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Bulgarian Journal of Agricultural Science, 12 (2006), 315-319 National Centre for Agrarian Sciences

Molecular-Biological Analysis of the Parasite Capillaria sp. of the Liver of Barbel (Barbus meridionalis petenyi Heck.) in Lake Ohrid

L. VELKOVA-JORDANOSKA SI Hydrobiological Institute, MC-6000 Ohrid, Macedonia

Abstract

L. VELKOVA-JORDANOSKA, 2006. Molecular-biological analysis of the parasite Capillaria sp. of the liver of barbel (Barbus meridionalis petenyi Heck.) in Lake Ohrid. Bulg. J. Agric. Sci.,12: 315-319

The analysis of parasites molecular variation has important implications on the taxonomy, phylogenic and population genetics studies. Polymerase chain reaction (PCR) can have signifi- cant advantages over other morphological, cytological and biochemical methods. The aim of our investigations was a development of molecular diagnostic methods, utilization of PCR for detec- tion of the zoonoses. We investigated presence of parasite Capillaria sp. in the barbel liver tissue in Ohrid Lake. Key words: polymerase chain reaction, zoonoses, Capillaria sp., Ohrid Lake

Introduction organisms (Zarlenga et al., 1999; Zarlenga and La Rosa, 2000; Zarlenga et al., 2001). Very often phenotype differences in dif- Introduction of PCR methods provoke ferent organisms can not be markers for revolution in molecular-biological investi- differences in their genotype. In fact, in gations, PCR methods are especially con- the final formation of some phe- venient for analysing the parasite, because notype, except genetical factors, is im- they use small quantity of material, and pacted by the environmental factors too. cultivation of great quantity of parasites is On the account of this, more variations of not necessary. Detection of mutations in the phenotype in just one species are pos- parasite genome with PCR method can sible. have significant advantages over some In order to make a precise diagnosis of currently used DNA approaches for the the parasite diseases, it is necessary to analysis of allelic and mutational sequence make rapid and exact identification of para- variations in parasites (Gasser, 1998). sites. The newest contributions in molecu- In the literature there is data about us- lar biology can be used in determination ing allozyme electrophoresis for identifi- of parasites and in phylogeny of the living cation of helminth species (Beveridge, 316 L. Velkova-Jordanoska

1998), and using the technique of multilocus subacute hepatitis, then eosinophilia and enzyme electrophoresis addressing the is- hepatomegalia (Kelsey, 1997). sues related to systematic of parasites In the hepatic tissue of barbel (Barbus (Andrews and Chilton, 1999). meridionalis petenyi Heck.) in Lake Molecular investigations of eel-pout, Ohrid eggs of Capillaria sp. were identi- cod and herring from Baltic Sea were per- fied (Velkova-Jordanoska, 2002; formed using multilocus allozyme electro- Roganovic-Zafirova et al., 2003). phoresis and genomic DNA analysis based on PCR technique (Szostakowska et al., Material and Methods 2001). The parasite Capillaria petruchewskii lives in liver parenchyme In the period from May to August 2002, and in fish spleen. The presence of only 41 samples of barbel (Barbus meridio- several individuals of Capillaria nalis petenyi Heck.) were collected wich petruchewskii is benignous, but more se- adult form of parasite Capillaria sp. were rious infestation is manifested with great isolated. We tried to introduce the method weakening of the fish (Klinger and Floyd, of molecular detection on the 1998). Capillaria sp. by using PCR amplifica- The artificial infestation of parasite tion at ITS-2 region on rDNA. Parasite in experimental rats DNA was isolated from two sources: gave significant results about the influence 1. Paraffin sections from conserved of parasite eggs on liver parenchyme. liver tissue from the samples with high Histopatological picture displayed septal level of parasite infestation; fibrosis in all , with focal lesions in 2. Adult worms on Capillaria sp. from parenchyme, necrosis and sometimes fresh liver tissue on barbel. granulomatous inflammation. Lesions of The isolation of DNA was made with liver displayed a linear proportion to inocu- the method NaCl/ hloroform extraction and lum sizes (Oliveira and Andrade, 2001). ethanol precipitation. DNA isolated with Infestation with Capillaria hepatica simply release method without isolation, of is discovered with many animals, however adult worms and parasite’s eggs was also it is also present with monkeys and hu- used for amplification. man population. The presence of this para- PCR mix: (10x PCR buffer; 25 mM site in the liver of monkey (Callithrix MgCl2 (1.5 mM each); 2.5 mM d NTP jacchus) provokes destruction of the tis- mixture (0.2mM each); 50 µM primers sue parenchyme. Microscope analysis dis- (1.25 µM each); Taq DNA polymerase (5 played lesions invaded by fibrous tissue, U/µM); 2 µl template DNA). monocites, and in same cases gigant cells. PCR protocol: Denaturing: 3 min – Parasite Capillaria brochieri is the first 94ºC. 35 cycles: denaturing: 94ºC - 1 min; species of the Capillaria discov- annealing: 55ºC - 1 min; primer extension: ered in the chimpanzee intestine. Because 72ºC - 1 min; Single 10 - min step at 72 ºC. of the close analogies between chim- panzee’s and man’s helminthes, it is pos- Forward primer: sible that Capillaria brochieri is present 5'-ACGTCTGGTTCAGGGTTGTT-3' in human pathology. The presence of the Reverse primer: parasite gives clinical picture like acute or 5'-TTAGTTTCTTTTCCTCCGCT-3' Molecular-Biological Analysis of the Parasite Capillaria sp... 317

Amplification was made on Perkins M 1 2 3 4 5 6 Elmer Termocycler, and used TaKaRa Amplification Kit. The PCR products were checked on 2% agarose gels in 0.5 x TBE buffer stained with ethidium bromide.

Results and Discussion

On Figure 1 positive result, i.e. PCR product is displayed only on the second position. This is DNA extracted from two adult individuals by release method. The fragment is about 400 bp long and under it one more fragment, which can be nonspe- cific amplification product or possibly marker for interspecies difference be- tween adult form of parasite, can be no- ticed. On the other positions there are no positive products, probably because the isolation process decreases the concen- Fig. 1. M-marker; 1 - negative control; tration of DNA and it is under the amplifi- 2 - PCR product of two adult worms (DNA cation level. We have amplification prod- release method); uct of DNA isolated on two adult forms, 3 - PCR product of one adult worms (DNA and we do not have a result of DNA on release method); one adult form by simultaneous isolation 4 - PCR product of parasite’s eggs (NaCl/ and some method. hloroform isolation); The used pair primers was use by 5,6 - PCR product of paraffin sections (NaCl/ Oliveros et al. (2000) for genetically hloroform isolation) charactersation of four species from the nose human diseases caused by helminth genus Trichuris (T. globulosa, T. leporis, parasites has improved, so our understand- T. suis and T. ovis) which is phylo- ing of the epidemiology and clinical mani- geneticaly very close to genus Capillaria. festation of these diseases has improved. Our experience with analysis of ITS-2 Humans can develop infection with helm- region of rDNA on Capillaria sp. im- inth parasites whose natural host is another poses necessity of additional investigations vertebrate. Potential reasons for these in- about the influence of fixation and paraf- fections are changes in social, dietary or fin conservation of the total quantity of cultural habits (McCarthy, 2000). Under- DNA. standing the epidemiology of zoonotic para- The research priorities include improve- sitic infection depends on availability of ment of diagnostic tests and development accurate and sensitive diagnostic tech- of molecular tests for investigation resis- niques. The development of molecular di- tance to antihelmintic drugs (Sangster, agnostic methods, which utilize PSR for 1999). The ability to recognize and diag- detection of zoonoses, offers us possibili- 318 L. Velkova-Jordanoska ties to identify and control the pathogens Kelsey, D. S., 1997. Enteric of by increasing the speed of diagnosis, speci- Lower Animals: Zoonoses. In: S. Baron (Edi- ficity and sensitivity (Watts and Kennedy, tor), Medical Microbiology. http://www. 1999; Morgan, 2000). gsbs.utmb.edu/microbook/ Klinger, R. E. and R. F. Floyd, 1998. Introduc- Conclusions tion to Freshwater Fish Parasites. Univer- sity of Florida. http://edis.ifas.ufl.edu/ The application of more modern meth- BODY_FA041 ods in the resolution of taxonomical prob- McCarthy, J. and T. A. Moore, 2000. Emerging lems, for instance molecular – genetic helminth zoonoses. International Journal analyses, will lead to obtaining a more ac- for Parasitology, 30 (12-13): 1351-1359. curate picture of the presence of certain Morgan, U. M., 2000. Detection and characteri- parasites, their taxonomical affiliation and sation of parasites causing emerging zoo- contribute to solve a greater number of noses. International Journal for Parasi- queries which cannot be explained by tology, 30 (12-13): 1407-1421. morphological analyses. Oliveros, C., C. Cutillas, M. de Rojas and P. Many authors have discussed molecu- Arias, 2000. Characterization of four spe- lar-biological characterization of parasites cies of Trichuris (Nematoda: ) by so far and the phylogenetic relationship of their second internal transcribed spacer ri- more species of parasites was examined bosomal DNA sequence. Parasitol. Res., by investigation of present sequence of 86: 1008-1013. DNA fragments. The ultimate objective Oliveira, R. F. and Z. A. Andrade, 2001. Worm of all the investigations is to find a relevant, Load and Septal Fibrosis of the Liver in stable marker or markers that would en- Capillaria hepatica-infected Rats. Memorias able a prompt and precise identification of do Instituto Oswaldo Cruz, 96 (7): 1001- the parasite and alongside that provide 1003. valid proofs for the taxonomy of them. Roganovic-Zafirova, D., M. Jordanova, S. Panov and L. Velkova-Jordanoska, 2003. References Hepatic capillariasis in the Mediterranean barbel (Barbus meridionalis petenyi Andrews, R. H. and N. B. Chilton, 1999. Heck.) from Lake Ohrid. Folia Veterinaria, Multilocus enzyme electrophoresis: avail- 47 (1): 35-37. able technique for providing answers to Sangster, N.C., 1999. Antihelmintic resistence: problems in parasite systematic. Interna- past, present and future. International tional Journal for Parasitology, 29 (2): Journal for Parasitology, 29 (1): 115-124. 213-253. Szostakowska, B., P. Myjak, J. Kur and T. Beveridge, I., 1998. Allozyme electrophoresis- Sywula, 2001. Molecular evaluation of difficulties encountered in studies on hel- Hysterothylacium auctum (Nematoda, minths. International Journal for Parasi- Ascaridida, Raphidascarididae) Tax- tology, 28 (6): 973-979. onomy from fish of the southern Baltic. Gasser, R. B., 1998. What’s in that band. In- Acta Parasitologica, 46 (3): 194-201. ternational Journal for Parasitology, 28 Velkova-Jordanoska, L., 2002. Histo- (6): 989-996. patological and moleculardiagnosti-cal Molecular-Biological Analysis of the Parasite Capillaria sp... 319

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