Genetic profile of scrapie codons 146, 211 and 222 in the PRNP gene locus in three breeds of dairy goats

Polymorphisms at PRNP gene locus have been associated with resistance against classical scrapie in goats. Genetic selection on this gene within appropriate breeding programs may contribute to the control of the disease. The present study characterized the genetic profile of codons 146, 211 and 222 in three dairy goat breeds in Greece. A total of 766 dairy goats from seven farms were used. Animals belonged to two indigenous Greek, Eghoria (n = 264) and Skopelos (n = 287) and a foreign breed, Damascus (n = 215). Genomic DNA was extracted from blood samples from individual animals. Polymorphisms were detected in these codons using Real-Time PCR analysis and four different Custom TaqMan® SNP Genotyping Assays. Genotypic, allelic and haplotypic frequencies were calculated based on individual animal genotypes. Chi-square tests were used to examine Hardy-Weinberg equilibrium state and compare genotypic distribution across breeds. Genetic distances among the three breeds, and between these and 30 breeds reared in other countries were estimated based on haplotypic frequencies using fixation index FST with Arlequin v3.1 software; a Neighbor-Joining tree was created using PHYLIP package v3.695. Level of statistical significance was set at P = 0.01. All scrapie resistance-associated alleles (146S, 146D, 211Q and 222K) were detected in the studied population. Significant frequency differences were observed between the indigenous Greek and Damascus breeds. Alleles 222K and 146S had the highest frequency in the two indigenous and the Damascus breed, respectively (ca. 6.0%). The studied breeds shared similar haplotypic frequencies with most South Italian and Turkish breeds but differed significantly from North-Western European, Far East and some USA goat breeds. Results suggest there is adequate variation in the PRNP gene locus to support breeding programs for enhanced scrapie resistance in goats reared in Greece. Genetic comparisons among goat breeds indicate that separate breeding programs should apply to the two indigenous and the imported Damascus breeds

PRNP genetic variability and molecular typing of natural goat scrapie isolates in a high number of infected flocks

One hundred and four scrapie positive and 77 negative goats from 34 Greek mixed flocks were analysed by prion protein gene sequencing and 17 caprine scrapie isolates from 11 flocks were submitted to molecular isolate typing. For the first time, the protective S146 variant was reported in Greece, while the protective K222 variant was detected in negative but also in five scrapie positive goats from heavily infected flocks. By immunoblotting six isolates, including two goat flockmates carrying the K222 variant, showed molecular features slightly different from all other Greek and Italian isolates co-analysed, possibly suggesting the presence of different scrapie strains in Greece

Genetic profile of scrapie codons 146, 211 and 222 in the PRNP gene locus in three breeds of dairy goats

Polymorphisms at PRNP gene locus have been associated with resistance against classical scrapie in goats. Genetic selection on this gene within appropriate breeding programs may contribute to the control of the disease. The present study characterized the genetic profile of codons 146, 211 and 222 in three dairy goat breeds in Greece. A total of 766 dairy goats from seven farms were used. Animals belonged to two indigenous Greek, Eghoria (n = 264) and Skopelos (n = 287) and a foreign breed, Damascus (n = 215). Genomic DNA was extracted from blood samples from individual animals. Polymorphisms were detected in these codons using Real-Time PCR analysis and four different Custom TaqMan® SNP Genotyping Assays. Genotypic, allelic and haplotypic frequencies were calculated based on individual animal genotypes. Chi-square tests were used to examine Hardy-Weinberg equilibrium state and compare genotypic distribution across breeds. Genetic distances among the three breeds, and between these and 30 breeds reared in other countries were estimated based on haplotypic frequencies using fixation index FST with Arlequin v3.1 software; a Neighbor-Joining tree was created using PHYLIP package v3.695. Level of statistical significance was set at P = 0.01. All scrapie resistance-associated alleles (146S, 146D, 211Q and 222K) were detected in the studied population. Significant frequency differences were observed between the indigenous Greek and Damascus breeds. Alleles 222K and 146S had the highest frequency in the two indigenous and the Damascus breed, respectively (ca. 6.0%). The studied breeds shared similar haplotypic frequencies with most South Italian and Turkish breeds but differed significantly from North-Western European, Far East and some USA goat breeds. Results suggest there is adequate variation in the PRNP gene locus to support breeding programs for enhanced scrapie resistance in goats reared in Greece. Genetic comparisons among goat breeds indicate that separate breeding programs should apply to the two indigenous and the imported Damascus breeds

Four types of scrapie in goats differentiated from each other and bovine spongiform encephalopathy by biochemical methods

Scrapie in goats has been known since 1942, the archetype of prion diseases in which only prion protein (PrP) in misfolded state (PrPSc) acts as infectious agent with fatal consequence. Emergence of bovine spongiform encephalopathy (BSE) with its zoonotic behaviour and detection in goats enhanced fears that its source was located in small ruminants. However, in goats knowledge on prion strain typing is limited. A European-wide study is presented concerning the biochemical phenotypes of the protease resistant fraction of PrPSc (PrPres) in over thirty brain isolates from transmissible spongiform encephalopathy (TSE) affected goats collected in seven countries. Three different scrapie forms were found: classical scrapie (CS), Nor98/atypical scrapie and one case of CH1641 scrapie. In addition, CS was found in two variants—CS-1 and CS-2 (mainly Italy)—which differed in proteolytic resistance of the PrPres N-terminus. Suitable PrPres markers for discriminating CH1641 from BSE (C-type) appeared to be glycoprofile pattern, presence of two triplets instead of one, and structural (in)stability of its core amino acid region. None of the samples exhibited BSE like features. BSE and these four scrapie types, of which CS-2 is new, can be recognized in goats with combinations of a set of nine biochemical parameters

DRUG - RESISTANCE'S STUDY OF STRAINS OF GENUS PASTEURELLA AGAINST ANTIMICROBIALDRUGS

ΣΤΗΝ ΕΡΓΑΣΙΑ ΑΥΤΗ ΜΕΛΕΤΑΤΑΙ ΤΑ ΦΑΙΝΟΜΕΝΟ ΤΗΣ ΕΠΙΚΤΗΤΗΣ ΑΝΘΕΚΤΙΚΟΤΗΤΑΣ ΣΕ ΣΤΕΛΕΧΗ ΤΟΥ ΓΕΝΟΥΣ PASTEURELLA ΠΟΥ ΜΕΧΡΙ ΤΗΝ ΔΕΚΑΕΤΙΑ ΤΟΥ 1970 ΕΙΧΑΝ ΕΥΡΥ ΦΑΣΜΑ ΕΥΑΙΣΘΗΣΙΑΣ ΣΤΑ ΑΝΤΙΜΙΚΡΟΒΙΑΚΑ ΦΑΡΜΑΚΑ ΜΕ ΑΠΟΤΕΛΕΣΜΑ Η ΘΕΡΑΠΕΙΑ ΤΩΝ ΛΟΙΜΩΞΕΩΝ ΠΟΥ ΠΡΟΚΑΛΟΥΣΑΝ ΝΑ ΕΙΝΑΙ ΕΥΚΟΛΗ. Η ΕΡΕΥΝΑ ΜΑΣ ΕΙΧΕ ΩΣ ΣΚΟΠΟ ΝΑ ΔΕΙΞΕΙ: Α) ΤΗΝ ΥΠΑΡΞΗ ΚΑΙ ΤΟ ΦΑΣΜΑ ΤΗΣ ΠΟΛΥΑΝΘΕΚΤΙΚΟΤΗΤΑΣ, ΓΙΑ ΤΑ ΣΤΕΛΕΧΗ ΤΟΥ ΓΕΝΟΥΣ PANTEURELLA. Β) ΤΗΝ ΙΚΑΝΟΤΗΤΑ ΑΛΛΑ ΚΑΙ ΤΟΝ ΒΑΘΜΟ ΙΚΑΝΟΤΗΤΑΣ ΩΣ ΠΡΟΣ ΤΗΝ ΑΝΑΔΥΣΗ ΑΝΘΕΚΤΙΚΩΝ ΣΤΕΛΕΧΩΝ. Γ) ΤΗΝ ΥΠΑΡΞΗ ΑΝΘΕΚΤΙΚΟΤΗΤΑΣ ΣΕ ΜΗ ΠΑΘΟΓΟΝΑ ΣΤΕΛΕΧΗ. ΜΕΛΕΤΗΘΗΚΑΝ ΔΥΟ ΟΜΑΔΕΣ ΣΤΕΛΕΧΩΝ, Η ΠΡΩΤΗ ΑΠΟ 30 ΣΤΕΛΕΧΗ ΠΟΥ ΑΠΟΜΟΝΩΘΗΚΑΝ ΑΠΟ ΥΓΙΗ ΖΩΑ (ΜΗ ΠΑΘΟΓΟΝΑ ΣΤΕΛΕΧΗ) ΚΑΙ Η ΔΕΥΤΕΡΗ ΑΠΟ 14 ΣΤΕΛΕΧΗ ΠΟΥ ΑΠΟΜΟΝΩΘΗΚΑΝ ΑΠΟ ΑΡΡΩΣΤΑ ΚΟΥΝΕΛΙΑ (ΠΑΘΟΓΟΝΑ ΣΤΕΛΕΧΗ). ΜΕΤΑ ΤΗΝ ΤΑΥΤΟΠΟΙΗΣΗ ΤΩΝ ΣΤΕΛΕΧΩΝ ΕΓΙΝΑΝ ΑΝΤΙΒΙΟΓΡΑΜΜΑΤΑ ΚΑΙ ΠΡΟΣΔΙΟΡΙΣΤΗΚΕ Η ΕΛΑΧΙΣΤΗ ΑΝΑΣΤΑΛΤΙΚΗ ΠΥΚΝΟΤΗΤΑ (MIC) ΩΣ ΠΡΟΣ ΤΑΑΝΤΙΜΙΚΡΟΒΙΑΚΑ ΦΑΡΜΑΚΑ ΣΤΡΕΠΤΟΜΥΚΙΝΗ, ΣΟΥΛΦΟΝΑΜΙΔΗ ΚΑΙ ΤΡΙΜΕΘΟΠΡΙΜΗ. ΣΤΗΝ ΣΥΝΕΧΕΙΑ ΕΓΙΝΕ ΣΥΖΕΥΞΗ (ΜΕ ΜΙΚΤΗ) ΚΑΛΛΙΕΡΓΕΙΑ ΑΝΘΕΚΤΙΚΩΝ ΣΤΕΛΕΧΩΝ ΤΟΥ ΓΕΝΟΥΣ PASTEURELLA ΜΕ ΤΟ ΕΥΑΙΣΘΗΤΟ ΣΤΕΛΕΧΟΣ E. COLI KIZF RC85 ΜΕ ΣΤΟΧΟ: Α) ΤΟΝ ΥΠΟΛΟΓΙΣΜΟ ΤΗΣ ΣΥΧΝΟΤΗΤΑΣ ΜΕΤΑΒΙΒΑΣΗΣ ΤΗΣ ΑΝΘΕΚΤΙΚΟΤΗΤΑΣ ΑΠΟ ΤΑ ΑΝΘΕΚΤΙΚΑ ΣΤΕΛΕΧΗ ΤΟΥΓΕΝΟΥΣ PASTEURELLA ΣΤΟ ΕΥΑΙΣΘΗΤΟ ΣΤΕΛΕΧΟΣ ΤΗΣ E. COLI ΜΕ ΑΝΤΙΜΙΚΡΟΒΙΑΚΑ ΕΠΙΛΟΓΗΣ ΣΤΡΕΠΤΟΜΥΚΙΝΗ, ΒΟΥΛΦΟΝΑΜΙΔΗ ΚΑΙ ΤΡΙΜΕΘΟΠΡΙΜΗ. Η ΣΥΧΝΟΤΗΤΑ ΚΥΜΑΝΘΗΚΕ ΑΠΟ 4Χ10^-7 ΕΩΣ 5,6Χ10^-4, ΑΠΟ 1,4Χ10^-5 ΕΩΣ 2,6Χ10^-3 ΚΑΙ ΑΠΟ (ΠΕΡΙΚΟΠΗ ΠΕΡΙΛΗΨΗΣ

Indigenous Lactic Acid Bacteria Isolated from Raw Graviera Cheese and Evaluation of Their Most Important Technological Properties

The aim of the present study was to characterize LAB isolates from raw-milk cheeses, to evaluate some of their technological properties and to select a few ‘wild’ LAB strains that could potentially be used as starter cultures. LAB strains were isolated and identified from raw milk, curd, and cheese at 30, 60, and 90 days of ripening. A total of 100 strains were isolated, 20 from each phase of ripening. All isolates were tested for acidification ability, curd formation, and aroma production at 32 °C and 42 °C after 24 and 48 h. Following the acidification test, 42 strains were selected for identification and characterization of their technological properties. A high proportion of lactic acid bacteria and Gram + cocci were found throughout the cheese-making process. Enterococci reached their maximum proportion on the 7th day of ripening while Lactobacilli increased significantly during the first month of ripening. Forty-two strains were identified by phenotypic, biochemical, and molecular techniques. Lactococci were predominant in raw milk and curd while Lactobacilli in the ripening of the cheese. Four LAB strains including one Leuconostoc pseudomenteroides, two Lacticaseibacillus paracasei subsp. paracasei and one Enterococcus hirae, were proposed for their potential use as starters or secondary cultures

Low fraction of the 222K PrP variant in the protease-resistant moiety of PrPres in heterozygous scrapie positive goats

The presence of lysine (K) at codon 222 has been associated with resistance to classical scrapie in goats, but few scrapie cases have been identified in 222Q/K animals. To investigate the contribution of the 222K variant to PrPres formation in natural and experimental Q/K scrapie cases, we applied an immunoblotting method based on the use of two different monoclonal antibodies, F99/97.6.1 and SAF84, chosen for their different affinities to 222K and 222Q PrP variants. Our finding that PrPres seems to be formed nearly totally by the 222Q variant provides evidence that the 222K PrP variant confers resistance to conversion to PrPres formation and reinforces the view that this mutation has a protective role against classical scrapie in goats

Polymorphisms of Codons 110, 146, 211 and 222 at the Goat PRNP Locus and Their Association with Scrapie in Greece

Scrapie is considered an endemic disease in both sheep and goats in Greece. However, contrary to sheep, in goats more than one prion protein (PrP) polymorphism has been recognized as a candidate for resistance breeding against the disease. For an impression, candidates which are circulating, (i) brain samples (n = 525) from scrapie-affected (n = 282) and non-affected (n = 243) animals within the national surveillance program, and (ii) individual blood samples (n = 1708) from affected (n = 241) and non-affected (n = 1467) herds, in a large part of mainland Greece and its islands, were collected and assayed. A dedicated Taqman method was used to test for amino acid polymorphisms 110T/P, 146N/S/D, 211R/Q, and 222Q/K. Highly prevalent genotypes were 110TT, 146NN, 211RR, and 222QQ. The frequencies of polymorphisms in blood and negative brain samples for codons 110P, 211Q, and 222K were 4.0%, 3.0%, and 1.9%, respectively, while 146D (0.7%) was present only on Karpathos island. Codon 110P was exclusively found in scrapie-negative brains, and homozygous 110P/P in two scrapie-negative goats. It is concluded that breeding programs in Karpathos could focus on codon 146D, while in other regions carriers of the 110P and 222K allele should be sought. Case-control and challenge studies are now necessary to elucidate the most efficient breeding strategies