Detection and measurement of fluoroacetate in plant extracts by 19FNMR

Examination of extracts from seeds and foliage of several species known to contain fluoroacetate, using¹³F NMR spectroscopy, has shown the presence of the characteristic FCH₂-signal in most of them and enabled quantitative determination of their fluoroacetate content. No other fluorine-containing plant metabolites were detected; fluoroacetate was not detected in the extracts of several non-toxic species. The limit of detection is estimated to beca 4 μg/g.     

Isolation of an Aldehyde Dehydrogenase Involved in the Oxidation of Fluoroacetaldehyde to Fluoroacetate in Streptomyces cattleya

Streptomyces cattleya is unusual in that it produces fluoroacetate and 4-fluorothreonine as secondary metabolites. We now report the isolation of an NAD⁺-dependent fluoroacetaldehyde dehydrogenase from S. cattleya that mediates the oxidation of fluoroacetaldehyde to fluoroacetate. This is the first enzyme to be identified that is directly involved in fluorometabolite biosynthesis. Production of the enzyme begins in late exponential growth and continues into the stationary phase. Measurement of kinetic parameters shows that the enzyme has a high affinity for fluoroacetaldehyde and glycoaldehyde, but not acetaldehyde.     

Purification and properties of fluoroacetate dehalogenase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> DSM 8341

The degradation of fluoroacetate by microorganisms has been established for some time, although only a handful of dehalogenases capable of hydrolyzing the stable C–F bond have been studied. Pseudomonas fluorescens DSM 8341 was originally isolated from soil and readily degrades fluoroacetate, thus it was thought that its dehalogenase might have some desirable properties. The enzyme was purified from cell-free extracts and characterised: it is a monomer of 32,500 Da, with a pH optimum of 8 and is stable between pH 4 and 10; its activity is stimulated by some metal ions (Mg²⁺, Mn²⁺ and Fe³⁺), but inhibited by others (Hg²⁺, Ag²⁺). The enzyme is specific for fluoroacetate, and the K _m for this substrate (0.68 mM) is the lowest determined for enzymes of this type that have been investigated to date.     

Fluoroacetate toxicity inThiobacillus neapolitanus and its relevance to the problem of obligate chemoautotrophy

Summary Fluoroacetate was extremely toxic toThiobacillus neapolitanus strainC, retarding growth even at 10^-9 m. Inhibition could be relieved by relatively high concentrations of acetate or propionate. Inhibited cultures eventually recovered from inhibition and grew in the presence of fluoroacetate over the concentration range 10^-9 to 10^-5 m. The recovery represented a recommencement of division of the total population, as it was shown that 60–100% of the organisms inoculated on to agar containing as much as 10^-3 m fluoroacetate formed colonies after lags as long as 37 days. Even longer lags occurred with more fluoroacetate, but fewer organisms survived. Fluoroacetate appeared specifically to inhibit the Krebs' cycle through fluorocitrate synthesis; this confirmed that the cycle is essential to the autotrophic metabolism.Fluoroacetate-resistant variants occurred spontaneously at a frequency of about 2 per million organisms. These grew at normal exponential rates even in the presence of 10^-2 m fluoroacetate. They appeared to differ from the wild type organism only in lacking acetyl coenzyme A synthetase and possibly having decreased permeability to acetate. The origin of acetyl coenzyme A for biosynthesis in these mutants, and the significance of the lack of heterotrophic enzymes from an obligate autotroph, are discussed.     

Persistent Colonization of Sheep by Escherichia coli O157:H7 and Other E. coli Pathotypes

Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness in humans. Ruminants appear to be more frequently colonized by STEC than are other animals, but the reason(s) for this is unknown. We compared the frequency, magnitude, duration, and transmissibility of colonization of sheep by E. coli O157:H7 to that by other pathotypes of E. coli. Young adult sheep were simultaneously inoculated with a cocktail consisting of two strains of E. coli O157:H7, two strains of enterotoxigenic E. coli (ETEC), and one strain of enteropathogenic E. coli. Both STEC strains and ETEC 2041 were given at either 10⁷ or 10¹⁰ CFU/strain/animal. The other strains were given only at 10¹⁰ CFU/strain. We found no consistent differences among pathotypes in the frequency, magnitude, and transmissibility of colonization. However, the STEC strains tended to persist to 2 weeks and 2 months postinoculation more frequently than did the other pathotypes. The tendency for persistence of the STEC strains was apparent following an inoculation dose of either 10⁷ or 10¹⁰ CFU. One of the ETEC strains also persisted when inoculated at 10¹⁰ CFU. However, in contrast to the STEC strains, it did not persist when inoculated at 10⁷ CFU. These results support the hypothesis that STEC is better adapted to persist in the alimentary tracts of sheep than are other pathotypes of E. coli.     

Genome-Wide Association Identifies SLC2A9 and NLN Gene Regions as Associated with Entropion in Domestic Sheep

Entropion is an inward rolling of the eyelid allowing contact between the eyelashes and cornea that may lead to blindness if not corrected. Although many mammalian species, including humans and dogs, are afflicted by congenital entropion, no specific genes or gene regions related to development of entropion have been reported in any mammalian species to date. Entropion in domestic sheep is known to have a genetic component therefore, we used domestic sheep as a model system to identify genomic regions containing genes associated with entropion. A genome-wide association was conducted with congenital entropion in 998 Columbia, Polypay, and Rambouillet sheep genotyped with 50,000 SNP markers. Prevalence of entropion was 6.01%, with all breeds represented. Logistic regression was performed in PLINK with additive allelic, recessive, dominant, and genotypic inheritance models. Two genome-wide significant (empirical P<0.05) SNP were identified, specifically markers in SLC2A9 (empirical P = 0.007; genotypic model) and near NLN (empirical P = 0.026; dominance model). Six additional genome-wide suggestive SNP (nominal P<1x10^-5) were identified including markers in or near PIK3CB (P = 2.22x10^-6; additive model), KCNB1 (P = 2.93x10^-6; dominance model), ZC3H12C (P = 3.25x10^-6; genotypic model), JPH1 (P = 4.68x20^-6; genotypic model), and MYO3B (P = 5.74x10^-6; recessive model). This is the first report of specific gene regions associated with congenital entropion in any mammalian species, to our knowledge. Further, none of these genes have previously been associated with any eyelid traits. These results represent the first genome-wide analysis of gene regions associated with entropion and provide target regions for the development of sheep genetic markers for marker-assisted selection.     

Stereochemical studies on a plasmid-coded fluoroacetate halidohydrolase

The stereochemical course of action of haloacetate halidohydrolase H-1 from Pseudomonas sp., strain A, which catalyzes the dehalogenation of fluoroacetate to glycolate, has been determined by enzymatic analysis of products from incubations with both enantiomers of 20-fluoropropionate, and by ¹H NMR analysis of the ester of (?)-α-methoxy-α-(trifluoromethyl)phenylacetic acid with phenacyl [2-²H₁]glycolate derived from the product of incubation with the (S)-monodeuterofluoroacetate. The results support a direct displacement mechanism for this enzyme, since they indicate that the reaction is catalyzed with inversion of configuration.     

Bacterial Population Adherent to the Epithelium on the Roo of the Dorsal Rumen of Sheep

By anaerobic procedures, the total number of adherent bacteria was determined on tissue samples obtained from the roof of the dorsal rumen of three sheep. After four washings, 1.91 × 10⁷, 0.34 × 10⁷, and 1.23 × 10⁷ bacteria per cm² were still attached to the rumen epithelium in sheep 1, 2, and 3, respectively. A total of 95 strains of bacteria were isolated from these three samples. Based on morphology, Gram stain, anaerobiosis, motility, and fermentation end products, they were presumptively identified as follows: Butyrivibrio fibrisolvens, 30 strains; atypical Butyrivibrio, 5 strains; Bacteroides ruminicola, 22 strains; Lactobacillus, 1 strain; and unknown Bacteroides species, 37 strains. For sheep 3, washing the rumen epithelium a total of 10 times reduced the adherent bacterial population by 93% (8.4 × 10⁵ bacteria per cm²). Of 30 strains isolated from this sample, 22 were presumptively identified as Butyrivibrio and Bacteroides types. These results suggest that the epithelium on the roof of the dorsal rumen is primarily colonized by two genera of bacteria, Butyrivibrio and Bacteroides. Most Butyrivibrio and Bacteroides ruminicola strains appeared to be similar to previously isolated rumen strains. However, the unknown Bacteroides species differed considerably from the three species of this genus which are commonly isolated from rumen contents.     

EFFECTS OF SODIUM FLUOROACETATE ON THE METABOLISM OF N-ACETYLASPARTATE AND ASPARTATE IN MOUSE BRAIN

A subconvulsant dose of sodium fluoroacetate inhibited the metabolic utilization of intracerebrally-administered N-acetyl-l -[U-¹⁴C]asparticacid and the labelling of glutamine from this precursor in mouse brain, but not the labelling of glutamate or aspartate. A convulsant dose also inhibited the utilization of l -[U-¹⁴C]aspartic acid. When intraperitoneal injection of a convulsant dose of sodium fluoroacetate was followed by intracerebral injection of N-acetyl-l -[U-¹⁴C]asparticacid, the levels of N-acetylaspartate, aspartate and glutamate in brain were lowered, while the glutamine content was increased. The specific radioactivity of glutamine relative to that of glutamate was much lower when these compounds were labelled from l -[U-¹⁴C]aspartic acid than when N-acetyl-l -[U-¹⁴C]aspartic acid was used as the precursor. Intracerebral injection of tracer amounts of l -[U-¹⁴C]aspartic acid reduced the content of N-acetylaspartate in brain and raised the glutamine content. Sodium fluoroacetate had no additional effect on the relative specific radioactivity of glutamine or the content of N-acetylaspartate, aspartate, glutamate or glutamine when l -[U-¹⁴C]aspartic acid was the precursor. We consider the results to be consistent with a selective inhibition both by sodium fluoroacetate and by exogenous aspartic acid of the tricarboxylic acid cycle in brain associated with the biosynthesis of glutamine. We suggest that the activity of this pathway may regulate the metabolism of N-acetylaspartate and aspartate.     

Simultaneous analysis of diphenylmethoxyacetic acid,a metabolite of diphenhydramine,and its deuterium-labeled stable isotope analog in ovine plasma and urine

Diphenylmethoxyacetic acid (DPMA) is a major metabolite of diphenhydramine in monkeys, dogs, and humans. The metabolic fate of diphenhydramine (DPHM) in sheep is not yet well understood; however, preliminary studies have demonstrated the presence of DPMA in the plasma and urine of sheep following an intravenous bolus of DPHM. Our current studies employ the simultaneous intravenous co-administration of DPHM and the stable isotope analog of DPHM to investigate the pharmacokinetics of DPHM in sheep. In these studies, in order to investigate the pharmacokinetics of the DPMA metabolite, measurement of both unlabeled and stable-isotope labeled DPMA is required. Thus, a stable isotope analog of DPMA ([²H₁₀]DPMA) was synthesized, characterized, and purified for use as an analytical standard. The quantitative method for the gas chromatography—electron-impact mass spectrometry (GC—EI-MS) analysis of DPMA and [²H₁₀]DPMA used a single step liquid-liquid extraction procedure using toluene for sample cleanup. The samples were derivatized with N-methyl-N-(tert.-butyldimethylsilyl) trifluoroacetamide. A 1.0-μl aliquot of the prepared sample was injected into the GC-MS system and quantitated using selected-ion monitoring (SIM). One ion was monitored for each compound, namely, m/z 165 for the internal standard diphenylacetic acid, m/z 183 for DPMA, and m/z 177 for [²H₁₀]DPMA. The ion chromatograms were free from chromatographic peaks co-eluting with the compound of interest. The calibration curve was linear from 2.5 ng/ml (limit of quantitation) to 250.0 ng/ml in both urine and plasma. The intra-day and inter-day variabilities of this assay method were within acceptable limits (below 20% at the limit of quantitation and below 10% at all other concentrations). This method was used to measure the concentration of DPMA and [²H₁₀]DPMA in plasma and urine samples from a ewe in which equimolar amounts of DPHM and [²H₁₀]DPHM were administered by an intravenous bolus dose via the femoral vein. DPMA appeared to persist longer in the plasma and the urine as compared to DPHM. This method is robust and reliable for the quantitation of DPMA and [²H₁₀]DPMA in biological samples obtained from sheep (e.g. plasma and urine).     

Differences in the regulation of RyR2 from human,sheep, and rat by Ca2+ and Mg2+ in the cytoplasm and in the lumen of the sarcoplasmic reticulum

Regulation of the cardiac ryanodine receptor (RyR2) by intracellular Ca²⁺ and Mg²⁺ plays a key role in determining cardiac contraction and rhythmicity, but their role in regulating the human RyR2 remains poorly defined. The Ca²⁺- and Mg²⁺-dependent regulation of human RyR2 was recorded in artificial lipid bilayers in the presence of 2 mM ATP and compared with that in two commonly used animal models for RyR2 function (rat and sheep). Human RyR2 displayed cytoplasmic Ca²⁺ activation (K_a = 4 µM) and inhibition by cytoplasmic Mg²⁺ (K_i = 10 µM at 100 nM Ca²⁺) that was similar to RyR2 from rat and sheep obtained under the same experimental conditions. However, in the presence of 0.1 mM Ca²⁺, RyR2s from human were 3.5-fold less sensitive to cytoplasmic Mg²⁺ inhibition than those from sheep and rat. The K_a values for luminal Ca²⁺ activation were similar in the three species (35 µM for human, 12 µM for sheep, and 10 µM for rat). From the relationship between open probability and luminal [Ca²⁺], the peak open probability for the human RyR2 was approximately the same as that for sheep, and both were ∼10-fold greater than that for rat RyR2. Human RyR2 also showed the same sensitivity to luminal Mg²⁺ as that from sheep, whereas rat RyR2 was 10-fold more sensitive. In all species, modulation of RyR2 gating by luminal Ca²⁺ and Mg²⁺ only occurred when cytoplasmic [Ca²⁺] was <3 µM. The activation response of RyR2 to luminal and cytoplasmic Ca²⁺ was strongly dependent on the Mg²⁺ concentration. Addition of physiological levels (1 mM) of Mg²⁺ raised the K_a for cytoplasmic Ca²⁺ to 30 µM (human and sheep) or 90 µM (rat) and raised the K_a for luminal Ca²⁺ to ∼1 mM in all species. This is the first report of the regulation by Ca²⁺ and Mg²⁺ of native RyR2 receptor activity from healthy human hearts.     

K Fluxes in Frog Skin

A method has been developed for measuring K influx into the epithelial cells of frog skin from the inside solution. Diffusion delay in the connective tissue has been taken into account. Ninety-four per cent of skin K was found to exchange with K⁴² in the inside solution with a single time constant. K influx showed saturation with increasing K concentration, was not altered by imposing a potential difference of ±200 mv across the skin, and was inhibited by dinitrophenol, fluoroacetate, and ouabain. Relatively low concentrations of dinitrophenol (5 x 10^-5 M) and fluoroacetate (10^-10 M) had no effect on k influx but caused a 40 per cent decrease in net Na flux. There was no correlation between the rate of K uptake at the "inner barrier" and the rate of net Na transport. Reduction of net Na transport by lowering Na concentration in the outside solution caused little change in K uptake. These observations indicate that there is not a significant Na-K exchange involved in active transport of Na across the skin. K influx was found, however, to require Na in the inside bathing solution.     

Epidermal Growth Factor-Mediated Mitogen-Activated Protein Kinase3/1 Pathway Is Conducive to In Vitro Maturation of Sheep Oocytes

Epidermal growth factor (EGF) has been shown to facilitate the in vitro maturation of sheep oocytes, and enhance embryo’s capability for further development. However, such kind of molecular mechanism has not yet been elucidated. In the present study, we investigated the effect of EGF-mediated mitogen-activated protein kinases 3 and 1 (MAPK3/1) pathway on in vitro maturation of sheep oocytes. U0126, a specific inhibitor of MEK (MAPK kinase), was added into the maturation culture medium to block the EGF-mediated MAPK3/1 pathway with different doses. Then, the nuclear maturation of sheep oocytes was examined. Additionally, the effect of EGF-mediated MAPK3/1 on cytoplasmic maturation was examined though in vitro fertilization and embryonic development. The rate of germinal vesicle breakdown (GVBD) after 6 h of culture with 10⁻⁴ mol/l of U0126 (50.4%) was significantly decreased compared with control (67.2%, p < 0.05), and the first polation body (PB1) extrusion rate after 22 h of culture in drug treatment was also significantly inhibited compared with control (28.6% vs. 48.4%, p < 0.05). However, 10⁻⁶ mol/l U0126 had slight effect on oocyte nuclear maturation. The normal distribution rate of α-tubulin in the oocytes after 22 h of in vitro maturation was significantly decreased in the 10⁻⁴ mol/l U0126 group (54%) compared with control (68%, p < 0.05). After in vitro fertilization, the cleavage rate in drug treatments (56.8% in 10⁻⁶ mol/l U0126 group and 42.6% in 10⁻⁴ mol/l U0126 group) was significantly decreased compared with control (72.3%, p < 0.01). The blastocyst rate in 10⁻⁴ mol/l U0126 group (17.6%) was also significantly decreased compared with control (29.9%, p < 0.05). Collectively, these results suggest that EGF-mediated MAPK3/1 pathway is conducive to in vitro maturation of sheep oocytes.     

A genome-wide scan study identifies a single nucleotide substitution in ASIP associated with white versus non-white coat-colour variation in sheep (Ovis aries)

In sheep, coat colour (and pattern) is one of the important traits of great biological, economic and social importance. However, the genetics of sheep coat colour has not yet been fully clarified. We conducted a genome-wide association study of sheep coat colours by genotyping 47 303 single-nucleotide polymorphisms (SNPs) in the Finnsheep population in Finland. We identified 35 SNPs associated with all the coat colours studied, which cover genomic regions encompassing three known pigmentation genes (TYRP1, ASIP and MITF) in sheep. Eighteen of these associations were confirmed in further tests between white versus non-white individuals, but none of the 35 associations were significant in the analysis of only non-white colours. Across the tests, the s66432.1 in ASIP showed significant association (P=4.2 × 10⁻¹¹ for all the colours; P=2.3 × 10⁻¹¹ for white versus non-white colours) with the variation in coat colours and strong linkage disequilibrium with other significant variants surrounding the ASIP gene. The signals detected around the ASIP gene were explained by differences in white versus non-white alleles. Further, a genome scan for selection for white coat pigmentation identified a strong and striking selection signal spanning ASIP. Our study identified the main candidate gene for the coat colour variation between white and non-white as ASIP, an autosomal gene that has been directly implicated in the pathway regulating melanogenesis. Together with ASIP, the two other newly identified genes (TYRP1 and MITF) in the Finnsheep, bordering associated SNPs, represent a new resource for enriching sheep coat-colour genetics and breeding.     

The Highly Prolific Phenotype of Lacaune Sheep Is Associated with an Ectopic Expression of the B4GALNT2 Gene within the Ovary

Prolific sheep have proven to be a valuable model to identify genes and mutations implicated in female fertility. In the Lacaune sheep breed, large variation in litter size is genetically determined by the segregation of a fecundity major gene influencing ovulation rate, named FecL and its prolific allele FecL^L. Our previous work localized FecL on sheep chromosome 11 within a locus of 1.1 Mb encompassing 20 genes. With the aim to identify the FecL gene, we developed a high throughput sequencing strategy of long-range PCR fragments spanning the locus of FecL^L carrier and non-carrier ewes. Resulting informative markers defined a new 194.6 kb minimal interval. The reduced FecL locus contained only two genes, insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) and beta-1,4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2), and we identified two SNP in complete linkage disequilibrium with FecL^L. B4GALNT2 appeared as the best positional and expressional candidate for FecL, since it showed an ectopic expression in the ovarian follicles of FecL^L/FecL^L ewes at mRNA and protein levels. In FecL^L carrier ewes only, B4GALNT2 transferase activity was localized in granulosa cells and specifically glycosylated proteins were detected in granulosa cell extracts and follicular fluids. The identification of these glycoproteins by mass spectrometry revealed at least 10 proteins, including inhibin alpha and betaA subunits, as potential targets of B4GALNT2 activity. Specific ovarian protein glycosylation by B4GALNT2 is proposed as a new mechanism of ovulation rate regulation in sheep, and could contribute to open new fields of investigation to understand female infertility pathogenesis.     

Age-related intramuscular pharmacokinetics of cefquinome in sheep

The pharmacokinetic profile of cefquinome was studied in one, six-months and one year old sheep following a single intramuscular doses of 1 and 10 mg kg⁻¹ b.wt. Cefquinome concentrations in serum were determined by microbiological assay technique using Micrococcus luteus (ATCC 9341) as test organism. Following intramuscular administration of cefquinome, the absorption half-lives (t_0.5(ab)) were 1.540, 1.037 and 0.664 h at a dose of 1 mg kg⁻¹ b.wt. and 1.844, 1.290 and 1.605 h at a dose of 10 mg kg⁻¹ b.wt. in the three ages, respectively. After the two doses, the maximum serum concentrations (C_max) of 0.732, 1.145, 1.205 and 3.525, 5.088, 4.576 μg ml⁻¹ were attained after (t_max) of 3.812, 3.029, 2.174 and 3.785, 2.824, 3.095 h in the three ages, respectively. The elimination half-life (t_0.5(el)) and MRT values of cefquinome were longer in one-month old sheep compared to six-months old and yearling sheep. The absorption and elimination processes were delayed in newborn sheep of one-month old in contrary to six-month and yearling animals. The in vitro serum protein-binding tendencies were 8.254%, 11.586% and 13.002%, for one, six-months and one year old sheep, respectively. Based on this study and economically, an optimal intramuscular dosage regimen of cefquinome would be 1 mg kg⁻¹ once daily in one-month, six-months and one-year old sheep to achieve and maintain the therapeutic serum levels within safe limits.     

Studies of haemoglobin types in barbary sheep (Ammotragus lervia)

1. Two haemoglobin types, haemoglobins Amm-C and Amm-B, were observed in five Barbary sheep (Ammotragus lervia). One animal was homozygous for haemoglobin Amm-C, a second was homozygous for haemoglobin Amm-B, and three were heterozygous for both. 2. Amino acid analyses of the globin from haemoglobin Amm-B showed that this type was related to, but not identical with, haemoglobin B of the domestic sheep. 3. The β-chain of haemoglobin Amm-C was found to be composed of 141 amino acid residues. Its amino acid composition differed from that of the β^C-chain of the anaemic domestic sheep in at least 14 residues. The Amm-β^C-chain contained one isoleucyl residue. 4. The amino acid compositions of tryptic peptides T-1, T-2, T-13 and T-14 of the Amm-β^C-chain were similar to those of the sheep β^C-chain. Peptides T-3, T-4, T-6, T-7, T-8, T-11 and T-15 were the same as the corresponding peptides of the sheep β^A- and β^C-chains. Peptide T-5 and to a smaller extent peptide T-9 resembled the corresponding peptides of the sheep β^A-chain, and peptide T-10 was identical with peptide γT-10 of sheep haemoglobin F. Peptide T-12 was not recovered. 5. The results of these investigations were interpreted as being indicative that the structural Amm-β^C-gene is closely related to the β^C-gene of sheep, from which through domestication the present domestic sheep originated.     

Experimental studies on the cadmium accumulation in the cestode Moniezia expansa (Cestoda: Anoplocephalidae) and its final host (Ovis aries)

The tapeworm Moniezia expansa and naturally infected sheep were investigated with respect to their cadmium accumulation. Cadmium chloride (CdCl₂, 0.2 g) was added to 10 ml of distilled water and administered orally to the sheep every day for a period of 1 week. The cadmium content of M. expansa was lower than that in the liver tissues of sheep, although this difference was not significant. The highest mean cadmium concentrations were found in the liver of sheep infected with M. expansa (24.5 ± 11.5 mg kg⁻¹ dry weight). The mean cadmium concentration measured in M. expansa was 21.5 ± 19.2 mg kg⁻¹ dry weight, which was 31 and 1.5 times higher than levels determined in the muscle and kidney of the host, respectively, but 0.9 times lower than levels determined in the liver of host. Sheeps with M. expansa infection always had higher cadmium concentrations in the tissues (with the exception of the blood) than their uninfected conspecifics.     

Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10⁴–10⁹ cells per g tissue) than those with H or VFR feet.     

Duddingtonia flagrans: centrifugal flotation technique with magnesium sulphate for the quantification and qualification of chlamydospores in sheep faeces

Duddingtonia flagrans is a nematode-trapping fungus responsible for attacking larval stages of helminths in pasture, which has potential as a biological control method. The aim of this study was to test the magnesium sulphate centrifugal flotation technique for the quantification of D. flagrans chlamydospores in sheep faeces and to verify their morphological viability. In this experiment one sheep received an oral dose of 4.5 × 10⁶ chlamydospores/day during 20 days. Fecal samples were collected between days 15 and 20 and analyzed by the centrifugal flotation technique with magnesium sulphate. Densities of 1.23, 1.27 and 1.31 g mL⁻¹ recovered 1.45 × 10⁵, 3.87 × 10⁵ and 1.65 × 10⁵ chlamydospores from the faeces, respectively. Based upon the results it was concluded that this is an efficient technique for the chlamydospores quantification in ovine faeces. Moreover, it allowed more accurate visualization of chlamydospore morphology.