Phenotypic characterization of equine Dermatophilus congolensis field isolates

In 1993 and 1994 a highly increased occurrence of equine dermatophilosis was observed, and a study was initiated to determine phenotypic heterogeneity among 120 clinical isolates using biochemistry, antibiotic resistance profiles, membrane protein profiles and Western blotting. The biochemical examinations contained 1% equine serum in medium. Moreover, the API ZYM-test from bioMérieux was used. The biochemical reactions were suited to identify Dermatophilus congolensis but did not allow a differentiation among the various isolates. Antibiotic resistance in one or more isolates was observed against polymyxin B, enrofloxacin, oxacillin, neomycin and trimethoprim-sulfamethoxazol. All isolates were sensitive penicillin G, ampicillin, streptomycin, gentamicin, lincomycin, erythromycin, tetracycline, oxytetracycline, bacitracin and ceftiofur. The evaluation of silver-stained and immuno-stained membrane protein profiles showed minor differences among several isolates. In total, all isolates appeared to be closely related and the minor differences observed did not correlate with the geographic origin of the respective isolates.     

Detection of extracellular proteases from microorganisms on agar plates

We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin) incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that these components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity of proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced protease liberation. Our results indicate that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.     

Controlled-release products for the control of the estrus cycle in cattle, sheep, goats, deer, pigs, and horses

This paper describes the estrus cycles of a number of livestock breeds and reviews the controlled-release drug delivery systems that are currently available for the purpose of controlled breeding. The bovine estrus cycle is reviewed in detail, and the estrus cycles of other species are described in a manner that highlights similarities and differences between species. Pertinent formulation and pharmacokinetic information about current drug delivery systems is presented and discussed, and recent advances in this area are also described.     

Characterization of Anaplasma isolates from eland (Taurotragus oryx). Pathogenicity in cattle and sheep and DNA profiles analysis

Two eland Anaplasma isolates, AnapE1, from Kenya, and AnapE2, from South Africa were characterised. Their characterization was based on their pathogenicity to intact and splenectomized cattle and sheep and also their DNA profiles. Their DNA profiles were analysed and compared to Anaplasma marginale, A. ovis and A. centrale after endonuclease restrictions and probing with Anaplasma DNA probes, AC5-12 and AC-1. The results of the pathogenicity trials showed AnapE1 to be similar to A. ovis and AnapE2 an isolate of A. marginale. On DNA profiles, AnapE1 was close to A. ovis, with differences that occur even in same Anaplasma species isolates from different locations. On the other hand, AnapE2, resembled one of the A. marginale isolates known to occur in South Africa. The DNA profiles correlated well with the pathogenicity results. It is concluded that elands are carriers of both A. marginale and A. ovis parasites and are therefore important reservoirs that need attention in epidemiology of anaplasmosis.     

Involvement of both caspase-like proteases and serine proteases in apoptotic cell death induced by ricin, modeccin, diphtheria toxin, and pseudomonas toxin

We investigated the involvement of caspases and serine proteases in apoptotic cell death induced by ricin, modeccin, diphtheria toxin, and Pseudomonas toxin in U937 cells. We found that caspase-3- and caspase-6-like activities, but not caspase-1-like activity, increased during toxin-induced apoptosis. Z-D-CH2-DCB, a caspase-like inhibitor, completely inhibited the generation of caspase-3- and caspase-6-like activities and blocked all features of apoptosis induced by toxins: nuclear morphological changes, DNA fragmentation, and cytotoxicity. However, three caspase-specific inhibitors, Ac-YVAD-CHO, Ac-DEVD-CHO, and Ac-VEID-CHO, had no effect, even though Ac-DEVD-CHO and Ac-VEID-CHO inhibited the increased caspase-3- and caspase-6-like activity, respectively. These results suggest that the generation of caspase-3- and caspase-6-like activities is redundant, and other caspases distinct from caspase-3 and -6 may be important in toxin-induced apoptosis. Furthermore, serine protease inhibitor, 3,4-dichloroisocoumarine (DCI), abolished the apoptotic cell death and DNA fragmentation caused by toxins, without affecting the increased caspase-3- and caspase-6-like activities. Our results suggest that multiple proteases with different preferences for apoptotic substrates participate in toxin-induced apoptotic death of U937 cells.     

An outbreak of a mixed infection of Dermatophilus congolensis and Microsporum gypseum in camels (Camelus dromedarius) in Saudi Arabia

We isolated and identified the major protein present in corneas with granular dystrophy (GCD). We compared Coomassie-blue-stained protein bands obtained on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) from the extracts of corneas with GCD, corneas with other disorders, and normal human corneal tissue. After SDS-PAGE and transfer to a polyvinylidene difluoride membrane, bands of interest were analyzed by amino acid sequencing and by Western blotting. Corneas with GCD were also examined immunohistochemically. On SDS-PAGE a 63-kd band just below albumin was present in extracts of all corneas. The albumin/63-kd ratio was normally approximately 3:1, suggesting that the protein is a dominant constituent of the cornea. This band was much more plentiful than normal in corneas with GCD. Amino-terminal sequence analysis of the protein revealed a Gly-Pro-Ala-Lys-Ser-Pro-Tyr-Gln-Leu-Val-Leu-Gln-His-Ser-Arg sequence indistinguishable from an amino-terminal protein sequence deduced from a cDNA clone designated beta ig-h3, and it as well as the abnormal accumulations in GCD cross-reacted with beta ig-h3 antiserum. The presence of excessive beta ig-h3 in human corneas with GCD together with reported mutations in the beta ig-h3 gene in GCD suggests that the mutated gene product is a fundamental constituent of the characteristic corneal accumulations in GCD.     

Experimental contact of bighorn sheep (Ovis canadensis) with horses and cattle, and comparison of neutrophil sensitivity to Pasteurella haemolytica cytotoxins

Peripheral blood neutrophils from horses, cattle, and Rocky Mountain bighorn sheep (Ovis canadensis canadensis) were evaluated for susceptibility to cytotoxin-dependent lysis of different biotypes and serotypes of Pasteurella haemolytica of domestic sheep, cattle, bighorn sheep, or mountain goat (Oreamnos americana) origin utilizing a cytotoxicity assay which measures the degree of bacteria cytotoxin-killing of neutrophils. All isolates of P. haemolytica (biotypes A and T) were noncytotoxic to horse neutrophils. Thirteen of 18 R haemolytica biotype A isolates were cytotoxic (> 50% neutrophil death in vitro) to bighorn sheep neutrophils, and four of 10 P. haemolytica biotype A isolates were cytotoxic to neutrophils of cattle; P. haemolytica biotype T (= Pasteurella trehelosi) isolates were noncytotoxic to neutrophils of bighorn sheep and cattle. When six bighorn sheep were pastured with three horses, only P. haemolytica biotype T isolates were recovered from the bighorn sheep throughout the study; Pasteurella spp. organisms were not isolated from the three horses. At initiation of a study where five bighorn sheep were pastured with three cattle, P. haemolytica biotype A, serotype 1, 2 was isolated from all three cattle, and only P. haemolytica biotype T isolates were recovered from the bighorn sheep. One bighorn sheep died in each of the horse and cattle copasturing experiments. Pasteurella haemolytica was not isolated from the bighorn sheep which died in the horse copasturing experiment. A noncytotoxic P. haemolytica biotype A, serotype 2 was isolated at necropsy from the bighorn which died in the cattle contact experiment. Based on these experiments, we believe bighorn sheep and horse association would not be detrimental to bighorns due to P. haemolytica induced pneumonia.     

Characterization of proteolytic activities of rumen bacterial isolates from forage-fed cattle

The proteolytic activities of eight strains of ruminal bacteria isolated from New Zealand cattle were characterized with respect to their cellular location, response to proteinase inhibitors and hydrolysis of artificial proteinase substrates. The Streptococcus bovis strains had predominantly cell-bound activity, which included a mixture of serine and cysteine-type proteinases which had high activity against leucine p-nitroanilide (LPNA). The Eubacterium strains had a mainly cell-associated activity with serine and metallo-type proteinases which showed high activity against the chymotrypsin substrate, N-succinyl alanine alanine phenylalanine proline p-nitroanilide (NSAAPPPNA) and some LPNA activity. A Butyrivibrio strain, C211, had a cell-bound mixture of cysteine and metallo-proteinase activities and strongly hydrolysed NSAAPPPNA and LPNA while the high activity Butyrivibrio-like strain, B316, had a cell-bound, mainly serine proteinase activity which strongly hydrolysed NSAAPPPNA. A Prevotella-like strain, C21a, had a mixture of cysteine, serine and metallo-proteinase activities which were cell-bound and hydrolysed LPNA. The activities of these strains did not match those of the bacterial fraction of rumen fluid, which contained activities mainly of the cysteine type with specificity towards the substrate N-succinyl phenylalanine p-nitroanilide. The contribution of these strains to proteolysis in the rumen is discussed.     

Applicability of cultured hepatocytes derived from goat, sheep and cattle in comparative drug metabolism studies

1. Using trimethoprim (TMP), scoparone (SCOP), ethylmorphine (EtM), 1-naphthol (1-N) and phenol red (PhR) as test substrates, biotransformation activities were investigated in cultured hepatocytes from male and female rat, male and female goat, and female sheep and cattle. 2. As compared with rat hepatocytes, the total culture cytochrome P450 content was relatively well maintained in ruminant hepatocytes. In 72 h, it decreased to approximately half the initial content, whereas in rat hepatocytes only 30% was maintained. In ruminant hepatocytes, sulphation of 1-N remained fairly stable, glucuronidation of PhR decreased gradually, and glucuronidation of 1-N increased during the 72-h culture period. 3. Oxidative metabolism of TMP was rapid in goat and sheep hepatocytes, as compared with rat hepatocytes, reflecting species differences in TMP pharmacokinetics in vivo. In contrast with rat hepatocytes, 6-O-demethylation was by far the major pathway of scoparone metabolism in ruminant hepatocytes. The glucuronidation and sulphation activities were similar among the species. 4. In goat liver cells, sex differences in some oxidative biotransformations were observed, females being more active than males. In rat hepatocytes, a reverse sex difference was observed. 5. In conclusion, cultured hepatocytes from agricultural target species appear a useful in vitro model to study comparative metabolism of veterinary drugs and other xenobiotics. Comparing rat and ruminant, sex and species differences and similarities in drug metabolism can be observed that reflect the in vivo situation.     

Inactivation of Clostridium botulinum toxin by ruminal microbes from cattle and sheep

Toxin from Clostridium botulinum type C was rapidly inactivated during incubation in vitro with ruminal contents from either a cow or a sheep. Fractions of ruminal contents from which cells had been removed by high-speed centrifugation did not inactivate toxin. Inactivation was associated with fractions containing bacteria, whereas fractions containing protozoa and relatively few bacteria were much less active. This activity may help explain the relatively greater tolerance by ruminants to oral doses of botulinum toxin than to toxin administered by other routes. The results are also pertinent to assays for botulinum toxin from gastrointestinal samples obtained postmortem.     

Testicular acid phosphatases in cattle, sheep, guinea pig, and rabbit

Acid phosphatase activities were measured with five different substrates in the total homogenates as well as after gel filtration on Sepharose 6B and cellulose chromatography of bull, guinea pig, rabbit, and ram testes. The response of the hydrolysis rate to NaF (5 mmol/l), Co2+ (5 mmol/l) and Zn2+ (5 mmol/l) was also tested. In the total homogenate the hydrolysis of p-nitrophenyl phosphate was markedly activated by Co2+, while in the presence of Zn2+ an activation was recorded in guinea pig and some inhibition in the bull, rabbit, and ram testes. NaF caused a decline in the total acid phosphatase activity, particularly in guinea pig and ram. The gel filtration resulted in three separate activity peaks with p-NPP and beta-NP as substrates. N-ASBI-P, alpha-NP, and Tym-P gave only two peaks. After subsequent cellulose chromatography of the activities only peak II gave rise to two further activities. Peak I of gel filtration (enzyme I) was able to hydrolyze all substrates tested and was highly sensitive to NaF. Peak I of cellulose chromatography (enzyme II) also hydrolyzed p-NPP and beta-NP. It was rather resistant to NaF but sensitive to Zn2+. It was slightly activated by Co2+. Peak II of cellulose chromatography (enzyme IV) hydrolyzed only p-NPP and was markedly activated by Co2+ and Zn2+. The adult testes of bull, guinea pig, rabbit, and ram have a closely similar testicular acid phosphatase pattern. Due to relative differences in the concentrations of the four enzymes in the tissue, varying activity levels are recorded in the presence of different substrate and modifier combinations.     

Mechanism of action of serine proteases: tetrahedral intermediate and concerted proton transfer

Stopped-flow spectrophotometry and proton inventory experiments have been used to define the reaction pathway for hydrolysis of a specific peptide substrate, Ac-L-Ala-L-Pro-L-Ala p-nitroanilide, by the serine proteases elastase and alpha-lytic protease. The stopped-flow studies reveal the existence and buildup of a tetrahedral adduct between the active site serine hydroxyl group and the sensitive carbonyl group of the substrate. The decomposition of this tetrahedral intermediate to the acyl enzyme and p-nitroaniline is the rate-limiting step for the hydrolytic reaction. The proton inventory data suggest the simultaneous transfer of two protons (presumably from the catalytic carboxyl of Asp-102 to N pi of the catalytic imidazole of His-57 and from N pi of the imidazole to the anilide NH) in the transition state leading to breakdown of the tetrahedral complex. That these proton transfers occur in a concerted, rather than stepwise, process attests to the ability of enzymes to lower the enthalpy of activation most effectively when the precise alignment of a highly specific substrate and catalytic groups minimizes the entropy of activation.     

Isolation of Coxiella burnetii from dairy cattle and ticks, and some characteristics of the isolates in Japan

Coxiella burnetii was isolated from raw milk (36/214, 16.8%) and uterus swab samples (13/61, 21.3%) originating from dairy cattle with reproductive disorders, aborted bovine fetus samples (2/4, 50%), mammary gland samples (4/50, 8%) originating from healthy dairy cattle, and tick samples (4/15, 26.7%) originating from 2 pastures. Fifty-nine strains had various degrees of pathogenicity, high (8; 13.6%), moderate (28; 47.5%) and low (23; 39%), for guinea pigs. The results of isolation suggested a high prevalence of Coxiella infection in dairy cattle with reproductive problems in Japan. Twelve strains (7, 2 and 3 strains from cattle, ticks and humans, respectively) and the reference Nine Mile strain of phases I and II were propagated in both yolk sacs of embryonated hen eggs and Buffalo green monkey (BGM) cell cultures. Protein profiles of these strains were similar to those of the reference strain of phase I. Lipopolysaccharide (LPS) profiles of 12 strains were similar to those of the reference strain of phase I and different from those of the reference strain of phase II. The LPS profiles of 12 strains suggested that these strains are associated with an acute form of Q fever.     

Purification and characterisation of an extracellular beta-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum

An extracellular beta-glucosidase from Fusarium oxysporum was purified to homogeneity by gel-filtration and ion-exchange chromatographies. The enzyme, a monomeric protein of 110 kDa, was maximally active at pH 5.0-6.0 and at 60 degrees C. It hydrolysed 1-->4-linked aryl-beta-glucosides and 1-->4-linked, 1-->3-linked and 1-->6-linked beta-glucosides. The apparent Km and kcat values for p-nitrophenyl beta-D-glucopyranoside (4-NpGlcp) and cellobiose were 0.093 (Km), 1.07 mM (kcat) and 1802 (Km), 461.5 min-1 (kcat), respectively. Glucose and gluconolactone inhibited the enzyme competitively with Ki values of 2.05 mM and 3.03 microM, respectively. Alcohols activated the enzyme; butanol showed maximum effect (2.2-fold at 0.5 M) while methanol increased the activity by 1.4-fold at 1 M. The enzyme catalysed the synthesis of methylglucosides, ethylglucoside and propylglucosides, as well as trisaccharides in the presence of different alcohols and disaccharides, respectively. In addition, the enzyme hydrolysed the unsubstituted and methylumbelliferyl cello-oligosaccharides [MeUmb(Glc)n] but the rate of hydrolysis decreased with increasing chain length. Analysis of products released from MeUmb(Glc)n as a function of time revealed that the enzyme attacked these substrates in a stepwise manner and from both ends. Thus, beta-glucosidase from F. oxysporum, with the above interesting properties, could be of commercial interest.     

5,5-trans lactone-containing inhibitors of serine proteases: identification of a novel, acylating thrombin inhibitor

Synthesis of a variety of 5,5-trans fused lactones, related to compounds found in extracts of Lantana camara, has provided a series of novel acylating inhibitors of human thrombin, trypsin, chymotrypsin and human leucocyte elastase. The most effective thrombin inhibitor is 7 with an IC50 of 130 nM and a Kobs/[1] of 4,000 M-1 s-1.     

Survey of antimicrobial resistance in bacterial isolates from diseased cattle in France

Adrenaline and noradrenaline are released from adrenal medullary chromaffin cells by regulated exocytosis from stored secretory granules. Many aspects of the mechanisms by which exocytosis is activated in chromaffin cells are now understood in detail and these cells have provided an important model for the study of neuroendocrine secretion in general. Exocytosis is triggered by Ca2+ influx which activates a multistep process involving at least two Ca(2+)-binding proteins with distinct Ca2+ affinities. Several cytosolic and membrane proteins have been implicated by functional studies as components of the exocytotic machinery. The likely roles of these proteins in exocytosis are discussed in this review and the questions that remain for the understanding of the molecular basis of catecholamine release are highlighted.     

Polymorphonuclear leukocytes induce PDGF release from IL-1beta-treated endothelial cells: role of adhesion molecules and serine proteases

Polymorphonuclear leukocytes (PMNs) and endothelial cells interact at sites of vascular injury during inflammatory response and during the development of atherosclerotic lesions. Such close proximity leads to the modulation of several of the biological functions of the 2 cell types. Because we have shown previously that PMNs enhance release of growth factors from resting endothelial cells, we decided to evaluate whether coincubation of PMNs with interleukin-1beta (IL-1beta)-stimulated human umbilical vein endothelial cells (HUVEC) could further modulate mitogen release from HUVEC. We found that PMN-HUVEC coincubation resulted in a 10-fold increase in mitogen release, compared with HUVEC alone (14+/-6 versus 1.3+/-0.1). When PMNs were incubated with IL-1beta-treated HUVEC, a further increase in mitogen release (up to 35-fold) was observed. The mitogenic activity was immunologically related to platelet-derived growth factor (PDGF) because the activity was abolished by an anti-PDGF antibody. PDGF-AB antigen, detected in low concentrations in conditioned medium from HUVEC alone, was increased 4-fold when IL-1beta or PMNs were incubated with HUVEC and dramatically upregulated (up to 40-fold) when PMNs were cocultured with IL-1beta-treated HUVEC. The presence of the protease inhibitor eglin C abolished mitogenic activity generation, suggesting a role for PMN-derived elastase and cathepsin G. Indeed, purified elastase and cathepsin G mimicked PMN-induced mitogen release from HUVEC. Because PMNs firmly adhered to IL-1beta-treated HUVEC, we investigated the role of cell-cell adhesion in mitogen release. Adhesion and PDGF release were inhibited by approximately 60% in the presence of anti-CD11a/CD18 and anti-intercellular adhesion molecule-1 monoclonal antibodies. This study suggests a new role for PMNs and their interaction with endothelium in pathological conditions in which intimal hyperplasia is a common feature.     

Incidence in Tanzania of CF antibody to Coxiella burneti in sera from man, cattle, sheep, goats and game

A total of 3735 sera were examined for the presence of CF antibody to Coxiella burneti. Of the samples tested 724 sera were from humans, 1507 from cattle, 525 from sheep, 575 from goats and 404 from game collected in the Serengeti National Park or Selous Game Reserve. It has been demonstrated that Q fever is well established in the above mentioned species in Tanzania (humans 3-9 per cent, cattle 13-3 per cent, sheep 17-1 per cent, goats 13-6 per cent and in game mainly in ruminants). Possible transmission links in the maintenance cycle of the disease are discussed.     

Pharmacokinetics and pharmacodynamics of tilmicosin in sheep and cattle

Tilmicosin is a long-acting macrolide antibiotic currently approved for treatment of bovine respiratory disease in the USA. Serum pharmacokinetics of tilmicosin were compared between cattle (major species) and sheep (minor species) after subcutaneous injection in order to evaluate a new potential application of the drug in currently nonapproved species. There were no significant differences in the elimination rates, maximum serum concentrations, half-lives (t1/2), areas under the curve (AUC), areas under the first-moment curve (AUMC), and mean residence times. Volume of distribution (Vd(area)) and clearance (Cl), when normalized by body weight, were also similar. The only significantly different parameter was time at which maximum drug concentration was reached (tmax), with sheep having the tmax of 3.9 h, compared to 0.5 h in cattle. Although macrolides are considered to be one of the safest anti-infective drugs, adverse cardiovascular effects of several macrolides have been reported. The cardiopulmonary effects of tilmicosin were monitored in healthy adult sheep after receiving a single subcutaneous (s.c.) injection of tilmicosin at the dose of 10 mg/kg, and no significant changes were found. The study indicates that tilmicosin can be used safely and effectively in sheep at the given dose, with no adverse cardiopulmonary effects.