Standardization of a SNP panel for parentage verification and identification in the domestic cat (Felis silvestris catus).

The domestic cat (Felis silvestris catus) is a valued companion animal throughout the world. Over 60 different cat breeds are accepted for competition by the cat fancy registries in different countries. Genetic markers, including short tandem repeats and SNPs, are available to evaluate and manage levels of inbreeding and genetic diversity, population and breed structure relationships, and individual identification for forensic and registration purposes. The International Society of Animal Genetics (ISAG) hosts the Applied Genetics in Companion Animals Workshop, which supports the standardization of genetic marker panels and genotyping for the identification of cats via comparison testing. SNP panels have been in development for many species, including the domestic cat. An ISAG approved core panel of SNPs for use in cat identification and parentage analyses is presented. SNPs (n = 121) were evaluated by different university-based and commercial laboratories using 20 DNA samples as part of the ISAG comparison testing procedures. Different SNP genotyping technologies were examined, including DNA arrays, genotyping-by-sequencing and mass spectroscopy, to select a robust and efficient panel of 101 SNPs as the ISAG core panel for cats. The SNPs are distributed across all chromosomes including two on the X chromosome and an XY pseudo-autosomal sexing marker (zinc-finger XY; ZFXY). A population study demonstrated that the markers have an average polymorphic information content of 0.354 and a power of exclusion greater than 0.9999. The SNP panel should keep testing affordable while also allowing for the development of additional panels to monitor health, phenotypic traits, hybrid cats and highly inbred cats.

FTO gene variants are associated with growth and carcass traits in cattle.

An important aim in animal breeding is the improvement of growth and meat quality traits. Previous studies have demonstrated that genetic variants in the fat mass and obesity associated (FTO) gene have a relatively large effect on human obesity as well as on body composition in rodents and, more recently, in livestock. Here, we examined the effects of the FTO gene variants on growth and carcass traits in the Slovenian population of Simmental (SS) and Brown (SB) cattle. To validate and identify new polymorphisms, we used sequencing, PCR-RFLP analysis and TaqMan assays in the SS breed and FTO gene variants data from the Illumina BovineSNP50 v1 array for the SB breed. Sequencing of the eight samples of progeny-tested SS sires detected 108 single nucleotide polymorphisms (SNPs) in the bovine FTO gene. Statistical analyses between growth and carcass traits and 34 FTO polymorphisms revealed significant association of FTO variants with lean meat percentage in both breeds. Additionally, FTO SNPs analyzed in SS cattle were associated with fat percentage, bone weight and live weight at slaughter. The FTO gene can thus be regarded as a candidate gene for the marker-assisted selection programs in our and possibly other populations of cattle. Future studies in cattle might reveal novel roles for the FTO gene in shaping carcass traits in livestock species as well as body composition control in other mammals.

Relative quantification of beta-casein expression in primary goat mammary epithelial cell lines.

Primary mammary epithelial cell cultures were established from mammary tissue of lactating and non-lactating goats to assess the expression of beta-casein (CSN2) in vitro. Primary cell cultures were established by enzymatic digestion of mammary tissue and characterized using antibodies against cytokeratin 14, cytokeratin 18, and vimentin. The established primary cell lines in the second passage were grown in basal medium on plastic and in hormone-supplemented (lactogenic) medium on plastic and on an extracellular matrix-covered surface, respectively. CSN2 gene expression was evaluated using quantitative reverse transcription PCR. The presence of CSN2 transcripts was detected in all samples, including cells originating from non-lactating goat, grown in basal medium. The presence of CSN2 protein was confirmed using immunofluorescence. Response to the hormonal treatment and cell morphology differed between the cell lines and treatments. In 2 cell lines supplemented with lactogenic hormones in the medium, CSN2 expression was increased, while CSN2 levels in one of the cell lines remained constant, regardless of the treatment. Addition of extracellular matrix showed positive effects on CSN2 transcription activity in 1 of the cell lines, while in the other 2 showed no statistically significant effects. CSN2 expression appeared to depend on subtle differences in physiological state of the starting tissue material, growth conditions, cell types present in the culture, and methods used for cell culture establishment. Further studies are necessary to identify factors that determine hormone-responsiveness and transcriptional activity of milk protein genes in goat primary mammary cell cultures.

Multilocus sequence analysis for Mycoplasma synoviae molecular genotyping.

The aim of this study was to identify molecular techniques which enable clear discrimination between Mycoplasma synoviae isolates for improved epidemiology. Multilocus sequence analysis (MLSA) of 6 M. synoviae loci was conducted for genotyping of isolates with previously determined 5'-vlhA sequences. Sequencing of three polymorphic genes (5'-vlhA, cysP and nanH) enables good discrimination between isolates with different genotypes. Such a genotyping scheme revealed 10 distinct genotypes, which were confirmed by sequencing of an additional three loci of the M. synoviae genome. Epidemiologically linked strains formed clusters with the same genotypes which clearly differed between clusters. MLSA used in this study is a promising tool for epidemiology of M. synoviae isolates, but it should be evaluated by further investigations using a much higher number of M. synoviae strains.

Population structure and genetic differentiation of livestock guard dog breeds from the Western Balkans.

Livestock guard dog (LGD) breeds from the Western Balkans are a good example of how complex genetic diversity pattern observed in dog breeds has been shaped by transition in dog breeding practices. Despite their common geographical origin and relatively recent formal recognition as separate breeds, the Karst Shepherd, Sarplaninac and Tornjak show distinct population dynamics, assessed by pedigree, microsatellite and mtDNA data. We genotyped 493 dogs belonging to five dog breeds using a set of 18 microsatellite markers and sequenced mtDNA from 94 dogs from these breeds. Different demographic histories of the Karst Shepherd and Tornjak breeds are reflected in the pedigree data with the former breed having more unbalanced contributions of major ancestors and a realized effective population size of less than 20 animals. The highest allelic richness was found in Sarplaninac (5.94), followed by Tornjak (5.72), whereas Karst Shepherd dogs exhibited the lowest allelic richness (3.33). Similarly, the highest mtDNA haplotype diversity was found in Sarplaninac, followed by Tornjak and Karst Shepherd, where only one haplotype was found. Based on FST differentiation values and high percentages of animals correctly assigned, all breeds can be considered genetically distinct. However, using microsatellite data, common ancestry between the Karst Shepherd and Sarplaninac could not be reconstructed, despite pedigree and mtDNA evidence of their historical admixture. Using neighbour-joining, STRUCTURE or DAPC methods, Sarplaninac and Caucasian Shepherd breeds could not be separated and additionally showed close proximity in the NeighborNet tree. STRUCTURE analysis of the Tornjak breed demonstrated substructuring, which needs further investigation. Altogether, results of this study show that the official separation of these dog breeds strongly affected the resolution of genetic differentiation and thus suggest that the relationships between breeds are not only determined by breed relatedness, but in small populations even more importantly by stochastic effects.

Genome-wide in silico screening for microRNA genetic variability in livestock species.

MicroRNAs are a class of non-coding RNAs that post-transcriptionally regulate target gene expression. Previous studies have shown that microRNA gene variability can interfere with its function, resulting in phenotypic variation. Polymorphisms within microRNA genes present a source of novel biomarkers for phenotypic traits in animal breeding. However, little is known about microRNA genetic variability in livestock species, which is also due to incomplete data in genomic resource databases. Therefore, the aim of this study was to perform a genome-wide in silico screening of genomic sources and determine the genetic variability of microRNA genes in livestock species using mirna sniper 3.0 (http://www.integratomics-time.com/miRNA-SNiPer/), a new version of our previously developed tool. By examining Ensembl and miRBase genome builds, it was possible to design a tool-based generated search of 16 genomes including four livestock species: pig, horse, cattle and chicken. The analysis revealed 65 polymorphisms located within mature microRNA regions in these four species, including 28% within the seed region in cattle and chicken. Polymorphic microRNA genes in cattle and chicken were further examined for mapping to quantitative trait loci regions associated with production and health traits. The developed bioinformatics tool enables the analysis of polymorphic microRNA genes and prioritization of potential regulatory polymorphisms and therefore contributes to the development of microRNA-based biomarkers in livestock species. The assembled catalog and the developed tool can serve the animal science community to efficiently select microRNA SNPs for further quantitative and molecular genetic evaluations of their phenotypic effects and causal associations with livestock production traits.

On the origin of the Slovenian Cika cattle.

In 2002, it was discovered that several Cika cattle in the mountain areas of Slovenia had escaped the official policy of cross-breeding. Here, we report a genetic characterization to assess their status as autochthonous breed. We compared genotypes for 14 microsatellite markers in 150 Cika cattle individuals with data from 16 Central European cattle breeds. We show that Cika cattle are genetically as diverse as other Eastern Alpine breeds, are more diverse than Austrian Simmental but less than the Balkan Busha cattle. STRUCTURE analysis showed Pinzgauer admixture in several individuals but also indicated a unique genetic identity for Cika. This analysis also allowed a selection of the most genetically pure Cika individuals as assessed by the panel of microsatellites. These original Cika cattle form an Eastern Alpine breed cluster together with Pinzgauer and Pustertaler cattle. Cika cattle should be considered as an authentic and valuable genetic resource, which offers clear opportunities for sustainable agriculture and landscape conservation in marginal and mountain areas.

Distribution of chicken cathepsins B and L, cystatin and ovalbumin in extra-embryonic fluids during embryogenesis.

1. Concentrations of chicken cathepsin B, cathepsin L, cystatin and ovalbumin were determined in the allantoic fluid, amniotic fluid and extracts of chorioallantoic membranes during days 6 to 12 of embryogenesis. 2. Similar trends for cystatin and ovalbumin were observed in the allantoic fluid with maximum concentrations of cystatin on day 7 (12 ± 4 µg/ml) and ovalbumin on day 8 (∼19 ± 2.5 µg/ml) of embryonic development. The highest concentrations of cathepsin B was found on day 7 and of cathepsin L on day 10, but were significantly lower than those of cystatin and ovalbumin. 3. In the allantoic fluid, especially on day 7, considerable proportions of cystatin and ovalbumin were phosphorylated and contained phosphorylated serine. 4. Concentrations of cathepsin B and L, cystatin and ovalbumin in the amniotic fluid were variable but were comparable to those in allantoic fluid.

Comparative genomics approach to identify candidate genetic loci for male fertility.

The article presents multi-species, genome-wide, comparative approach to review male fertility-associated loci to contribute to the development of new genetic markers that could be of interest for functional studies and have the potential to be implemented in farm animal breeding programmes. We reviewed 835 male fertility-associated candidate loci from seven species and presented them as bovine orthologues where possible. The candidate loci were identified exploiting seven different research approaches: (i) data from animal models: mouse transgenics and knock-outs (569 genes) and random chemical mutagenesis of mouse genome (31); (ii) animal QTL (69); (iii) genes differentially expressed between fertile and subfertile phenotype in humans and mouse (95); (iv) DNA sequence variations that show specific allele-phenotype interactions (43 in human and 13 in farm animals); (v) germ line-specific small non-coding RNAs (47); (vi) testes expressed genes controlling complex differentiation process of mammalian spermatogenesis (6); and (vii) epigenetically regulated genes (4). According to the number of different research approaches reporting effects of individual genes, we selected 33 most promising candidate genes, which were further in silico analysed for expression levels in testes, genetic variability and top biological functions in functional networks. The aim of this study was to review systematically male fertility-associated candidate loci using integrated information from different study approaches and species, which will further facilitate development of novel genetic markers for selection towards improved fertility in domestic animals.

Topics and trends in Mountain Livestock Farming research: a text mining approach.

Pasture-based and small-scale livestock farming systems are the main source of livelihood in the mountain primary sector, ensuring socioeconomic sustainability and biodiversity in rural communities throughout Europe and beyond. Mountain livestock farming (MLF) has attracted substantial research efforts from a wide variety of scientific communities worldwide. In this study, the use of text mining and topic modelling analysis drew a detailed picture of the main research topics dealing with MLF and their trends over the last four decades. The final data corpus used for the analysis counted 2 679 documents, of which 92% were peer-reviewed scientific publications. The number of scientific outputs in MLF doubled every 10 years since 1980. Text mining found that milk, goat and sheep were the terms with the highest weighed frequency in the data corpus. Ten meaningful topics were identified by topic analysis: T1-Livestock management and vegetation dynamics; T2-Animal health and epidemiology; T3-Methodological studies on cattle; T4-Production system and sustainability; T5-Methodological studies; T6-Wildlife and conservation studies; T7-Reproduction and performance; T8-Dairy/meat production and quality; T9-Land use and its change and T10-Genetic/genomic studies. A hierarchical clustering analysis was performed to explore the interrelationships among topics, and three main clusters were identified: the first focused on sustainability, conservation and socioeconomic aspects (T4; T6 and T9), the second was related to food production and quality (T7 and T8) and the last one considered methodological studies on mountain flora and fauna (T1; T2; T3; T5 and T10). The 10 topics identified represent a useful and a starting source of information for further and more detailed analysis (e.g. systematic review) of specific research or geographical areas. A truly holistic and interdisciplinary research approach is needed to identify drivers of change and to understand current and future challenges faced by livestock farming in mountain areas.

Database of cattle candidate genes and genetic markers for milk production and mastitis.

A cattle database of candidate genes and genetic markers for milk production and mastitis has been developed to provide an integrated research tool incorporating different types of information supporting a genomic approach to study lactation, udder development and health. The database contains 943 genes and genetic markers involved in mammary gland development and function, representing candidates for further functional studies. The candidate loci were drawn on a genetic map to reveal positional overlaps. For identification of candidate loci, data from seven different research approaches were exploited: (i) gene knockouts or transgenes in mice that result in specific phenotypes associated with mammary gland (143 loci); (ii) cattle QTL for milk production (344) and mastitis related traits (71); (iii) loci with sequence variations that show specific allele-phenotype interactions associated with milk production (24) or mastitis (10) in cattle; (iv) genes with expression profiles associated with milk production (207) or mastitis (107) in cattle or mouse; (v) cattle milk protein genes that exist in different genetic variants (9); (vi) miRNAs expressed in bovine mammary gland (32) and (vii) epigenetically regulated cattle genes associated with mammary gland function (1). Fourty-four genes found by multiple independent analyses were suggested as the most promising candidates and were further in silico analysed for expression levels in lactating mammary gland, genetic variability and top biological functions in functional networks. A miRNA target search for mammary gland expressed miRNAs identified 359 putative binding sites in 3'UTRs of candidate genes.

Identification of chromosomal regions associated with growth and carcass traits in an F(3) full sib intercross line originating from a cross of chicken lines divergently selected on body weight.

An F(3) resource population originating from a cross between two divergently selected lines for high (D+ line) or low (D- line) body weight at 8-weeks of age (BW55) was generated and used for Quantitative Trait Locus (QTL) mapping. From an initial cross of two founder F(0) animals from D(+) and D(-) lines, progeny were randomly intercrossed over two generations following a full sib intercross line (FSIL) design. One hundred and seventy-five genome-wide polymorphic markers were employed in the DNA pooling and selective genotyping of F(3) to identify markers with significant effects on BW55. Fifty-three markers on GGA2, 5 and 11 were then genotyped in the whole F(3) population of 503 birds, where interval mapping with GridQTL software was employed. Eighteen QTL for body weight, carcass traits and some internal organ weights were identified. On GGA2, a comparison between 2-QTL vs. 1-QTL analysis revealed two separate QTL regions for body, feet, breast muscle and carcass weight. Given co-localization of QTL for some highly correlated traits, we concluded that there were 11 distinct QTL mapped. Four QTL localized to already mapped QTL from other studies, but seven QTL have not been previously reported and are hence novel and unique to our selection line. This study provides a low resolution QTL map for various traits and establishes a genetic resource for future fine-mapping and positional cloning in the advanced FSIL generations.

Polymorphisms in the kappa casein (CSN3) gene in horse and comparative analysis of its promoter and coding region.

The major parts of the coding region and promoter of the equine kappa casein (CSN3) gene were sequenced and compared among several species. Four SNPs were identified in the CSN3 gene: two in exon 1 and two in exon 4. The SNPs were genotyped in six Slovenian horse breeds using RFLP and two different PCR-based methods. The highest variation in genotype frequencies was found in the Slovenian cold-blood breed. The SNPs in exon 4 may cause a change in the amino acid sequence and may alter chemical/functional properties of the protein. Using horse-specific primers, we obtained 400 bp of exon 4 sequence from zebra and donkey. Two SNPs within the zebra exon 4 sequence were discovered; both presumably caused amino acid substitutions. Within the equine promoter sequence, 15 SNPs were found and 12 of them could be involved in the gain/loss of potential transcription factor (TF) binding sites. Using a comparative genomics approach, we obtained 1482 bp of the promoter sequence from zebra and donkey. Sequence alignment revealed highly conserved blocks of promoter sequence among nine species (sheep, goat, cow, zebra, donkey, horse, chimp, macaque and human) and clustered these species in three distinct groups. Consensus binding sites for TFs STAT5, C/EBP, NF1 and STAT6, previously demonstrated to be associated with expression, were located within conserved regions. Four promoter regions were tested for specific binding of TFs using electrophoretic mobility shift assays. Predicted binding sites for C/EBP and NF1 were confirmed and one conserved region was specifically detected by a yet-uncharacterized TF.

Evidence for two transferrin loci in the Salmo trutta genome.

To determine the organization of transferrin (TF) locus in the Salmo trutta genome, partial DNA and cDNA sequencing, fluorescent in situ hybridization (FISH) and Salmo salar BAC analysis were performed. TF expression levels and copy number prediction were assessed using real-time PCR. In addition to two previously reported DNA TF variant sequences of S. trutta and Salmo marmoratus (TF1), two novel variant sequences (TF2) were revealed in both species. Variant-specific sequence tags, characterizing two variants for each TF type (TF1 and TF2), were identified in genomic clones from each of the F1 hybrids between S. trutta and S. marmoratus. These clearly documented double heterozygote status at the TF loci. The real-time PCR data showed that each of the two TF types (TF1 and TF2) existed in one copy only and that the transcription of TF2 was considerably lower compared with TF1. Using FISH, hybridization signals were observed on two medium-sized acrocentric chromosomes of S. trutta karyotype. A TF type-specific PCR followed by a restriction analysis revealed the presence of two TF loci in the majority of analysed BAC clones. It was concluded that the TF gene is duplicated in the genome of S. trutta, and that the two TF loci are located adjacent to one another on the same chromosome. The differing transcription levels of TF1 and TF2 appear to depend on the corresponding promoter activity, which at least for TF2 seems to vary between different Salmo congeners.

Cellular reprogramming in farm animals: an overview of iPSC generation in the mammalian farm animal species.

Establishment of embryonic stem cell (ESC) lines has been successful in mouse and human, but not in farm animals. Development of direct reprogramming technology offers an alternative approach for generation of pluripotent stem cells, applicable also in farm animals. Induced pluripotent stem cells (iPSCs) represent practically limitless, ethically acceptable, individuum-specific source of pluripotent cells that can be generated from different types of somatic cells. iPSCs can differentiate to all cell types of an organism's body and have a tremendous potential for numerous applications in medicine, agriculture, and biotechnology. However, molecular mechanisms behind the reprogramming process remain largely unknown and hamper generation of bona fide iPSCs and their use in human clinical practice. Large animal models are essential to expand the knowledge obtained on rodents and facilitate development and validation of transplantation therapies in preclinical studies. Additionally, transgenic animals with special traits could be generated from genetically modified pluripotent cells, using advanced reproduction techniques. Despite their applicative potential, it seems that iPSCs in farm animals haven't received the deserved attention. The aim of this review was to provide a systematic overview on iPSC generation in the most important mammalian farm animal species (cattle, pig, horse, sheep, goat, and rabbit), compare protein sequence similarity of pluripotency-related transcription factors in different species, and discuss potential uses of farm animal iPSCs. Literature mining revealed 32 studies, describing iPSC generation in pig (13 studies), cattle (5), horse (5), sheep (4), goat (3), and rabbit (2) that are summarized in a concise, tabular format.

Long-term study of Chlamydophilosis in Slovenia.

Immune reactivity for Chlamydophila (C.) psittaci in Slovenia was monitored in parrots, canaries, finches and nine species of recently captured free-living birds (house sparrows, Eurasian goldfinches, tree sparrows, chaffinches, European greenfinches, European serines, Eurasian siskins, Eurasian linnets and Eurasian bullfinches) in the period from 1991 to 2001. In subsequent years, specific IgG antibodies were found using immunofluorescence in parrots (0.7-53.6%), canaries (0.0-3.5%), finches (0.0-5.7%) and in captured free-living birds (33.3% of Eurasian goldfinches in 1994). An experimental infection with C psittaci was performed in order to study clinical signs and pathological changes in canaries and finches. The C. psittaci strain used for experimental infection was isolated from a cockatiel (Nymphicus hollandicus). Chlamydial DNA was extracted from clinical material followed by RFLP-PCR analysis. Infection of canaries and finches was confirmed in organ smears by direct immunofluorescence and a modified Gimenez staining method. In addition, serological tests of indirect immunofluorescence and complement fixation were applied. However, in spite of positive immunological reaction there were no clinical signs three weeks after infection. The present study includes results of a serological survey of persons belonging to the most important risk groups (breeders, pet shopkeepers and veterinarians). The results of microimmunofluorescence to identify the presence of specific antibodies and correlation between appearance of infection in birds and important risk groups are presented. Out of 143 persons belonging to the high-risk group we found 10 (7%) persons who were immunologically positive. Testing of two successive samples was used to demonstrate an increase in IgG and IgA titres in human sera. However, IgM, which is indicative of acute infection, could not be detected.

Allelic differences in bovine kappa - CN gene which may regulate gene expression.

The most common kappa casein (κ-CN) variants, κ-CN A and κ-CN B, are synthesised differentially in the lactating mammary gland of heterozygous animals (κ-CN AB). In this study we evaluated several approaches for quantification of allele specific mRNA transcripts. The most consistent results were obtained using allele specific RT-PCR and capillary electrophoresis. On average, 13.4% more allele B specific than A specific transcripts were found. DNA sequencing of the proximal promoter region in several homozygous animals (κ-CN AA, BB, EE) did not reveal any allele specific polymorphisms. Using the EMSA and DNase I footprinting we confirmed functional binding sites for three transcription factors (AP-2, NF1 and MGF) within the κ-CN proximal promoter region. Sequence analysis of the 3'-UTR of the κ-CN gene revealed seven allele specific sites. Two of these allelic differences were close to previously identified 3'-end regulatory sequences. In addition, allele specific differences in length between mRNAs of both variants were found. The two later findings suggest a possible post translational control determining content differences of κ-CN in milk.

Sequence polymorphisms within the pMGA genes and pMGA antigenic variants in Mycoplasma gallisepticum.

Antigenic variants of Mycoplasma gallisepticum major surface lipoprotein, pMGA, are encoded by a large gene family. In this study sequence analyses of the PCR-amplified pMGA genes showed two types of sequences similar to the pMGA1.2 gene in M. gallisepticum strains. They differed in the sequence encoding a proline-rich region (PRR) at the N-terminus of the pMGA protein. The type A genes had sequences similar to the published pMGA1.2 gene sequence of strain S6, whereas the type B genes lacked the second repetitive segment encoding PTPN sequence within PRR and were similar to the published sequence of PG31 strain. Low in vitro passages of M. gallisepticum strains isolated recently in Slovenia from four avian species showed very different expression patterns of pMGA1.2 and pMGA1.9 genes. Among isogenic populations of S6(B) and IHB1 strains a high frequency of pMGA antigenic variants lacking an epitope for monoclonal antibody (mAb) 71 was found. Strain IHB1 clones, which synthesized pMGA recognized by mAb 71, transcribed pMGA genes whose partial sequence encoded the amino acid sequence (262)TNGDEPRSVS of the mAb 71 epitope. Other IHB1 clones synthesized pMGA variants with different isoelectric points, lacking the epitope for mAb 71, but expressing downstream epitopes for other mAbs. Our study suggests that a molecular basis for pMGA antigenic variation lies in the corresponding changes at the DNA level.

Recombinant DNA probes and polymerase chain reaction for detection of Mycoplasma gallisepticum strains.

Two recombinant DNA clones, pMG286.2 and pMG301.1, were isolated from the partial genomic library of Mycoplasma gallisepticum strain S6. Recombinant M. gallisepticum specific fragments were used as probes in Southern hybridisation with 10 M. gallisepticum strains whose DNA was digested by EcoRI, HindIII, BglII, RsaI and BamHI. The 1.5 kb fragment pMG301.1 did not show polymorphism in hybridisation patterns with M. gallisepticum strains, while the 3.5 kb fragment pMG286.2 enabled differentiation of M. gallisepticum strains into clusters. The DNA sequence of pMG301.1 was used to design a pair of 27-mer oligonucleotides flanking a 1.3 kb genomic region. These two primers directed specific in vitro amplification of all M. gallisepticum strains assayed giving an expected 1.3 kb product. Digestion of polymerase chain reaction products by DdeI enabled simple differentiation between clusters of M. gallisepticum strains and may be useful for improved epizootiological studies of M. gallisepticum infections in poultry.

The characterization of Mycoplasma synoviae EF-Tu protein and proteins involved in hemadherence and their N-terminal amino acid sequences.

An abundant cytoplasmic 43-kDa protein from Mycoplasma synoviae, a major pathogen from poultry, was identified as elongation factor Tu. The N-terminal amino acid sequence (AKLDFDRSKEHVNVGTIGHV) has 90% identity with the sequence of the Mycoplasma hominis elongation factor Tu protein. Monoclonal antibodies reacting with the M. synoviae elongation factor Tu protein also reacted with 43-kDa proteins from the avian Mycoplasma species Mycoplasma gallinarum, Mycoplasma gallinaceum, Mycoplasma pullorum, Mycoplasma cloacale, Mycoplasma iners and Mycoplasma meleagridis, but not with the proteins from Mycoplasma gallisepticum, Mycoplasma imitans or Mycoplasma iowae. In addition, two groups of phase variable integral membrane proteins, pMSA and pMSB, associated with hemadherence and pathogenicity of M. synoviae strains AAY-4 and ULB925 were identified. The cleavage of a larger hemagglutinating protein encoded by a gene homologous to the vlhA gene of M. synoviae generates pMSB1 and pMSA1 proteins defined by mAb 125 and by hemagglutination inhibiting mAb 3E10, respectively. The N-terminal amino acid sequences of pMSA proteins (SENKLI ... and SENETQ ...) probably indicate the cleavage site of the M. synoviae strain ULB 925 hemagglutinin.