Genomic heterogeneity of Dichelobacter nodosus within and between UK sheep flocks and between age groups within a flock.

BACKGROUND: Footrot and interdigital dermatitis are endemic infectious diseases in all sheep farming regions, impairing welfare and production. The development of efficacious vaccines against the primary causative pathogen has been hampered by the extensive antigenic diversity of Dichelobacter nodosus. Understanding the heterogeneity of the pathogen within and between flocks is essential if the feasibility of bespoke vaccine production is to be assessed for use in the U.K. RESULTS: In this study 56 ewe and lamb isolates from 9 flocks were compared by D. nodosus serogroup and Multi Locus Sequence Type which provides significantly enhanced discriminatory power for molecular epidemiology. Serogroup heterogeneity between flocks ranged from two to five unique serogroups per flock. Three flocks contained isolates of two serogroups, two flocks contained isolates of three serogroups and one flock included isolates of five serogroups. Analysis of 25 isolates from one flock with high prevalence of lameness, identified that serogroup and sequence type was significantly correlated with age. Significantly higher proportion of lambs were infected with serogroup B (principally ST85) as opposed to serogroup H (principally ST86), which predominated amongst adult sheep. CONCLUSIONS: Genomic heterogeneity of the pathogen was significantly lower within flock compared to heterogenicity observed between flocks. Furthermore, this study indicates that within a flock, the host-pathogen dynamics and susceptibility to particular D. nodosus strains may be age dependent.

Molecular Epidemiology of Streptococcus uberis Clinical Mastitis in Dairy Herds: Strain Heterogeneity and Transmission.

Multilocus sequence typing was successfully completed on 494 isolates of Streptococcus uberis from clinical mastitis cases in a study of 52 commercial dairy herds over a 12-month period. In total, 195 sequence types (STs) were identified. S. uberis mastitis cases that occurred in different cows within the same herd and were attributed to a common ST were classified as potential transmission events (PTEs). Clinical cases attributed to 35 of the 195 STs identified in this study were classified PTE. PTEs were identified in 63% of the herds. PTE-associated cases, which include the first recorded occurrence of that ST in that herd (index case) and all persistent infections with that PTE ST, represented 40% of all the clinical mastitis cases and occurred in 63% of the herds. PTE-associated cases accounted for >50% of all S. uberis clinical mastitis cases in 33% of the herds. Nine STs (ST-5, -6, -20, -22, -24, -35, -233, -361, and -512), eight of which were grouped within a clonal complex (sharing at least four alleles), were statistically overrepresented (OVR STs). The findings indicate that 38% of all clinical mastitis cases and 63% of the PTEs attributed to S. uberis in dairy herds may be caused by the nine most prevalent strains. The findings suggest that a small subset of STs is disproportionally important in the epidemiology of S. uberis mastitis in the United Kingdom, with cow-to-cow transmission of S. uberis potentially occurring in the majority of herds in the United Kingdom, and may be the most important route of infection in many herds.

Inter-laboratory ring trial to compare four quantitative polymerase chain reaction assays employed for detection of Mycobacterium avium subspecies paratuberculosis.

Johne's disease is an infectious enteric disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) affecting ruminant species worldwide. In Project 1, an independent performance comparison ring trail was conducted between three different commercial MAP quantitative polymerase chain reaction (qPCR) assay services (B, C, and D) currently marketed in Great Britain by three separate laboratories against each other and against a fourth assay (A) not available commercially in Great Britain. A total of 205 individual ovine and bovine samples from five farms were analyzed to give 41 sets of pooled results (pool size five) from each laboratory according to their specific protocols. The numbers of positive pools for assays A-D were 18, 12, 11, and 1 (43.9%, 29.2%, 26.8%, and 2.4%), respectively. Assessment of interrater reliability produced a Fleiss' kappa coefficient of 0.15, indicating very poor overall agreement between the four laboratories. Laboratories A-D diagnosed 4, 3, 2, and 1 flocks at the farm level, respectively, as MAP positive. In Project 2, 38 pooled ovine samples from 10 flocks were analyzed to compare the performance of laboratories A and B. The numbers of positive results for laboratories A and B were 24 (63.1%) and 17 (44.7%), respectively (Cohen's kappa 0.54), indicating that laboratory A was more sensitive than B in line with results from Project 1. Variation between laboratories offering MAP qPCR assays is a significant concern, and further work is warranted to validate and standardize the performance of assays between laboratories for both ovine and bovine samples.IMPORTANCEOur study reports the findings of an inter-laboratory ring trial comparing the performance of four different quantitative polymerase chain reaction (qPCR) assay services for detecting Mycobacterium avium subspecies paratuberculosis (MAP) infection in cattle and sheep. MAP is the causative agent of Johne's disease (also known as paratuberculosis), a significant production-limiting disease in livestock populations with a worldwide distribution. The content of this paper is significant and novel as it is the first to highlight the marked variation between the diagnostic sensitivity and reproducibility of the three principal commercial laboratories offering MAP qPCR diagnostic and screening services in Great Britain. The low sensitivity and high variability between the laboratories are of great concern and relevance to veterinary practitioners and livestock producers.

Role of pulmonary infection in the development of chronic lung disease of prematurity.

We studied the role of ante- and post-natal infection in the development of chronic lung disease (CLD) of prematurity. 192 newborn infants (61 term and 131 pre-term of <34 weeks gestation: 88 with respiratory distress syndrome, 35 developed CLD and eight died) were recruited. 16S ribosomal RNA (rRNA) genes were identified by PCR of DNA isolated from 840 gastric and lung fluid samples. Ureaplasma spp. were also cultured. Presence of 16S rRNA genes (OR 1.6, 95% CI 1.2-2.2) and Ureaplasma spp. (OR 3.6, 95% CI 1.7-7.7) was significantly associated with the development of CLD. This association remained if the 16S rRNA genes and Ureaplasma spp. were first identified within the first 3 days of life (OR 2.4 (95% CI 1.4-4.1) and 3.8 (95% CI 1.4-10.0), respectively) or if first identified after 3 days of age (OR 1.7 (95% CI 1.1-2.8) and OR 5.1 (95% CI 1.3-19.8), respectively). Peak lung fluid interleukin (IL)-6 and IL-8 were significantly associated with presence of microbes (p<0.0001 and p=0.0001, respectively) and development of CLD (p=0.003 and 0.001, respectively). Both early and late microbial presence in neonatal lung fluid samples was significantly associated with the development of CLD suggesting that both ante- and post-natal infection play a role in the development of CLD.

The nonhelical structure of antifreeze protein type III.

Antifreeze proteins (AFPs) are present in the blood of some marine fishes and inhibit the growth of ice crystals at subzero temperatures by adsorption to the ice lattice. The solution structure of a Type III AFP was determined by two-dimensional nuclear magnetic resonance spectroscopy. These measurements indicate that this 66-residue protein has an unusual fold in which eight beta strands form two sheets of three antiparallel strands and one sheet of two antiparallel strands, and the triple-stranded sheets are packed orthogonally into a beta sandwich. This structure is completely different from the amphipathic, helical structure observed for Type I AFPs.

Alignment of rat cardionatrin sequences with the preprocardionatrin sequence from complementary DNA.

Mammalian atria contain peptides that promote the excretion of salt and water from the kidney. When rat atrial tissue is extracted under conditions known to inhibit proteolysis, four natriuretic peptides, cardionatrins I to IV, are consistently isolated. These peptides derive from a common precursor, preprocardionatrin, of 152 amino acids, whose sequence was determined by DNA sequencing of a complementary DNA clone. Amino acid sequencing located the start points of cardionatrins I, III, and IV in the overall sequence. Cardionatrin IV most closely resembles procardionatrin because it begins immediately after the signal sequence at residue 25. Cardionatrin III begins at residue 73, and cardionatrin I, sequenced previously, begins at residue 123. Compositional analysis indicated that each of these cardionatrins extends up to tyrosine at position 150 but lacks the terminal two arginine residues.

Sialic acid synthase: the origin of fish type III antifreeze protein?

Fish type III antifreeze protein is homologous to the C-terminal region of mammalian sialic acid synthase. Similarity is greatest in the protein core and the flat ice-binding region. This relationship adds to the growing list of links between ice-binding proteins (antifreezes) and proteins that interact with sugars and polysaccharides.

Pollution signature for temperate reef biodiversity is short and simple.

Pollution increasingly impacts healthy functioning of marine ecosystems globally. Here we quantify concentrations of major pollutant types (heavy metals/sewage/petrochemicals/plastics) as accumulated within marine sediments on and/or immediately adjacent to shallow reefs for 42 sites spanning coastal population centres across south-eastern Australia. Gradients in pollutants were revealed, but few pollutants co-varied, while increasing wave exposure ostensibly diluted concentrations of all pollutants except microplastics. Examination of reef biodiversity indicators revealed that maximum size of fauna and flora, a key life-history parameter summarised by the Community shortness index, plus declining functional and species richness, were the most sensitive bioindicators of pollutants - for which heavy metals and nutrient-enrichment were most pervasive. Results indicate that assemblages of biogenic habitat formers and associated fauna collapse from "long and complicated" to "short and simplified" configurations in response to increasing pollution, and this community signature may form an effective bioindicator to track human-driven degradation.

Flounder antifreeze protein synthesis under heat shock control in transgenic Drosophila melanogaster.

The gene coding for the most abundant antifreeze protein (AFP) in the winter flounder was placed downstream of the Drosophila melanogaster hsp70 promoter and introduced into the D. melanogaster germ line by P-element-mediated transformation. In each of six transgenic strains tested, heat shock treatment induced the expression of two major AFP gene transcripts and one minor one. All three transcripts were spliced despite the lack of an obvious D. melanogaster internal intron-splicing sequence. The variation in transcript length was caused by selection of different polyadenylation sites. Western blots showed the presence of immunoreactive AFP in hemolymph from heat-shocked transformants. The immunoreactive material had a molecular weight of 6,200, which is consistent with the loss of the signal sequence from the primary translation product and the retention of the pro sequence. Thus, all the signals for flounder pre-mRNA and preprotein processing were recognized in D. melanogaster.

Wolffish antifreeze protein genes are primarily organized as tandem repeats that each contain two genes in inverted orientation.

The antifreeze protein genes of the wolffish (Anarhichas lupus) constitute a large multigene family of 80 to 85 copies, which can be classified into two sets. One-third of the genes were linked but irregularly spaced. The other two-thirds were organized as 8-kilobase-pair (kbp) tandem direct repeats that each contained two genes in inverted orientation; DNA sequence analysis suggests that both genes are functional. Except for a single region specific to each gene, the genes and their immediate flanking sequences were 99.2% identical. This degree of identity ended soon after a putative transcription termination sequence; as the 3' ends of the genes were only 1.3 kbp apart, these sequences might confer mutual protection from interference by transcriptional runoff. A Southern blot of wolffish DNA restricted with enzymes that do not cut within the tandem repeats indicated that the repeats were clustered in groups of six or more. The organization of antifreeze protein genes in the wolffish was very similar to that in the unrelated winter flounder, which produces a completely different antifreeze. This similarity might reflect common dynamics by which their progenitors adapted to life in ice-laden sea water.

Antifreeze proteins.

Antifreeze proteins comprise a structurally diverse class of proteins that inhibit the growth of ice. Recently, new AFP types have been discovered; more active AFPs have been isolated; antecedents have been recognized supporting the notion of recent, multiple origins; and detailed structures have emerged leading to models for their adsorption to ice.

The antifreeze potential of the spruce budworm thermal hysteresis protein.

Antifreeze proteins (AFP) inhibit ice growth by surface adsorption that results in a depression of the freezing point below the melting point. The maximum level of this thermal hysteresis shown by the four structurally unrelated fish AFP is approximately 1.5 degrees C. In contrast, hemolymph and crude extracts from insects can have 5 degrees to 10 degrees C of thermal hysteresis. Based on the isolation, cloning, and expression of a thermal hysteresis protein (THP) from spruce budworm (Choristoneura fumiferana), the vastly greater activity is attributable to a 9 kDa protein. This novel, threonine- and cysteine-rich THP has striking effects on ice crystal morphology, both before and during freezing. It is also 10 to 30 times more active than any known fish AFP, offering the prospect of superior antifreeze properties in cryoprotective applications.

Refined solution structure of type III antifreeze protein: hydrophobic groups may be involved in the energetics of the protein-ice interaction.

BACKGROUND: Antifreeze proteins are found in certain fish inhabiting polar sea water. These proteins depress the freezing points of blood and body fluids below that of the surrounding sea water by binding to and inhibiting the growth of seed ice crystals. The proteins are believed to bind irreversibly to growing ice crystals in such a way as to change the curvature of the ice-water interface, leading to freezing point depression, but the mechanism of high-affinity ice binding is not yet fully understood. RESULTS: The solution structure of the type III antifreeze protein was determined by multidimensional NMR spectroscopy. Twenty-two structures converged and display a root mean square difference from the mean of 0.26 A for backbone atoms and 0.62 A for all non-hydrogen atoms. The protein exhibits a compact fold with a relatively large hydrophobic core, several short and irregular beta sheets and one helical turn. The ice-binding site, which encompasses parts of the C-terminal sheet and a loop, is planar and relatively nonpolar. The site is further characterized by the low solvent accessibilities and the specific spatial arrangement of the polar side-chain atoms of the putative ice-binding residues Gln9, Asn14, Thr15, Thr18 and Gln44. CONCLUSIONS: In agreement with the adsorption-inhibition mechanism of action, interatomic distances between active polar protein residues match the spacing of water molecules in the prism planes (¿10&1macr;0¿) of the hexagonal ice crystal. The particular side-chain conformations, however, limit the number and strength of possible proten-ice hydrogen bonds. This suggests that other entropic and enthalpic contributions, such as those arising from hydrophobic groups, could play a role in the high-affinity protein-ice adsorption.

A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands.

BACKGROUND: THP12 is an abundant and extraordinarily hydrophilic hemolymph protein from the mealworm Tenebrio molitor and belongs to a group of small insect proteins with four highly conserved cysteine residues. Despite their sequence homology to odorant-binding proteins and pheromone-binding proteins, the function of these proteins is unclear. RESULTS: The first three-dimensional structure of THP12 has been determined by multidimensional NMR spectroscopy. The protein has a nonbundle helical structure consisting of six alpha helices. The arrangement of the alpha helices has a 'baseball glove' shape. In addition to the hydrophobic core, electrostatic interactions make contributions to the overall stability of the protein. NMR binding studies demonstrated the binding of small hydrophobic ligands to the single hydrophobic groove in THP12. Comparing the structure of THP12 with the predicted secondary structure of homologs reveals a common fold for this new class of insect proteins. A search with the program DALI revealed extensive similarity between the three-dimensional structure of THP12 and the N-terminal domain (residues 1-95) of recoverin, a member of the family of calcium-binding EF-hand proteins. CONCLUSIONS: Although the biological function of this new class of proteins is as yet undetermined, a general role as alpha-helical carrier proteins for small hydrophobic ligands, such as fatty acids or pheromones, is proposed on the basis of NMR-shift perturbation spectroscopy.

Catfish vitellogenin and its messenger RNA are smaller than their chicken and Xenopus counterparts.

Injection of male bullheads (Ameiurus nebulosus) with estradiol induced the production of a major serum phosphoprotein of molecular weight 145,000. This protein was immunoprecipitable by antisera raised against lipovitellin from bullhead eggs and was absent from the serum of control males. Production of this serum protein coincided with changes in the liver mRNA population, which suggested that estradiol had induced the synthesis of additional mRNA sequences in the high-frequency class. Agarose gel electrophoresis in the presence of methyl mercury hydroxide showed that this mRNA population contained at least one species which was not present in the liver of uninjected males. This new RNA was the major polyadenylated species present in the total cellular RNA and its size relative to ribosomal RNAs and locust vitellogenin mRNA was estimated as 5800 nucleotides. When the liver total RNAs were translated in the mRNA-dependent rabbit reticulocyte lysate system the major translation product from the induced fish had the same molecular weight (145,000) as the serum phosphoprotein and was immunoprecipitable by antilipovitellin antisera. This translation product was not coded for by RNA from control fish. These observations are consistent with the induction of vitellogenesis by estradiol as reported in other egg-laying vertebrates and they show that bullhead vitellogenin and its mRNA are significantly smaller than their avian and amphibian counterparts.

Divergence of protamine gene sequences in fish.

Complementary DNA to trout protamine mRNA was hybridized to excess genomic DNA from trout, salmon and yellow perch. Although there was extensive hybridization of the cDNA to trout DNA, no cross-reaction with yellow perch DNA was observed and the hybridization to salmon DNA was noticeably less than in the homologous reaction. To confirm these results, yellow perch protamine mRNA was purified and compared directly to trout protamine mRNA. Yellow perch protamine mRNA was shorter than trout protamine mRNA, when measured by agarose gel electrophoresis in the presence of methyl mercury hydroxide. The two mRNAs did not cross-react in cDNA/RNA hybridizations, although the homologous reactions went to 90% of completion. This lack of sequence homology was confirmed when the oligopyrimidine tracts from the cDNAs were compared. No sequences longer than tetranucleotides were common to both species. Trout protamine cDNA contained oligopyrimidines of composition C7T4, C4T2, C3T2, C2T3, C1T5 and C1T4 whereas yellow perch protamine cDNA contained C6T3 and C4T3.

Recent and rapid amplification of the sperm basic nuclear protein genes in winter flounder.

The high molecular weight basic nuclear proteins (HMrBNPs), which are tightly bound to sperm chromatin in winter flounder, are made up of imperfect reiterations of simple peptide sequences that contain phosphorylatable DNA-binding motifs. Genomic Southern blots hybridized with probes to the coding and non-coding regions of HMrBNP mRNA showed that HMrBNP sequences form a complex multi-gene family. Previously, one gene (2B) was used to establish an evolutionary link between histone H1 and the HMrBNPs. Further examination of this complex, multi-gene family has now revealed that the majority of the HMrBNP genes are linked as 4.5 kb direct tandem repeats that each contain a 2.8 kb coding region and a 1.7 kb intergenic region (IR). These findings, combined with the cloning of the IR, established that the tandemly repeated genes lack introns and code for the abundant 3 kb HMrBNP mRNAs that produce the prominent 110 kDa HMrBNP. Southern blotting of DNAs from other righteye flounder species showed that HMrBNP multi-gene families were present in closely related species, though with substantial differences in restriction patterns and band intensities, but were not detected in more distantly related flounders. These observations are consistent with recent and rapid elaboration of the HMrBNP gene family.

Isolation and characterization of an antifreeze protein precursor from transgenic Drosophila: evidence for partial processing.

Transgenic Drosophila melanogaster that contain a winter flounder antifreeze protein (AFP) gene fused to the transcriptional and translational control sequences of the host heat shock protein 70 gene express the transgene under heat shock conditions. They secrete into the hemolymph small quantities of a protein that reacts with antisera to AFP and is of a similar size to the proAFP precursor. To facilitate purification and characterization of this precursor, transformed fly lines homozygous for inserts on the 2nd, 3rd and X chromosomes were crossed together to generate a line with five and six AFP genes present in males and females, respectively. AFP production in the multi-gene line was approximately equal to the sum of that observed in the three starting lines and was just sufficient to perturb the growth of ice crystals. The AFP component was purified from heat-denatured hemolymph of this line by cation- and anion-exchange chromatography, followed by reverse-phase HPLC. Edman degradation sequencing of the purified protein showed that its N-terminus began two amino acids in from the predicted signal peptide cleavage point. An additional amino acid sequence was present that began two amino acids further into the 'pro' sequence. These AFP products are consistent with processing of the proAFP in Drosophila by a type IV dipeptidyl aminopeptidase, as has been suggested for processing in flounder.

Molecular cloning and bacterial expression of cDNA for rat calpain II 80 kDa subunit.

The complete cDNA of 3.2 kb for rat calpain II large subunit has been constructed from library- and polymerase chain reaction-derived fragments, and sequenced. The cDNA encodes a protein of 700 amino acids having 93% sequence identity with human calpain II, and 61% identity with human calpain I. The gene possesses 21 exons, of which exons 3-21 have been mapped over 33 kb of the rat genome. A new phagemid expression vector was created from pT7-7 by insertion of the f1 origin and mutation of an NdeI to an NcoI site. Rat calpain II cDNA ligated into this vector expressed in Escherichia coli an 80 kDa protein identical in size to highly purified rat calpain II; this protein was specifically recognized on immunoblots by an affinity-purified anti-rat calpain II antibody. This is the second mammalian calpain II large subunit to be fully sequenced, and the first to be artificially expressed.

P-glycoprotein genes in the winter flounder, Pleuronectes americanus: isolation of two types of genomic clones carrying 3' terminal exons.

In mammals, P-glycoprotein (P-gp) is encoded by two or more highly conserved genes that differ in their abilities to transport drugs. One isoform class (class I) is consistently associated with the multidrug resistance phenotype, while the other (class III) is not. This study was designed to enumerate the P-gp genes in fish and determine how they are related to the two functional classes already defined in mammals. Southern blot analysis using a conserved single exon from the 3' terminal region of hamster P-gp cDNA (pEX1-172) as a probe indicated that there were two P-gp genes in right-eye flounders. Subsequently, two sets of clones were isolated from a winter flounder genomic library that correspond to the 3' ends of the two flounder P-gp genes. Sequence analysis was done on two key areas: the 3' ATP binding site and the 3' terminal exon, both of which were found to be homologous with their mammalian counterparts. Despite high levels of sequence identity in the predicted coding regions of the gene fragments it has not been possible to use these sequences to relate the homologs to particular mammalian classes of P-gp genes, perhaps because of gene conversion between mammalian P-gp genes. These cloned sequences are the first set of P-gp genes reported in lower vertebrates and will be useful for delineating the expression of P-gp genes in fish and understanding the role of P-gp in fish physiology.