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Increasing use of Mycoplasma gallisepticum (MG) live vaccines has led to a need for the differentiation of MG strains. The MG strains MK-7, MS-16, S6, FS-9 and R strains and the MG live vaccine strain F were compared by random amplification of polymorphic DNA (RAPD) in this study. Using RAPD, different patterns were found among the MG strains. In addition to this, we examined the differentiating potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) primers targeted at the crmA, crmB, crmC, gapA, mgc2 and pvpA genes encoding cytadherence-related surface proteins. These proteins may take part in the pathogenesis of MG-induced disease. Differentiation of strain F is based on the identification of restriction enzyme sites in the PCR amplicons. Using HphI enzyme, crmC PCR amplicons produced different RFLP patterns. Digestion of amplicons of gapA-specific PCR with MboI enzyme also produced distinct patterns. Differences were observed among strains R and F by digestion of mgc2 PCR amplicons with HaeIII and VspI enzymes and digestion of pvpA PCR amplicons with AccI, PvuII and ScrFI endonucleases. This method can be used for the rapid differentiation of vaccine strain from wild strains. Differentiation of MG strains is a great advantage for diagnosticians or practitioners and it is useful for epidemiological studies.

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The aim of this study was to detect different alleles of the prolactin receptor (PRLR) gene and to examine their effects on the litter size of the indigenous Hungarian pig, the Mangalica. G1789A single nucleotide polymorphism (SNP) was investigated as a candidate for litter size. Samples from 80 purebred Mangalica sows and data of their 335 litters were provided by Olmos & Tóth Ltd. Hair follicles were used to isolate the required DNA. Allelic discrimination was performed by means of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method using the AluI restriction enzyme and agarose gel electrophoresis. In the population examined, the A allele was found to be preferable in the Mangalica breed group. The most advantageous AA genotype was the least prevalent (8.75%), while the frequencies of AB and BB were 40% and 51.25%, respectively. Remarkably, the average number of piglets born alive per litter was 1.11 ± 0.39 higher in sows with AA as compared to those with BB genotype. By raising the frequency of the AA genotype, the litter size is likely to increase. However, the effect of PRLR genotypes can differ among pig breeds and even lines. Further studies may be required to observe and estimate possible pleiotropic effects of this polymorphism on other traits.

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Objectives: The aim of this work was to investigate the prevalence of TNF-a -308 polymorphism among the 29 members of a family with RA and the association between the MHC-linked biallelic HSP70-2 gene and the TNF-a polymorphism. Five of the members with RA were diagnosed by using the revised 1987 ACR criteria, and 1 member suffered from SLE. Methods: The variations in the TNF-a and the HSP70-2 genotypes were analyzed by PCR-RFLP, using NcoI and PstI restriction enzymes. Results: Two of the 29 members were homozygotes for allele A, 18 were heterozygotes (TNF A/G) and 9 of them were homozygotes for allele G. Nineteen of the 29 were heterozygotes for HSP70-2 (A/G), 10 of them were homozygotes for the G allele, and none were homozygotes for allele A. Four of the 5 the RA patients carried the A allele for TNF-a all 5 were heterozygotes for HSP70-2 genotypes. Conclusion: The carriage of the A allele for TNF-a of -308 SNP in 4 of the 5 RA patients, and the high prevalence (68.0%) of TNF A allele carriers in this family confirms the important role of this candidate gene in the pathomechanism of RA, and might be of prognostic value for future clinical observations. Further, to test for association a much larger set of genetically independent patients and controls is needed. 

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Acta Physiologica Hungarica
Authors: S. Flores-Martínez, J. Martínez, M. Machorro-Lazo, A. García-Zapién, L. Salgado-Goytia, E. Cruz-Quevedo, M. Morán-Moguel, and J. Sánchez-Corona

The analysis of polymorphic markers within or closely linked to the cystic fibrosis transmembrane regulator (CFTR) gene is useful as a molecular tool for carrier detection of known and unknown mutations. To establish the association between mutations in the CFTR gene in western Mexican cystic fibrosis (CF) patients, the distribution of XV2c/KM19 haplotypes was analyzed by PCR and restriction enzyme digestion in 384 chromosomes from 74 CF patients, their unaffected parents, and normal subjects.The haplotype analysis revealed that haplotype B was present in 71.9% of CF chromosomes compared to 0% of non-CF chromosomes. The F508del and G542X mutations were strongly associated with haplotype B (96.7% and 100% of chromosomes, respectively). The haplotype distribution of the CF chromosomes carrying other CFTR mutations had a more heterogeneous background.Our results show that haplotype B is associated with CFTR mutations. Therefore, haplotype analysis is a suitable alternate strategy for screening CF patients with a heterogeneous clinical picture from populations with a high molecular heterogeneity where carrier detection programs are not available. In addition, it may be a helpful diagnostic tool for genetic counseling and carrier detection in the relatives of CF patients and in couples who are planning to have children.

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In PI466495, a powdery mildew resistance source of wild barley ( Hordeum vulgare ssp. spontaneum ), one gene conferring powdery mildew resistance was identified in the Mla locus. In this paper, the RGH1a gene sequence was used as source for the development of a cleaved amplified polymorphic sequence (CAPS) marker. Co-segregation between this marker and powdery mildew resistance was analysed by specific DNA fragments associated with each allele of the gene using 286 F 2 plants derived from a cross between winter barley ( H. vulgare L.) variety ‘Tiffany’ and PI466495. For the co-dominant marker RGH1aI1a , three fragments, 370 bp, 82 bp and 59 bp in size, were amplified from F 2 plants exhibiting resistance reaction types 0 and 0–1 to powdery mildew; whereas two fragments, 429 bp and 82 bp in size, were amplified in susceptible plants. Simple procedures based on polymerase chain reaction and restriction enzyme digestion allowed for identifying the plants susceptible to powdery mildew ( Blumeria graminis f. sp. hordei ) and plants homozygous or heterozygous for the resistance allele. The RGH1aI1a marker was positioned 0.85 cM to the resistance gene and the efficiency of marker-assisted selection (MAS), evaluated as the probability of crossing-over between the marker and the targeted gene, was 99%. The CAPS marker RGH1aI1a is a valuable candidate for MAS and gene transfer into barley varieties susceptible to powdery mildew.

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An account is given using typing methods and detection of virulence genes of different serotypes of Escherichia coli isolated in Hungary. By hybridization using SLT-I and SLT-II probes and PCR method using stx1-2, eae and ehx primers we could differentiate O157 strains of different serotypes into eight  (stx, eae, ehxA positive; stx, eae positive; stx, ehxA positive; stx positive; eae, ehxA positive; eae positive; ehxA positive; stx, eae, ehxA negative) types. The discriminatory power of phage typing proves to be much higher than that of the plasmid profile. RAPD typing with different primers could confirm or exclude the subtypes identity of the isolated E. coli O157 serotypes. Escherichia coli O157:HNM isolates could be sorted in six different phage types and six different RAPD types with ERIC-1, in five RAPD types with ERIC-2 and in seven types with M13 primers. Escherichia coli O157:H7 showed six different phage types and three RAPD types with ERIC-1 and ERIC-2 and five types with M13 primers. According to our results the standard PFGE protocol [32] gives the opportunity to differentiate epidemiologically independent but evolutionary related or unrelated isolates, but the practical value of PFGE method for epidemiological purposes must be confirmed by other or more restriction enzymes or using an other protocol. Summarizing our results we suggest the use of phage and RAPD typing and in doubtful cases the PFGE method.

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Acta Veterinaria Hungarica
Authors: Zsuzsanna Varga, Boglárka Sellyei, Petra Paulus, Melitta Papp, Kálmán Molnár, and Csaba Székely

The objective of this study was to survey the incidence of Flavobacterium columnare in wild and cultured freshwater fish species in Hungary. This bacterium usually causes disease in waters of more than 25 °C temperature. However, with the introduction of intensive fish farming systems, infected fish exposed to stress develop disease signs also at lower temperatures; in addition, the temperature of natural waters rises to the critical level due to global warming. Twenty-five isolates from wild and cultured freshwater fishes were identified as F. columnare by specific PCR, although both the fragment lengths and the results of PCRRFLP genotyping with BsuRI (HaeIII) and RsaI restriction enzymes raised doubts regarding this species classification. Sequencing of the 16S ribosomal RNA gene revealed that 23 isolates belonged to the species F. johnsoniae and two represented Chryseobacterium spp. The isolates were found to have high-level multidrug resistance: all were resistant to ampicillin and polymyxin B, the 23 F. johnsoniae strains to cotrimoxazole, 88% of them to gentamicin, and 72% to chloramphenicol. The majority of the 25 isolates were sensitive to erythromycin (88%), furazolidone (76%), and florfenicol (68%).

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Six strains (Bi11, Bi30, Bi36, Bi50, Bi52 and Bi55) isolated from bio-yoghurts and two strains (KD10 and KD11) derived from human faeces were identified by genus- and species specific polymerase chain reaction (PCR) with reference to the type strains of B. animalis subsp. lactis DSM 10140 and B. animalis subsp. animalis DSM 20104. The isolates were differentiated by using Bcu I ( Spe I), Xba I and Dra I endonucleases for subsequent pulsed field gel electrophoresis (PFGE) technique and by API 50 CHL tests.All the isolates tested were classified to B. animalis subsp. lactis species. The reliable identification as B. animalis subsp. lactis (by PCR with Bflact2/Bflact5 primers), however, required confirmation by a negative result of B. animalis subsp. animalis -specific PCR.Differentiation of the B. animalis subsp. lactis isolates with PFGE method enabled to distinguish KD11 strain with all the restriction enzymes applied, and Bi11 and Bi30 — exclusively with Dra I and Spe I enzymes, respectively. The biochemical tests, however, revealed that all the strains tested were characterised by a unique fermentation pattern. It was concluded that differentiation of the B. animalis subsp. lactis strains should be carried out on the basis of both genetic and phenotypic features.

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Orvosi Hetilap
Authors: János Megyesi, Anna Biró, László Wigmond, Jenő Major, and Anna Tompa

Bevezetés: A comet assay a DNS száltöréseinek kimutatására alkalmas fluoreszcens mikroszkópos módszer. Jelentősége az alacsony sejtszámú mintáknál mutatkozik, képes számokban kifejezni a DNS-károsodást a nem proliferáltatható sejtekben. Célkitűzés: Genotoxikus, illetve oxidatív DNS-károsodásokat mértünk foglalkozásuk során benzollal, policiklusos aromás szénhidrogénekkel, illetve sztirollal exponált csoportokban. Célunk volt annak megvizsgálása, hogy a módszer használható-e genotoxikológiai monitorhatás markereként. Módszer: A comet assay alaplépései mellett az enzimkezelt mintát formamido-pirimidin-DNS glikoláz restrikciós enzimmel kezeljük, ami az oxidatív DNS-károsodás mértékére utal. Eredmények: A kezeletlen (genotoxikus DNS-károsodás) és a kezelt (oxidatív DNS-károsodás) minták esetében emelkedés volt tapasztalható a csóvahosszokat illetően minden csoporton belül a kontrollhoz képest. Következtetések: Megállapítható, hogy a környezeti (munkahelyi) expozíció valószínűsíthető a vizsgált csoportokban. A comet assay kitűnő hatásmarker, illetve kiegészítő módszer lehet egy monitorrendszerben, amelynek adatai tájékoztatást adhatnak a munkahelyeken emelkedett genotoxikus hatások jelenlétéről vagy hiányáról. Orv. Hetil., 2014, 155(47), 1872–1875.

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Orvosi Hetilap
Authors: Katalin Csép, Márta Vitay, Gyöngyi Dudutz, László Rosivall, and László Korányi

Célkitűzés: A FABP2 (intestinal fatty acid–binding protein)-gén a vékonybél hámsejtjeiben található és a zsírsavak metabolizmusában vélhetően fontos szerepet betöltő fehérjét kódol. A gén anyagcserezavarokra hajlamosító A54T-polimorfizmusának kapcsolatát vizsgáltuk az IDF által 2005-ben ajánlott kritériumok alapján diagnosztizált metabolikus szindrómával a marosvásárhelyi populációnál. Anyag és módszer – Eset-kontroll tanulmányt végeztünk 144 metabolikus szindrómás betegnél és 73 hasonló korú és életmódot folytató egészséges személynél. Az inzulinérzékenységet HOMA- és QUICKI-indexek számítása révén ítéltük meg, míg a génpolimorfizmust PCR és ezt követő Hha I restrikciós enzimmel történő hasítás segítségével vizsgáltuk. Eredmények: A T54 gyakrabban fordult elő a metabolikus szindrómás betegeknél, mint az egészséges kontrollcsoportnál (35,71% vs. 28,08%, p < 0,05). A T54-allél jelenlétében tapasztalt megbetegedési kockázat enyhe, de statisztikailag szignifikáns, és kifejezettebb homozigóták esetében (TT vs. AT + OR = 4,31, CI 95% 1,21–5,29, p = 0,015 és TT vs. OR = 4,61, CI95%: 1,24–7,03, p = 0,0195). Nem tapasztaltunk statisztikailag szignifikáns különbséget a követett anyagcsere-paraméterekben a három eltérő genotípusnak megfelelően a két vizsgálati csoportban. Következtetések: Eredményeink alapján a FABP2-gén T54-allélja egy poligénes rendszer keretén belül szerepet játszhat a metabolikus szindrómára való hajlam kialakításában.

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