 | 1042 | [19] |
S. miescheriana | SmieF | Forward | ACGCTGTATGCACCACTGAG | 56 | 45 | 658 | Present study |
SmieR | Reverse | CTGAACAGCGCTACAAATGC | |||||
2nd round | |||||||
S. bertrami | GsSberF | Forward | GTATGAACTGTCAACGGATGGAGTA | 65 | 35 | 482 | Present study |
GsSberR | Reverse | TCAACATTAGCGAGGTAAATACTATC | |||||
S. miescheriana | GsSmieF | Forward | GTTCCTCGGTATTAGCAGCGTACT | 65 | 35 | 509 | Present study |
GsSmieR | Reverse | AGTTAAATATTTTAGTGCCCGTTGGA | |||||
Primers for semi-nested PCR | |||||||
S. bovifelis | VoboF | Forward | GATCGGTATTACTGTTGCACTCATT | 58 | 45 | 701 | Present study |
VoboR | Reverse | AGGCCACATCATTGGTGCTTA | |||||
GaBfEF | Forward | ATCAACTTCCTAGGTACAGCGGTATT | 57 | 35 | 521 | [11] | |
S. tenella | VoteF1 | Forward | AGCGGTGAACTTCTTAGGAACC | 59 | 35 | 526 | Present study |
VoteR | Reverse | AATAATCCGCTGTTAACGTATGC | Present study | ||||
VoteF2 | Forward | CATTGTAATGCTCCTCGACGATA | 59 | 30 | 401 | Present study | |
S. capracanis | VocaF | Forward | GTAAACTTCCTGGGTACTGTGCTGT | 60 | 35 | 526 | Present study |
VocaR1 | Reverse | CCAGTAATCCGCTGTCAAGATAC | Present study | ||||
VocaR2 | Reverse | AGTACCCATCACGGTGCCTATC | 63 | 35 | 500 | Present study | |
Species-specific primers for nested PCR | |||||||
S. bovifelis | V2bo1 | Forward | AACTTCCTAGGTACAGCGGTATTCG | 60 | 40 | 556 | Present study |
V2bo2 | Reverse | TGAACAGCAGTACGAAGGCAAC | Present study | ||||
V2bo3 | Forward | ATATTTACCGGTGCCGTACTTATGTT | 60 | 30 | 410 | Present study | |
V2bo4 | Reverse | GCCACATCATTGGTGCTTAGTCT | Present study | ||||
S. cruzi | V2cr1 | Forward | TACAATGTGCTGTTTACGCTCCA | 57 | 50 | 776 | Present study |
V2cr2 | Reverse | GCAATCATGATAGTTACGGCAGA | Present study | ||||
V2cr3 | Forward | ACCATCCTGTTCTGTGGTGCTATG | 65 | 30 | 298 | Present study | |
V2cr4 | Reverse | AAACTACTTTACTGCCTACGGTACTC | Present study | ||||
S. hirsuta | V2hi5 | Forward | TATGTTGGTTCTGCCGAAGTCAT | 60 | 45 | 686 | Present study |
V2hi6 | Reverse | GGTATGGCAATCATTATGGTTACAG | Present study | ||||
V2hi7 | Forward | GCACCGTAATATTTCAGGGATGT | 60 | 30 | 299 | Present study | |
V2hi8 | Reverse | AACCTGCTTGCCGGAGTAAGTA | Present study | ||||
S. arieticanis | V2arie1 | Forward | CTCTTTGCCGTAGATTCGCTAGTTA | 63 | 55 | 884 | Present study |
V2arie2 | Reverse | CAAAGATCGGTAGATATCCAATGC | Present study | ||||
V2arie3 | Forward | TAGTTCTTGGCCTGGCTATTCTT | 59 | 25 | 371 | Present study | |
V2arie4 | Reverse | CTGACCTCCAAAAACTGGCTTAC | Present study | ||||
S. tenella | V2te1 | Forward | GAGCGGTGAACTTCTTAGGAACC | 60 | 40 | 537 | Present study |
V2te2 | Reverse | CCCAATAATCCGCTGTTAACGTA | Present study | ||||
V2te3b | Forward | ATTGTAATGCTCCTCGACGATATG | 57 | 30 | 314 | Present study | |
V2te4 | Reverse | ATAGTCACGGCAGAGAAGTAGGAC | Present study | ||||
S. capracanis | VocaF | Forward | GTAAACTTCCTGGGTACTGTGCTGT | 60 | 40 | 531 | Present study |
VocaR1 | Reverse | CCAGTAATCCGCTGTCAAGATAC | Present study | ||||
V2cap3 | Forward | ATACCGATCTTTACGGGAGCAGTA | 63 | 30 | 330 | Present study | |
V2cap4 | Reverse | GGTCACCGCAGAGAAGTACGAT | Present study | ||||
S. bertrami | V2ber1 | Forward | GTATGAACTGTCAACGGATGGAGTA | 58 | 60 | 883 | Present study |
V2ber2 | Reverse | AGAAGCCATGTTCGTGACTACC | Present study | ||||
V2ber3 | Forward | GTACTACCTCCTTCCAGTCGGTTC | 57 | 40 | 600 | Present study | |
V2ber4 | Reverse | CGGGTATCCACTTCAAGTCCAG | Present study | ||||
S. miescheriana | V2mie1 | Forward | TGCTGCGGTATGAACTATCTACCT | 61 | 60 | 922 | Present study |
V2mie2 | Reverse | GCCCAGAGATCCAAATCCAG | Present study | ||||
V2mie3 | Forward | CTTGGTTCAACGTTACTCCTCCA | 61 | 30 | 474 | Present study | |
V2mie4 | Reverse | CTTCGATCCAGCTGAACTAAAGC | Present study |
Method | Sporocysts Isolation | gDNA Extraction | Number of Water Samples (n) | Positive Samples * | Cases | Species Detected **** | |
---|---|---|---|---|---|---|---|
Positive ** | False Positive *** | ||||||
Direct PCR (species-specific primers) | Sucrose gradient | “GeneJET Genomic DNA Purification Kit” | 20 | 0 (0.0%) | 0 | 0 | 0 |
Nested PCR (species-specific primers only in 2nd round of PCR) | Sucrose gradient | “GeneJET Genomic DNA Purification Kit” | 35 | 8 (22.9%) | 12 | 2 | 2 |
Sedimentation | “Genomic DNA Purification Kit” | 35 | 3 (8.6%) | 4 | 1 | 2 | |
Filtration | “GeneJET Genomic DNA Purification Kit” | 35 | 14 (40.0%) | 15 | 1 | 3 | |
Multiplex-nested PCR (species-specific primers only in 2nd round of PCR) | Filtration | “GeneJET Genomic DNA Purification Kit” | 35 | 7 (20.0%) | 10 | 3 | 2 |
Semi-nested PCR (species-specific primers) | Filtration | “GeneJET Genomic DNA Purification Kit” | 35 | 0 (0.0%) | 0 | 0 | 0 |
Nested PCR (species-specific primers in both rounds) | Filtration | “GeneJET Genomic DNA Purification Kit” | 35 | 34 (97.1%) | 84 | 0 | 8 |
Species | GenBank Acc. No. (Length, bp) | Sequence Similarity, % | ||
---|---|---|---|---|
Comparing Obtained Sequences with Each Other | Comparing Obtained Sequences and Those of the Same Species Available in GenBank | Comparing Obtained Sequences with Those of Most Closely Related Species | ||
S. bovifelis | ON211315-18 (361) | 99.7–100 | 99.5–100 | S. bovini 93.1–94.5 |
S. cruzi | ON211319-22 (248) | 98.8–100 | 98.0–99.6 | S. alceslatrans 85.1–86.8 |
S. hirsuta | ON211323-26 (254) | 99.6–100 | 98.4–100 | S. buffalonis 92.8–93.2 |
S. arieticanis | ON211327-30 (325) | 99.7–100 | 92.3–99.4 | S. hircicanis 86.2–87.4 |
S. tenella | ON211331-34 (263) | 99.2–100 | 96.2–100 | S. capracanis 91.3–93.2 |
S. capracanis | ON211335-38 (284) | 99.7–100 | 96.8–99.7 | S. tenella 90.4–92.9 |
S. bertrami | ON211339-42 (554) | 99.1–99.8 | 97.5–99.8 | S. bovifelis 77.0–78.4 |
S. miescheriana | ON211343-46 (428) | 99.5–100 | 93.9–99.5 | S. rangiferi 79.6–80.2 |
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Strazdaitė-Žielienė, Ž.; Baranauskaitė, A.; Butkauskas, D.; Servienė, E.; Prakas, P. Molecular Identification of Parasitic Protozoa Sarcocystis in Water Samples. Vet. Sci. 2022, 9, 412. https://doi.org/10.3390/vetsci9080412
Strazdaitė-Žielienė Ž, Baranauskaitė A, Butkauskas D, Servienė E, Prakas P. Molecular Identification of Parasitic Protozoa Sarcocystis in Water Samples. Veterinary Sciences. 2022; 9(8):412. https://doi.org/10.3390/vetsci9080412
Chicago/Turabian StyleStrazdaitė-Žielienė, Živilė, Agnė Baranauskaitė, Dalius Butkauskas, Elena Servienė, and Petras Prakas. 2022. "Molecular Identification of Parasitic Protozoa Sarcocystis in Water Samples" Veterinary Sciences 9, no. 8: 412. https://doi.org/10.3390/vetsci9080412