Investigation of Babesia bovis and Babesia bigemina in cattle by Real Time PCR and molecularcharacterization of the isolates
Öz
This study was carried out to investigate the prevalence of Babesia bovis and B. bigemina in cattle in Erzurum region by Real Time PCR and to characterize the isolates molecularly. Totally 300 whole blood samples from clinically healthy cattle in different ages, sex and breeds were collected into the tubes with EDTA in 2011-2012. Following DNA extraction, Sybergreen and TaqMan probe based Real Time PCR analyses were carried out with B. bovis and B. bigemina primers amplifying msa-2c and rap-1 gene regions. Out of 300 samples, 28 (9.3%) and 17 (5.7%) were found to be infected with B.bovis and B. bigemina. No mix infection was found in the samples. Four of B. bovis and three of B. bigemina isolates were sequenced with respect to msa-2c and rap-1 gene regions for phylogenetic analyses. According to pairwise comparisons of B. bovis isolates, genetic distances were found as 0.6%, 2.3%, and 7.5%, among each other, some other isolates in Turkey, and in the world, respectively. In the phylogenetic analyses of B.bigemina, genetic differences were detected as 0.3% and 0.5% among each other and the other isolates in the world. In the statistical analyses of B. bovis and B. bigemina positive samples according to age, sex and breed of cattle no statistical significance (p>0.05) was founded according to age and breed while the statistical difference according to sex was found significant for B. bigemina (p<0.05), and not significant for B. bovis (p>0.05). In conclusion, the molecular characterization and the prevalence of B. bovis and B. bigemina in cattle in Erzurum region were determined by Real Time PCR. This is the first report on the molecular characterizations of B. bovis and B. bigemina in Erzurum region
Anahtar Kelimeler
Babesia bigemina Babesia bovis cattle Erzurum molecular characterization Real Time PCR
Kaynakça
- Almeria S, Castella J, Ferrer D, Gutierrez JF, Estrada- Pena A, Sparagano O (2002): Reverse line blot hybridization used to identify hemoprotozoa in Minorcan cattle. Ann N Y Acad Sci, 969, 78-82.
- Altay K, Aydin MF, Dumanli N, Aktas M (2008): Molecular detection of Theileria and Babesia infections in cattle. Vet Parasitol, 158: 295-301.
- Berens SJ, Brayton KA, Molloy JB, Bock RE, Lew AE, McElwain TF (2005): Merozoite surface antigen 2 proteins of Babesia bovis vaccine breakthrough isolates contain a unique hypervariable region composed of degenerate repeats. Infect Immun, 73, 7180-7189.
- Bilgin Z (2007): Trakya’da sığırlarda bulunan Theileria ve Babesia türlerinin ve bunların sığırlarda yaygınlığının reverse line blooting (RLB) tekniği ile araştırılması, Doktora tezi, İstanbul Üniversitesi Sağlık Bilimleri Enstitüsü, İstanbul.
- Bock R, Jackson L, de Vos A, Jorgensen W (2004): Babesiosis of cattle. Parasitology, 129, 247-269.
- Borgonio V, Mosqueda J, Genis AD, Falcon A, Alvarez JA, Camacho M, Figueroa JV (2008): msa-1 and msa-2c gene analysis and common epitopes assessment in Mexican Babesia bovis isolates. Ann N Y Acad Sci, 1149, 145-148.
- Cao S, Aboge GO, Terkawi MA, Yu L, Kamyingkird K, Luo Y, Li Y, Goo YK, Yamagishi J, Nishikawa Y, Yokoyama N, Suzuki H, Igarashi I, Maeda R, Inpankaew T, Jittapalapong S, Xuan X (2012): Molecular detection and identification of Babesia bovis and Babesia bigemina in cattle in northern Thailand. Parasitol Res, 111, 1259-1266.
- Criado-Fornelio A, Buling A, Asenzo G, Benitez D, Florin-Christensen M, Gonzalez-Oliva A, Henriques G, Silva M, Alongi A, Agnone A, Torina A, Madruga CR (2009): Development of fluorogenic probe-based PCR assays for the detection and quantification of bovine piroplasmids. Vet Parasitol, 162, 200-206.
- Criado-Fornelio A (2007): A review of nucleic-acid-based diagnostic tests for Babesia and Theileria, with emphasis on bovine piroplasms. Parassitologia, 49, 39-44.
- Dominguez M, Echaide I, Echaide ST, Mosqueda J, Cetrá B, Suarez CE, Florin-Christensen M (2010): In silico predicted conserved B-cell epitopes in the merozoite surface antigen-2 family of B. bovis are neutralization sensitive. Vet Parasitol, 167, 216-226.
- Drummond AJ, Ashton B, Buxton S: Geneious v5.5. http://www.geneious.com. (15.10.2013).
- Düzlü Ö, İnci A, Yıldırım A (2011): Karadeniz Bölgesi'ndeki sığırlardan elde edilen Babesia bovis suşlarının moleküler karakterizasyonu. ERÜ Sağ Bil Derg, 20, 18-28.
- Düzlü Ö, İnci A, Yildirim A (2012): Türkiye’de evcil ruminantlarda babesiosis. Turkiye Klinikleri J Vet Sci, 2, 27-34.
- Genis AD, Mosqueda JJ, Borgonio VM, Falcón A, Alvarez A, Camacho M, de Lourdes Muñoz M, Figueroa JV (2008): Phylogenetic analysis of Mexican Babesia bovis isolates using msa and ssrRNA gene sequences. Ann N Y Acad Sci, 1149, 121-125.
- Gubbels MJ, De Vos S, Van Der Weide M, Viseras J, Schouls LM, De Vries E, Jongejan F (1999): Simultaneous detection of bovine Theileria and Babesia species using reverse line blotting hybridization. J Clin Microbiol, 37, 1782-1789.
- Ica A, Vatansever Z, Yildirim A, Duzlu O, Inci A (2007): Detection of Theileria and Babesia species in ticks collected from cattle. Vet Parasitol, 148, 156-160.
- İça A, İnci A, Yıldırım A (2007): Parasitological and molecular prevalence of bovine Theileria and Babesia species in the vicinity of Kayseri. Turk J Vet Anim Sci, 31, 33-38.
- Kim C, Iseki H, Herbas MS, Yokoyama N, Suzuki H, Xuan X, Fujisaki K, Igarashi I (2007): Development of TaqMan-based real-time PCR assays for diagnostic detection of Babesia bovis and Babesia bigemina. Am J Trop Med Hyg, 77, 837-841,.
- Lau AO, Tibbals DL, McElwain TF (2007): Babesia bovis: the development of an expression oligonucleotide microarray. Exp Parasitol, 117: 93-98.
- Machado RZ, McElwain TF, Pancracio HP, Freschi CR, Palmer GH (1999): Babesia bigemina: immunization with purified rhoptries induces protection against acute parasitemia. Exp Parasitol, 93, 105-108.
- Petrigh R, Ruybal P, Thompson C, Neumann R, Moretta R,Wilkowsky S, Draghi G, Echaide I, de Echaide ST, Farbera M (2008): Improved molecular tools for detection of Babesia bigemina. Animal biodiversity and emerging diseases. Ann N YAcad Sci, 1149, 155-157.
- Ramos CA, Araşjo FR, Souza II, Bacanelli G, Luiz HL, Russi LS, Oliveira RH, Soares CO, Rosinha GM, Alves LC (2011): Real-time polymerase chain reaction based on msa2c gene for detection of Babesia bovis. Vet Parasitol, 176, 79-83.
- Sam-Yellowe TY (1996): Rhoptry organelles of the apicomplexa: their role in host cell invasion and intracellular survival. Parasitol Today, 12, 308-316.
- Shebish E, Vemulapalli R, Oseto C (2012): Prevalence and molecular detection of Anaplasma marginale, Babesia bovis and Babesia bigemina in cattle from Puntarenas Province, Costa Rica. Vet Parasitol, 188, 164-167.
- Suarez CE, McElwain TF, Echaide I, Torioni de Echaide S, Palmer GH (1994): Interstrain conservation of babesial RAP-1 surface-exposed B-cell epitopes despite rap-1 genomic polymorphism. Infect Immun, 62, 3576- 3579.
- Suarez CE, Palmer GH, Hötzel I, McElwain TF (1998): Structure, sequence, and transcriptional analysis of the Babesia bovis rap-1 multigene locus. Mol Biochem Parasitol, 93, 215-222.
- Tanyüksel M, Vatansever Z, Karaer Z, Araz E, Haznedaroğlu T, Yukarı BA (2002): Sığır babesiosisinin epidemiyolojisi ve zoonotik önemi. T Parazitol Derg, 26, 42-47.
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011): MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol, 28, 2731-2739.
- Terkawi MA, Huyen NX, Shinuo C, Inpankaew T, Maklon K, Aboulaila M, Ueno A, Goo YK, Yokoyama N, Jittapalapong S, Xuan X, Igarashi I (2011): Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes in the northeast region of Thailand. Vet Parasitol, 178, 201-207.
- Vatansever Z, İça A, Deniz A, Nalbantoğlu S, Karaer Z, Çakmak A, Sparagano O (2003): Ankara yöresinde sığırlarda kene kaynaklı protozoon enfeksiyonlarının yayılışının reverse line blotting (RLB) ve indirek floresan antikor testi (IFAT) ile saptanması. 13. Ulusal Parazitoloji Kongresi, Tebliğ Özetleri. Konya, p. 194.
- Vatansever Z, Nalbantoğlu S, Çakmak A (2001): Çukurova bölgesinde sığır babesiosis’nin epidemiyolojisi. 12. Ulusal Parazitoloji Kongresi, Elazığ.
- Vidotto O, McElwain TF, Machado RZ, Perryman LE, Suarez CE, Palmer GH (1995): Babesia bigemina: identification of B cell epitopes associated with parasitized erythrocytes. Exp Parasitol, 81, 491-500.
- Wilkowsky SE, Farber M, Echaide I, Torioni de Echaide S, Zamorano PI, Dominguez M, Suarez CE, Florin-Christensen M (2003): Babesia bovis merozoite surface protein-2c (MSA-2c) contains highly immunogenic, conserved B-cell epitopes that elicit neutralization- sensitive antibodies in cattle. Mol Biochem Parasitol, 127, 133-141.
- Wilkowsky SE, Farber M, Gil G, Echaide I, Mosqueda J, Alcaraz E, Suarez CE, Florin-Christensen M (2008): Molecular characterization of Babesia bovis strains using PCR restriction fragment length polymorphism analysis of the msa2-a/b genes. Ann N Y Acad Sci, 1149, 141-144.
- Yavuz A, İnci A, Düzlü Ö, Bişkin Z, Yıldırım A (2011): Molecular characterization of Babesia bovis msa-2c gene. Turkiye Parazitol Derg, 35, 140-144.
- Yıldırım A, Düzlü Ö, İnci A, Önder Z, Çiloğlu A (2013): Sığırlarda Babesia bovis ve Babesia bigemina’nın Reverse Line Blotting, Nested PCR ve Real Time PCR Teknikleri ile Karşılaştırmalı Tanısı. Kafkas Univ Vet Fak Derg, 19, 895-902.
- Yokoyama N, Okamura M, Igarashi I (2006): Erythrocyte invasion by Babesia parasites: current advances in the elucidation of the molecular interactions between the protozoan ligands and host receptors in the invasion stage. Vet Parasitol, 138, 22-32. Geliş tarihi: 27.01.2013/ Kabul tarihi: 13.06.2014
Sığırlarda Babesia bovis ve Babesia bigemina'nın Real-Time PCR ile araştırılması ve izolatların moleküler karakterizasyonu
Öz
Bu çalışma, Erzurum yöresi sığırlarında Babesia bovis ve B. bigemina yaygınlığının Real Time PCR ile araştırılması ve saptanan izolatların moleküler karakterizasyonları amacıyla yapılmıştır. Bu amaçla 2011-2012 tarihleri arasında farklı yaş, cinsiyet ve ırklarda, klinik olarak sağlıklı görünümlü toplam 300 sığırdan EDTA’lı tüplere tam kan örnekleri toplanmıştır. DNA ekstraksiyonunu takiben B. bovis ve B.bigemina için sırasıyla msa-2c ve rap-1 gen bölgelerine özgü primerlerle Sybergreen ve TaqMan prob bazlı Real Time PCR analizleri gerçekleştirilmiştir. İncelenen 300 kan örneğinin 28’inde (%9,3) B. bovis, 17’sinde (%5,7) B. bigemina saptanmıştır. Örneklerde miks enfeksiyona rastlanmamıştır. B. bovis pozitiflerden 4’ünün, B. bigemina pozitiflerden 3’ünün msa-2c ve rap-1 gen bölgelerine göre sekans analizleri yapılmıştır. B. bovis izolatlarının pairwise kıyaslamalarında kendi aralarında, Türkiye’den diğer bazı ve dünyadaki diğer izolatlarla aralarında sırasıyla %0,6, %2,3 ve %7,5 genetik farklılık belirlenmiştir. B. bigemina izolatlarının kendi aralarında %0,3 ve dünyadaki diğer izolatlarla aralarında %0,5 genetik farklılık saptanmıştır. B. bovis ve B. bigemina yönünden pozitif belirlenen örneklerin parazit türü temel alınarak sığırların yaş, cinsiyet ve ırk özelliklerine göre istatistiksel analizlerinde; yaş ve ırk özelliklerine göre istatistiksel bir farklılık belirlenmezken (p>0,05), cinsiyete bağlı farklılık B. bovis için önemsiz (p>0,05), B. bigemina için ise önemli (p<0,05) bulunmuştur. Sonuç olarak bu çalışmayla Erzurum yöresi sığırlarında ilk kez Real Time PCR tekniği ile B. bovis ve B. bigemina moleküler olarak karakterize edilmiş ve yaygınlıkları ortaya konmuştur
Anahtar Kelimeler
Babesia bigemina Babesia bovis Erzurum moleküler karakterizasyon Real Time PCR sığır
Kaynakça
- Almeria S, Castella J, Ferrer D, Gutierrez JF, Estrada- Pena A, Sparagano O (2002): Reverse line blot hybridization used to identify hemoprotozoa in Minorcan cattle. Ann N Y Acad Sci, 969, 78-82.
- Altay K, Aydin MF, Dumanli N, Aktas M (2008): Molecular detection of Theileria and Babesia infections in cattle. Vet Parasitol, 158: 295-301.
- Berens SJ, Brayton KA, Molloy JB, Bock RE, Lew AE, McElwain TF (2005): Merozoite surface antigen 2 proteins of Babesia bovis vaccine breakthrough isolates contain a unique hypervariable region composed of degenerate repeats. Infect Immun, 73, 7180-7189.
- Bilgin Z (2007): Trakya’da sığırlarda bulunan Theileria ve Babesia türlerinin ve bunların sığırlarda yaygınlığının reverse line blooting (RLB) tekniği ile araştırılması, Doktora tezi, İstanbul Üniversitesi Sağlık Bilimleri Enstitüsü, İstanbul.
- Bock R, Jackson L, de Vos A, Jorgensen W (2004): Babesiosis of cattle. Parasitology, 129, 247-269.
- Borgonio V, Mosqueda J, Genis AD, Falcon A, Alvarez JA, Camacho M, Figueroa JV (2008): msa-1 and msa-2c gene analysis and common epitopes assessment in Mexican Babesia bovis isolates. Ann N Y Acad Sci, 1149, 145-148.
- Cao S, Aboge GO, Terkawi MA, Yu L, Kamyingkird K, Luo Y, Li Y, Goo YK, Yamagishi J, Nishikawa Y, Yokoyama N, Suzuki H, Igarashi I, Maeda R, Inpankaew T, Jittapalapong S, Xuan X (2012): Molecular detection and identification of Babesia bovis and Babesia bigemina in cattle in northern Thailand. Parasitol Res, 111, 1259-1266.
- Criado-Fornelio A, Buling A, Asenzo G, Benitez D, Florin-Christensen M, Gonzalez-Oliva A, Henriques G, Silva M, Alongi A, Agnone A, Torina A, Madruga CR (2009): Development of fluorogenic probe-based PCR assays for the detection and quantification of bovine piroplasmids. Vet Parasitol, 162, 200-206.
- Criado-Fornelio A (2007): A review of nucleic-acid-based diagnostic tests for Babesia and Theileria, with emphasis on bovine piroplasms. Parassitologia, 49, 39-44.
- Dominguez M, Echaide I, Echaide ST, Mosqueda J, Cetrá B, Suarez CE, Florin-Christensen M (2010): In silico predicted conserved B-cell epitopes in the merozoite surface antigen-2 family of B. bovis are neutralization sensitive. Vet Parasitol, 167, 216-226.
- Drummond AJ, Ashton B, Buxton S: Geneious v5.5. http://www.geneious.com. (15.10.2013).
- Düzlü Ö, İnci A, Yıldırım A (2011): Karadeniz Bölgesi'ndeki sığırlardan elde edilen Babesia bovis suşlarının moleküler karakterizasyonu. ERÜ Sağ Bil Derg, 20, 18-28.
- Düzlü Ö, İnci A, Yildirim A (2012): Türkiye’de evcil ruminantlarda babesiosis. Turkiye Klinikleri J Vet Sci, 2, 27-34.
- Genis AD, Mosqueda JJ, Borgonio VM, Falcón A, Alvarez A, Camacho M, de Lourdes Muñoz M, Figueroa JV (2008): Phylogenetic analysis of Mexican Babesia bovis isolates using msa and ssrRNA gene sequences. Ann N Y Acad Sci, 1149, 121-125.
- Gubbels MJ, De Vos S, Van Der Weide M, Viseras J, Schouls LM, De Vries E, Jongejan F (1999): Simultaneous detection of bovine Theileria and Babesia species using reverse line blotting hybridization. J Clin Microbiol, 37, 1782-1789.
- Ica A, Vatansever Z, Yildirim A, Duzlu O, Inci A (2007): Detection of Theileria and Babesia species in ticks collected from cattle. Vet Parasitol, 148, 156-160.
- İça A, İnci A, Yıldırım A (2007): Parasitological and molecular prevalence of bovine Theileria and Babesia species in the vicinity of Kayseri. Turk J Vet Anim Sci, 31, 33-38.
- Kim C, Iseki H, Herbas MS, Yokoyama N, Suzuki H, Xuan X, Fujisaki K, Igarashi I (2007): Development of TaqMan-based real-time PCR assays for diagnostic detection of Babesia bovis and Babesia bigemina. Am J Trop Med Hyg, 77, 837-841,.
- Lau AO, Tibbals DL, McElwain TF (2007): Babesia bovis: the development of an expression oligonucleotide microarray. Exp Parasitol, 117: 93-98.
- Machado RZ, McElwain TF, Pancracio HP, Freschi CR, Palmer GH (1999): Babesia bigemina: immunization with purified rhoptries induces protection against acute parasitemia. Exp Parasitol, 93, 105-108.
- Petrigh R, Ruybal P, Thompson C, Neumann R, Moretta R,Wilkowsky S, Draghi G, Echaide I, de Echaide ST, Farbera M (2008): Improved molecular tools for detection of Babesia bigemina. Animal biodiversity and emerging diseases. Ann N YAcad Sci, 1149, 155-157.
- Ramos CA, Araşjo FR, Souza II, Bacanelli G, Luiz HL, Russi LS, Oliveira RH, Soares CO, Rosinha GM, Alves LC (2011): Real-time polymerase chain reaction based on msa2c gene for detection of Babesia bovis. Vet Parasitol, 176, 79-83.
- Sam-Yellowe TY (1996): Rhoptry organelles of the apicomplexa: their role in host cell invasion and intracellular survival. Parasitol Today, 12, 308-316.
- Shebish E, Vemulapalli R, Oseto C (2012): Prevalence and molecular detection of Anaplasma marginale, Babesia bovis and Babesia bigemina in cattle from Puntarenas Province, Costa Rica. Vet Parasitol, 188, 164-167.
- Suarez CE, McElwain TF, Echaide I, Torioni de Echaide S, Palmer GH (1994): Interstrain conservation of babesial RAP-1 surface-exposed B-cell epitopes despite rap-1 genomic polymorphism. Infect Immun, 62, 3576- 3579.
- Suarez CE, Palmer GH, Hötzel I, McElwain TF (1998): Structure, sequence, and transcriptional analysis of the Babesia bovis rap-1 multigene locus. Mol Biochem Parasitol, 93, 215-222.
- Tanyüksel M, Vatansever Z, Karaer Z, Araz E, Haznedaroğlu T, Yukarı BA (2002): Sığır babesiosisinin epidemiyolojisi ve zoonotik önemi. T Parazitol Derg, 26, 42-47.
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011): MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol, 28, 2731-2739.
- Terkawi MA, Huyen NX, Shinuo C, Inpankaew T, Maklon K, Aboulaila M, Ueno A, Goo YK, Yokoyama N, Jittapalapong S, Xuan X, Igarashi I (2011): Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes in the northeast region of Thailand. Vet Parasitol, 178, 201-207.
- Vatansever Z, İça A, Deniz A, Nalbantoğlu S, Karaer Z, Çakmak A, Sparagano O (2003): Ankara yöresinde sığırlarda kene kaynaklı protozoon enfeksiyonlarının yayılışının reverse line blotting (RLB) ve indirek floresan antikor testi (IFAT) ile saptanması. 13. Ulusal Parazitoloji Kongresi, Tebliğ Özetleri. Konya, p. 194.
- Vatansever Z, Nalbantoğlu S, Çakmak A (2001): Çukurova bölgesinde sığır babesiosis’nin epidemiyolojisi. 12. Ulusal Parazitoloji Kongresi, Elazığ.
- Vidotto O, McElwain TF, Machado RZ, Perryman LE, Suarez CE, Palmer GH (1995): Babesia bigemina: identification of B cell epitopes associated with parasitized erythrocytes. Exp Parasitol, 81, 491-500.
- Wilkowsky SE, Farber M, Echaide I, Torioni de Echaide S, Zamorano PI, Dominguez M, Suarez CE, Florin-Christensen M (2003): Babesia bovis merozoite surface protein-2c (MSA-2c) contains highly immunogenic, conserved B-cell epitopes that elicit neutralization- sensitive antibodies in cattle. Mol Biochem Parasitol, 127, 133-141.
- Wilkowsky SE, Farber M, Gil G, Echaide I, Mosqueda J, Alcaraz E, Suarez CE, Florin-Christensen M (2008): Molecular characterization of Babesia bovis strains using PCR restriction fragment length polymorphism analysis of the msa2-a/b genes. Ann N Y Acad Sci, 1149, 141-144.
- Yavuz A, İnci A, Düzlü Ö, Bişkin Z, Yıldırım A (2011): Molecular characterization of Babesia bovis msa-2c gene. Turkiye Parazitol Derg, 35, 140-144.
- Yıldırım A, Düzlü Ö, İnci A, Önder Z, Çiloğlu A (2013): Sığırlarda Babesia bovis ve Babesia bigemina’nın Reverse Line Blotting, Nested PCR ve Real Time PCR Teknikleri ile Karşılaştırmalı Tanısı. Kafkas Univ Vet Fak Derg, 19, 895-902.
- Yokoyama N, Okamura M, Igarashi I (2006): Erythrocyte invasion by Babesia parasites: current advances in the elucidation of the molecular interactions between the protozoan ligands and host receptors in the invasion stage. Vet Parasitol, 138, 22-32. Geliş tarihi: 27.01.2013/ Kabul tarihi: 13.06.2014
Ayrıntılar
| Birincil Dil | İngilizce |
|---|---|
| Konular | Veteriner Cerrahi |
| Diğer ID | JA22PG24SY |
| Bölüm | Araştırma Makalesi |
| Yazarlar | |
| Yayımlanma Tarihi | 1 Mart 2015 |
| Yayımlandığı Sayı | Yıl 2015 |