Introduction. Species typing in leishmaniasis gains importance in diagnostics, epidemiology, and clinical studies. A restriction fragment length polymorphism (RFLP) assay of PCR amplicons from a partial heat-shock protein 70 gene (hsp70) had been described for the New World, allowing identification of some species. Methods. Based on an initial in silico analysis of 51 hsp70 sequences, most of which we recently determined in the frame of a phylogenetic study, species-specific restriction sites were identified. These were tested by PCR-RFLP on 139 strains from 14 species, thereby documenting both inter- and intra-species variability. Results. Our assay could identify Leishmania infantum, L. donovani, L. tropica, L. aethiopica, L. major, L. lainsoni, L. naiffi, L. braziliensis, L. peruviana, L. guyanensis, and L. panamensis by applying 2 subsequent digests. L. mexicana, L. amazonensis, and L. garnhami did not generate species-specific restriction fragment patterns. Conclusion. Currently no assay is available for global Leishmania species discrimination. We present a universal PCR-RFLP method allowing identification of most medically relevant Old and New World Leishmania species on the basis of a single PCR, obviating the need to perform separate PCRs. The technique is simple to perform and can be implemented in all settings where PCR is available.

Pseudorabies virus (PrV) infections appear to be more widely distributed in the European wild boar (Sus scrofa) population than assumed. In Europe, attempts to isolate and characterize the causative agents have been limited so far. We therefore collected and examined a total of 35 PrV isolates obtained from wild boar or hunting dogs in Germany, France, Spain, Italy, Slovakia and Hungary between 1993 and 2008. Restriction enzyme analysis of genomic DNA using BamHI showed that all isolates, except one, belonged to genogroup I but different subtypes were evident. For further investigations of the phylogenetic relationships, a 732-bp fragment of the glycoprotein C (gC) gene was amplified by PCR. Sequence analysis revealed about 40 variant positions within this fragment. Comparison of the nucleotide sequences supported the separation into a clade containing isolates from North-Rhine Westphalia, Rhineland-Palatinate (Germany), France and Spain (clade B) and an apparently more variable clade comprising isolates from Brandenburg, Baden-Wurttemberg, Saxony, Saxony-Anhalt (Germany), Slovakia, Hungary, Italy and France (clade A).

Two clones of East African Trypanosoma brucei, with distinct homozygous isoenzyme patterns for one of three enzymes examined, were cotransmitted through the tsetse fly vector Glossina morsitans centralis. Flies with mature infections were individually fed on mice and the subsequent bloodstream form populations analysed for the presence of hybrid trypanosomes by isoenzyme analysis. Several combinations have previously been detected using this approach (Schweizer, Tait & Jenni, 1988; Sternberg et al. 1989). Four clones were isolated from one of the hybrid-containing populations. They showed a hybrid phenotype, as would be expected for the F1 progeny in a diploid Mendelian system. The analysis of the progeny clones, using two gene probes which detect restriction fragment length polymorphisms between the two parental stocks, showed that alleles had segregated at each locus and given rise to three different non-parental combinations of alleles in the hybrid progeny. Characterization of the hybrid progeny clones by PFGE (pulsed field gradient gel electrophoresis) revealed that all progeny clones were recombinant for the intermediate size chromosomes. From the analysis of the segregation of the larger chromosomes, marked by P0K (phosphoglycerate kinase) and CP (cysteine protease) gene probes, it was inferred that the progeny clones did not result from a direct fusion of diploid cells. Results with the PGK probe fit into a classical system with meiosis and subsequent fusion of the nuclei to form diploid progeny. On the other hand, blots with the CP probe as well as some of the ethidium bromide stained PFGE gels revealed the existence of non-parental size chromosomes in some of the hybrid progeny. This phenomenon was observed previously (Gibson, 1989) and further investigation is required to elucidate the mechanism.

The genetic diversity of dinitrogen-fixing bacteria associated with rice (Oryza sativa) was assessed by a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) approach on the nifH gene amplified directly from DNA extracted from washed rice roots and rhizospheric soil. Restriction digestion with the enzymes MnlI and HaeIII was performed to characterize 54 cloned nifH PCR products. RFLP profiles were clustered and analysed with the UPGMA program. Eight pairs of similar RFLP patterns (similarity>50%) and two pairs of homologous RFLP patterns (100% identity) were found from the washed roots and the rhizospheric soil, respectively. Three specific diazotrophic patterns were found from rhizospheric soil and rice roots. The analyses have revealed the presence of different nifH types, which appear to be significant components of the diazotrophic community in paddy fields, indicating that some of the diazotrophs may colonize the inside and the surface of the rice roots.

In order to evaluate the genotype of the calpastatin (CAST) gene and its relationship to muscle histology and other post mortem traits in the Jinhua × Piétrain F2 pig family, 158 barrows and gilts were electrically stunned and exsanguinated. Both blood and muscle samples were collected, and both post mortem traits and meat qualities were recorded. Restriction fragment length polymorphism (RFLP) analysis, the periodic acid Schiff reaction (PAS) and myosin heavy-chain immunohistochemistry were employed to explore the relationship between genotype and muscle histology. Based on PAS reactivity, muscle fibres can be classified into three types: PAS (−), PAS (+) and PAS (++). Myosin heavy-chain immunohistochemistry can differentiate muscle fibres into either slow or fast fibres; the proportion of slow and fast fibres were 6% and 94%, respectively. When the amplification products of the CAST gene were digested with MspI, HinfI and RsaI, two different cleavage patterns could be discriminated from the endonuclease map detected using each enzyme. The results showed that the polymorphisms detected using these three endonucleases are identical. Only three genotypes (AA/CC/EE, AB/CD/EF and BB/DD/FF) were distinguished. Their frequencies were 0.1835, 0.5823 and 0.2342, respectively. Different genotypes had significant association with area and pH45m value of loin muscle, while showing no significant association with the water-holding capacity and conductivity of loin muscle. The results also revealed that the genotypes had a significant correlation with diameter, area, circularity and the aspect ratio of muscle fibres. It was also presented that the genotypes significantly correlated with the percentage of intramuscular connective tissue.

We partially sequenced the mitochondrial D-loop region in 47 individuals from eleven Spanish and foreign goat breeds. Phylogenetic analysis of these sequences allowed us to identify a particular D-loop haplotype shared by individuals from the Palmera, Majorera and Tinerfeña Canarian breeds. Genotyping of 281 goats from 17 different breeds by PCR-HpaII RFLP evidenced that the geographical distribution of this haplotype is restricted to the Canary Islands. This ancestral mitochondrial haplotype might originate in the domestic goat herds brought by the native Canarian inhabitants approximately 3000 years ago. Although we observed other miscellaneous D-loop haplotypes in the Palmera, Majorera and Tinerfeña breeds, any of them allowed us to group individuals from these three populations in a single cluster, a feature that suggests that these haplotypes might have diverse origins. The remarkable degree of phylogeographic structure of the Canary goat breeds with regard to other Spanish and foreign populations might be attributed to the isolation of these breeds in the Canary Islands for approximately 2500 years, without exposure to the migratory movements and commercial trading events that probably affected the genesis of most domestic goat breeds worldwide. The Canarian D-loop haplotype can be efficiently genotyped by using DNA isolated from milk and cheese samples, which paves the way for the future establishment of a Canary breed identity test for these dairy products.

Intergenic region typing by restriction analysis of the ribosomal internal transcribed spacer (ITS) and mini-exon provide diagnostic markers for some Leishmania. Here, we evaluate restriction analysis of these targets for genotyping and phylogenetic analysis within the Leishmania donovani complex (agents of visceral leishmaniasis). Each method was useful for genotyping of both L. donovani complex strains and Old World Leishmania species. The targets produced less robust groups than gp63 intergenic regions, but support the need for re-evaluation of the taxonomy of the L. donovani complex.

Riboprinting is one of several molecular methods that can generate comparative data independently of the complexity of the organism's morphology. Restriction fragment length polymorphism (RFLP) profiles derived from digestion of polymerase chain reaction (PCR) products of the ribosomal 18S from Leishmania spp. yields a typical ‘riboprint’ profile that can vary intraspecifically. A selection of 76 stocks of L. major and L. tropica, isolated from patients with cutaneous leishmaniasis, was analysed by riboprinting to assess divergence within and between species. L. major and L. tropica could be easily differentiated from each other. Analysis of PCR–RFLP profiles indicated that stocks of Leishmania spp. could be broadly partitioned into 2 species corresponding to L. major and L. tropica. To test if ribosomal 18S sequences were homogeneous within each species, several isolates of each of the Leishmania spp. were digested. Interpretation of the riboprint profiles of the 18S independently amplified by PCR, there would appear to be one restriction pattern present within each Leishmania spp. Homogeneity within copies of the ribosomal 18S within a single genome has, therefore, been demonstrated. The species designation established by riboprinting results were in agreement with the zymodeme analysis of the same isolates. The restriction patterns produced were simple, reproducible and easy to interpret.

Polymerase chain reaction (PCR) primers specific to exon 2 of the bovine lymphocyte antigen (BoLA)-DRB3 gene were used successfully to amplify the equivalent region in 34 Murrah and 36 Surti buffaloes selected at random. The 304 bp amplified product of the DRB3 gene was separately digested with BstγI, HaeIII and Rsal enzymes. Digestion with BstγI enzyme did not reveal any polymorphism and all animals showed a single restriction pattern, which corresponded exactly to the BstγI pattern ‘b’ previously described for cattle. Digestion with HaeIII enzyme resulted in five patterns, four of which corresponded to the Haelll patterns previously reported in cattle. The new HaeIII pattern was observed in both the breeds of buffaloes studied. The fragment analysis with RsaI revealed 13 different patterns. All of these RsaI patterns corresponded to the RsaI patterns previously described for cattle. The high degree of similarity in the restriction fragment length polymorphism (RFLP) patterns of cattle and buffalo observed in the present study provide evidence for the strong conservation amongst other bovine species, of restriction sites previously reported in cattle.

The positive effect of administration of growth hormone (GH) on milk production and its presence in selected dairy cattle lines of higher GH concentrations prompted an examination of the presence of restriction fragment length polymorphism (RFLP) in the growth hormone gene using the enzyme Taq/ and to investigate associations between this polymorphism and milk production traits. Blood was sampled from 251 Italian Holstein-Friesian cows. Three fragment bands, arbitrarily denoted A, B and E, of 6·2, 5·2 and 1·9 kilobase (kb), respectively, were observed. Their combinations exhibited six patterns, AA, AB, ABE, AE, BB and BE with frequencies of 64·5, 24·3, 2·4, 6·8, 1·4 and 0·4%, respectively. The statistical analysis was performed using linear mixed animal models. The results indicated an effect of the GHgene polymorphic Taq/restriction fragment on 305-day productions of milk, fat and protein; the low frequency pattern AE showed productions inferior to those for AA or AB patterns. Effect estimates of AA, AB and AE were, respectively, 200 (s.e. 215), 218 (s.e. 267) and -910 (s.e. 380) kg for milk production, 7·75 (s.e. 7·98), 16·10 (s.e. 9·79) and -22·14 (s.e. 14·42) kg for fat production, and 6·78 (s.e. 6·21), 8·57 (s.e. 7·58) and -20·74 (s.e. 11·14) kg for protein production. The average substitution effect estimates of E were -891 (s.e. 278), -26·56 (s.e. 10·16) and -24·50 (s.e. 9·43) kg for milk, fat and protein yields, respectively. In conclusion, these results suggest that the E fragment deserves further designed and specific study.

Precision of zygosity determination in twins can be improved by the use of modern methods of DNA analysis. The clinical application of 4 single- (SLS) and 2 multi-locus (MLS), and 6 PCR (polymerase chain reaction) systems for zygosity determination in 12 twin pairs with oral clefts was compared with regard to the quality and quantity of sample material required and the probability of error in monozygosity determination. PCR systems proved to be superior to SLS or MLS, as DNA sampling is much more convenient, while its level of accuracy still fulfils clinical requirements. For this reason, PCR systems should be considered a basic method in modern clinical twin research.

The use of DNA restriction fragment length polymorphisms (RFLP) to analyze variable number of tandem repeat (VNTR) sequences dispersed in the human genome, has become a powerful tool for the study of population genetics due to the very substantial polymorphism involved. Because the markers usually employed for twin zygosity determination (such as sex combination, placentation, HLA typing, blood group antigens, etc) may not be uniformly informative, we propose the use of synthetic olygonucleotides, representing VNTR “core” sequences, for the determination of zygosity in twins.