Protein kinase-C activity in phorbol myristate acetate-stimulated neutrophils from newborn and adult cattle.

Neutrophils from newborn calves have been shown to be deficient in ability to generate superoxide anion (O2-) after stimulation of the respiratory burst enzyme with the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This compound activates the O(2-)-generating enzyme of bovine neutrophils through a pathway involving protein kinase C (PKC). To investigate the biochemical basis underlying this functional difference between neutrophils from newborn and adult cattle, we measured and compared the activity of the enzyme PKC in nonstimulated and PMA-stimulated bovine neutrophils. Neutrophils from newborn calves (n = 5) and adult cows (n = 5) were stimulated with various concentrations of PMA (0, 10, 100, and 500 ng/ml) for 3 minutes, and PKC activity was assayed in the cytosolic and the membrane fractions. In nonstimulated cells, most PKC activity was detected in the cytosolic fraction of neutrophils from newborn and adult cattle. Activity of PKC in the cytosol was dependent on the presence of added calcium and phospholipids, whereas membrane-associated PKC in nonstimulated cells did not have such dependence. Significant differences in PKC activity were not observed between newborn and adult cattle in either the cytosolic or the membrane fractions from nonstimulated cells. Stimulation with PMA caused redistribution of PKC activity in the cell (translocation) in newborns and adults, consisting of decrease in cytosolic PKC activity and increase in membrane-associated PKC activity. Similar to that in nonstimulated cells, PKC activity in cytosolic fractions from PMA-stimulated neutrophils was dependent on the presence of cofactors (calcium and phospholipids), whereas PKC activity in the membrane did not have such requirement.(ABSTRACT TRUNCATED AT 250 WORDS)     

The probable role of Babesia argentina esterase in the in vitro activation of plasma prekallikrein

The purification of an esterase from B. argentina parasites, using affinity chromatography is described. Two fractions were obtained from crude “L” and “S” parasite extracts. The second fraction from each extract obtained after desorption of the column had a greatly enhanced esterase specific activity. When added to normal plasma and incubated at 37°C for 60 min, the second L₁ fraction activated between 12.1% and 72.3% of bovine plasma prekallikrein and the second S₁ fraction, 76.3% and 87.1%. Only slight activation occurred with fraction 1 of L₁ and S₁, and none with normal red cell components. The active fractions were associated with a fibrinogen-like substance and caused fibrin formation in vitro. The implications of these findings are discussed.     

Characterization and purification of cytosolic and membrane-bound protease(s) in adults of Haemonchus contortus

The activity of protease(s) was observed separately in cytosolic and membrane-bound protease(s) from male and female Hae monchus contortus (Nematoda: Trichostrongylidae). Different fractions showed optimum protease activity at 37 degrees C, pH 8.5 and 8.0 mg casein concentration. The female fractions had a particularly high activity of protease(s) in comparison with the male fractions, especially of membrane-bound enzymes in the anterior half. Inhibition, activation studies revealed the presence of four kinds of protease(s) in cytosolic and membrane-bound fractions. Protease(s) in different fractions are purified to a greater extent by higher concentrations of saturated ammonium sulphate solution, i.e. ranging from 50 to 65%. The purification study revealed the presence of multiple forms of protease(s) in cytosolic and membrane-bound extracts of H. contortus.     

Alkaline and Acid Phosphatase Activity, pH and Osmotic Pressure of Boar Semen

Alkaline phosphatase activity was recorded in forty ejaculates of the sperm rich fraction of boar semen as 9,790 ± 5,250 Klein-Babson-Read units per 100 ml. of seminal plasma. Acid phosphatase activity in the same ejaculates was 681 ± 304 Babson-Read units per 100 ml. of seminal plasma. No alkaline phosphatase activity was detected in the seminal plasma of vasectomized boars.

The pH of the sperm rich fractions was 7.69 ± 0.33 and the osmotic pressure was 313.56 ± 7.98 milliosmols.

     

Effect of growth hormone on estrogen receptor binding properties in the chick oviduct in vivo

The equilibrium dissociation constant (K_d) and the maximum binding capacity (B_max) of estrogen receptor in cytosolic and nuclear fractions in oviduct of pullets following various daily injections (1–10 times) of growth hormone (GH: 50 µg/chick) were examined by Scathchard analysis of specific [³H]estradiol‐17β (E₂) binding. The K_d values of receptor in both fractions decreased after three times of GH‐injection. In the B_max values, three times of the injection caused a marked decrease in that value in the cytosolic fraction with a concomitant increase in that in the nuclear fraction, whereas the total B_max (sum of B_max in the cytosolic and nuclear fractions) did not change. A similar relationship between the K_d values in the binding of the two fractions was also observed in 4–10 times of GH‐injection. However, in 4–10 times of GH‐injection the B_max of the both fractions and total B_max was greater than that in vehicle‐injection (control). When the chicks were injected with 6–10 times of GH‐injection, the weight of oviduct was increased. No change in the plasma concentrations of E₂ was found following GH‐ and vehicle‐injections into the chicks. The results suggest that growth hormone stimulates the growth of the chick oviduct by increasing the binding affinity and capacity of estrogen receptor.     

Microsomal enzymes in lambs and adult sheep,and their possible relationship to alveld

The difference in susceptibility to alveld between lambs and adult sheep may be caused by differences in the microsomal enzyme activities in their livers. There was no difference in NADPH-cytochrome c reductase or 7-ethoxyresorufin-O-deethylase (EROD) activity between ewes, control lambs and phenobarbitone-dosed lambs 3 weeks after dosing ceased. However, aldrin epoxidase activity was at that time significantly highest in the phenobarbitone-dosed lambs and significantly lowest in the ewes. The liver cytosolic glutathione transferase activity was significantly highest in the ewes and significantly lowest in the control lambs at the same time.     

The in vitro motility response to various anthelmintics of third-stage larvae of Oesophagostomum spp. from pigs

The in vitro activities of thiabendazole, levamisole, pyrantel, morantel and ivermectin against Oesophagostomum spp., the nodular worm of pigs, were determined and compared. The study was carried out using isolates of O. dentatum and O. quadrispinulatum, which had been defined in vivo. Infective larvae were exposed to the anthelmintics for 24 h and then placed in a micromotility meter. All the treatments significantly reduced the motility of the ensheathed L₃ larvae, but the micromotility meter was not able to differentiate between anthelmintic resistant and anthelmintic susceptible isolates.     

An Automated Method for Measuring Rhesus Monkey Erythrocyte and Plasma Cholinesterase

An automated colorimetric method using the substrate acetylthiocholine was modified to measure red blood cell (RBC) and plasma cholinesterase (ChE) in samples from rhesus monkeys. The kinetic parameters Km and V^max were calculated for ChE from both sources. Nonenzymatic hydrolysis of acetylthiocholine was uniform and small, and reproducibility of RBC and plasma ChE values was extremely high.

Hemolyzing RBC (by freezing, or by using water or a nonionic detergent as diluents) did not affect ChE activity significantly, but hemolysis did increase nonenzymatic activity. The anticoagulants heparin and EDTA had no effect on RBC or plasma ChE measurements.

A microdilution technique was devised for RBC ChE measurements using 13 microliters of RBC packed in a standard microhematocrit tube. Reproducibility was quite high, but values using this technique were significantly greater than in duplicate samples assayed using standard techniques. Since plasma ChE activity is much lower than that of RBC, the more efficient extrusion of plasma in RBC samples packed in microhematocrit tubes probably accounted for the higher values.

Normal levels of plasma and RBC ChE in adult rhesus monkeys were calculated. There were no significant differences between sexes.

     

Establishment of canine and feline cells expressing canine signaling lymphocyte activation molecule for canine distemper virus study

Antimicrobial therapy is a useful tool to control bovine mastitis caused by Staphylococcus aureus, as consequence an increase in staphylococci resistant cases has been registered. Alternative strategies are desirable and bacteriocins represent attractive control agents to prevent bovine mastitis. The aim of this work was to evaluate the activity of five bacteriocins synthesized by Bacillus thuringiensis against S. aureus isolates associated to bovine mastitis. Fifty S. aureus isolates were recovered from milk composite samples of 26 Holstein lactating cows from one herd during September 2007 to February 2008 in México and susceptibility of those isolates to 12 antibiotics and 5 bacteriocins from B. thuringiensis was evaluated. S. aureus isolates were mainly resistant to penicillin (92%), dicloxacillin (86%), ampicillin (74%) and erythromycin (74%); whereas susceptibility to gentamicin, trimethoprim and tetracycline was detected at, respectively, 92%, 88%, and 72%. All S. aureus isolates showed susceptibility to the five bacteriocins synthesized by B. thuringiensis, mainly to morricin 269 and kurstacin 287 followed by kenyacin 404, entomocin 420 and tolworthcin 524. Our results showed that S. aureus isolates had differences in the antimicrobial resistance patterns and were susceptible to bacteriocins produced by B. thuringiensis, which could be useful as an alternative method to control bovine mastitis.     

Relationship of nematode cholinesterase activity and nematode burdens to the development of resistance to trichostrongyle infections in sheep

The changes in nematode cholinesterase (ChE) activities were examined in relation to the development of resistance in (1) a flock of young grazing sheep, (2) grazing and penned sheep treated with dexamethasone and (3) penned sheep receiving a single mixed infection. Nematodes from grazing sheep with high faecal egg counts (FECs) had higher ChE activities than those from sheep with low FECs. Female nematodes tended to have higher ChE activities than males, and ChE activities in both tended to decline with increasing age of the sheep. The decline in female Trichostrongylus colubriformis ChE activity was associated with a decline in both worm length and in utero egg count. No decline in nematode ChE activity was observed when grazing sheep were treated with dexamethasone. ChE activity of T. colubriformis established in immunosuppressed penned sheep declined 10-20 fold 8 weeks after cessation of treatment. Nematode burdens in the small intestine and abomasum of grazing sheep were significantly correlated, and in individual species they were also correlated with ChE activities. The development of resistance in sheep and the elimination of adult nematode burdens is discussed in relation to gastrointestinal mucosal globule leucocyte numbers, mucus antiparasite activity and the impairment of nematode metabolic function.     

Albendazole enantiomeric metabolism and binding to cytosolic proteins in the liver fluke <Emphasis Type="Italic">Fasciola hepatica</Emphasis>

Fascioliasis causes important economic losses in ruminant species all over the world. Its control is largely based on the use of the flukicidal compound triclabendazole (TCBZ). However, its chemically related benzimidazole anthelmintic albendazole (ABZ) is being successfully used to control TCBZ-resistance flukes. This research gains some insights into the comparative molecular behaviour of both anthelmintics within the target fluke. The goals of the current work were: (i) to assess the competitive binding of ABZ and TCBZ to cytosolic proteins of F. hepatica, and (ii) to evaluate the enantioselective biotransformation of ABZ in microsomal fractions obtained from TCBZ-susceptible and TCBZ-resistant strains of the liver fluke. Cytosolic proteins from fluke specimens bound TCBZ with greater affinity (83%) than ABZ (44%) and the fraction of TCBZ bound to cytosolic proteins was not displaced by ABZ. The microsomes from both -susceptible and resistant flukes sulphoxidized ABZ into ABZ sulphoxide (ABZSO). However, this oxidative activity was 49% higher in microsomes from TCBZ-resistant flukes (P < 0.001) with a predominant production of the (+) ABZSO enantiomer. As earlier shown for TCBZ, the results reported here confirm an enhanced ability for ABZ oxidation in TCBZ-resistant flukes. While this enhanced oxidative metabolism of ABZ may cooperate to the resistance phenomenon, other pharmacodynamic-based mechanisms may be involved, which would explain why, although being chemically-related, ABZ remains efficacious against TCBZ resistant flukes under field conditions.     

外源H2O2对沙打旺抗黄矮根腐病的影响

本研究以沙打旺抗病和感病品种为材料,接种埃里砖格孢孢子悬浮液后分别施加外源H₂O₂及其清除剂AsA,通过统计发病率、病情指数及测定POD、PPO、CAT、SOD、Glu和Cht六种病程相关蛋白酶活性,研究H₂O₂对沙打旺抗黄矮根腐病的影响。结果表明,接种埃里砖格孢后2种沙打旺H₂O₂含量高于未接种植株,且感病品种在38 h H₂O₂含量最高,为929 μmol/g,抗病品种最高值出现在24 h,为986 μmol/g。抗病、感病沙打旺发病率、病情指数在H₂O₂处理后最低,施加AsA最高。H₂O₂处理各种酶活性都有不同程度的增加,且对抗病品种酶活性提高大于感病品种,表明外施H₂O₂可以减缓沙打旺黄矮根腐病的发生和侵染。表明H₂O₂与沙打旺抗病性紧密相关,并通过调节病程相关酶活性来增强沙打旺对黄矮根腐病的抗性,减少侵染率。     

Phosphatases in helminths: Effects of pH and various chemicals and anthelmintics on the enzyme activities

Phosphatases of four helminth species, Ascaridia galli, Centrorhynchus convi, Cotylophoron cotylophorum and Raillietina cesticillus have been analysed colorimetrically. The optimum pH for acid phosphatase activity was 5.4, 4.5, 4.7 and 5.0 in A. galli, C. convi, R. cesticillus and C. cotylophorum, respectively. The optimum pH for alkaline phosphatase activity was 9.1, 9.5, 8.7 and 8.4 in A. galli, C. corvi, R. cesticillus and C. cotylophorum, respectively. In A. galli and C. cotylophorum the acid phosphatase showed more activity than alkaline phosphatase, whereas the latter was more active in R. cesticillus and C. corvi.Effects of the various chemicals MgSO₄, CuSO₄, FeCl₃, KCN, NaF, sodium citrate, glycine and formaldehyde on the enzyme activities were studied. Variable degrees of inhibition of the enzyme activities were achieved following the addition of the anthelmintics Bilevon, Mansonil, Vermex, Zanil, Distodin and Carbon tetrachloride.     

Comparison of in vitro methods and faecal egg count reduction test for the detection of benzimidazole resistance in small strongyles of horses

The objective of the study was to compare the in vitro egg hatch test (EHT), larval development test (LDT) and in vivo faecal egg count reduction test (FECR test) for the detection of benzimidazole resistance in equine strongyles. The presence of resistant or susceptible strongyle populations was determined in 25 stud farms using the in vivo FECR test and in vitro EHT. On the basis of the FECR values, resistance to fenbendazole was detected on 15 of the 25 farms (60%). The ED₅₀ value (anthelmintic concentration producing 50% inhibition of hatching) for suspected resistant populations varied from 0.110 to 0.222 g/ml thiabendazole (TBZ). Final LD₅₀ values (anthelmintic concentration inhibiting development of 50% of eggs into L₃ infective larvae) above 0.029 g/ml TBZ in the in vitro larval development test on samples from 11 stud farms revealed the presence of populations of small strongyles suspected of being benzimidazole-resistant.     

Influence of environmental temperature on experimental infection of redfin perch (Perca fluviatilis) and rainbow trout (Oncorhynchus mykiss) with epizootic haematopoietic necrosis virus, an Australian iridovirus

SUMMARY Experimental transmission of epizootic haematopoietic necrosis virus (EHNV) to adult redfin perch Perca fluviatilis and juvenile rainbow trout Oncorhynchus mykiss was undertaken at different water temperatures using intraperitoneal (IP) and bath inoculation. Redfin perch were highly susceptible to EHNV by both routes of infection. Bath inoculation with as few as 0.08 TCID₅₀. _mL^-1 was lethal. The incubation period in redfin perch was about 11 days at a water temperature of 19–21°C but was longer at colder temperatures and disease did not occur at temperatures below 12°C. The longest incubation period recorded in redfin perch was 28 days. Rainbow trout were not susceptible to infection by bath inoculation but the disease was reproduced after IP inoculation with 10^5.6 TCID₅₀ at water temperatures ranging from 8–21°C. The incubation period was 3–10 days at 19–21°C, but was up to 32 days at 8–10°C. Persistent infection with EHNV was detected by virus isolation in a clinically unaffected rainbow trout after 63 days. The implications of these findings in the understanding of the epidemiology of EHNV infection are discussed.     

In vitro safety assessments and antimicrobial activities of Lactobacillus rhamnosus strains isolated from a fermented mare's milk

Safety and probiotic characteristics such as antimicrobial activities of three Lactobacillus rhamnosus strains, FSMM15, FSMM22 and FSMM26, previously isolated as potential probiotics from fermented mare's milk were investigated. The three FSMM strains were susceptible to ampicillin, gentamycin, kanamycin, streptomycin, tetracycline and chloramphenicol, whereas they were resistant to erythromycin (minimal inhibitory concentration (MIC) = 4?8 µg/mL) and clindamycin (MIC = 4 µg/mL); bioconversion of bile salts, hemolytic activity and mucin degradation activity were negative; enzymatic activities of α‐chymotrypsin and β‐glucosidase were detected, but those of α‐galactosidase, β‐glucuronidase and N‐acetyl‐β‐glucosaminidase, were undetectable. Among the strains, strain FSMM15 was chosen as a safer probiotic candidate due mainly to the lack of plasminogen binding ability. Despite lower acid production of strain FSMM15 than others, its cell‐free culture supernatant inhibited growths of Salmonella Typhimurium LT‐2, Shigella sonnei , Listeria monocytogenes , and Escherichia coli O157 with comparable levels of ampicillin, suggesting a favorable aspect of strain FSMM15 as a probiotic strain.     

Self-cure and immunity following infection and reinfection in ovine haemonchosis

Egg expulsion and post mortem examination were used to compare self-cure and resistance to reinfection with Haemonchus contortus in female Merino sheep, AA and BB hemoglobin types. Animals were infected by 500 or 5000 larvae of H. concortus once, or several times, before being challenged by 500 (low dose) or 5000 (high dose) larvae.In AA sheep infected once by 500 larvae self-cure was detected by a challenge of either 500 or 5000 larvae while in multi-infected sheep only a challenge of 5000 L₃ induced the self-cure. In BB sheep a mild self-cure was detected only by a challenge of 5000 larvae.In AA sheep receiving several infections, challenge by 500 larvae was not effective in producing resistance while 5000 L₃ was effective in producing a state of resistance for up to 35 days post challenge.At post mortem examination, a strong state of resistance was evidenced in multi-infected animals by the low number of L₄ and adults present in the abomasum up to the 35th day after challenge.In BB sheep no state of resistance was demonstrated by challenge with either 500 or 5000 L₃, even in multi-infected animals.     

Disposition of oral telithromycin in foals and in vitro activity of the drug against macrolide‐susceptible and macrolide‐resistant Rhodococcus equi isolates

Javsicas, LH., Giguère, S., Womble, AY. Disposition of oral telithromycin in foals and in vitro activity of the drug against macrolide‐susceptible and macrolide‐resistant Rhodococcus equi isolates. J. vet. Pharmacol. Therap. doi: 10.1111/j.1365‐2885.2009.01151.x. The objectives of this study were to determine the serum and pulmonary disposition of telithromycin in foals and to determine the minimum inhibitory concentration (MIC) of telithromycin against macrolide‐susceptible and macrolide‐resistant Rhodococcus equi isolates. A single dose of telithromycin (15 mg/kg of body weight) was administered to six healthy 6–10‐week‐old foals by the intragastric route. Activity of telithromycin was measured in serum, pulmonary epithelial lining fluid (PELF), and bronchoalveolar lavage (BAL) cells using a microbiological assay. The broth macrodilution method was used to determine the MIC of telithromycin, azithromycin, clarithromycin and erythromycin against R. equi. Following intragastric administration, mean ± SD time to peak serum telithromycin activity (T_max) was 1.75 ± 0.76 h, maximum serum activity (C_max) was 1.43 ± 0.37 μg/mL, and terminal half‐life (t_½) was 3.81 ± 0.40 h. Telithromycin activity, 4 h postadministration was significantly higher in BAL cells (50.9 ± 14.5 μg/mL) than in PELF (5.07 ± 2.64 μg/mL), and plasma (0.84 ± 0.25 μg/mL). The MIC₉₀ of telithromycin for macrolide‐resistant R. equi isolates (8 μg/mL) was significantly higher than that of macrolide‐susceptible isolates (0.25 μg/mL). The MIC of telithromycin for macrolide‐resistant isolates (MIC₅₀ = 4.0 μg/mL) was significantly lower than that of clarithromycin (MIC₅₀ = 24.0 μg/mL), azithromycin (MIC₅₀ =256 μg/mL) and erythromycin (MIC₅₀ = 24 μg/mL).     

Characterization and Purification of Cytosolic and Membrane‐Bound Protease(s) in Adults of Haemonchus contortus

The activity of protease(s) was observed separately in cytosolic and membrane‐bound protease(s) from male and female Haemonchus contortus (Nematoda: Trichostrongylidae). Different fractions showed optimum protease activity at 37°C, pH 8.5 and 8.0 mg casein concentration. The female fractions had a particularly high activity of protease(s) in comparison with the male fractions, especially of membrane‐bound enzymes in the anterior half. Inhibition/activation studies revealed the presence of four kinds of protease(s) in cytosolic and membrane‐bound fractions. Protease(s) in different fractions are purified to a greater extent by higher concentrations of saturated ammonium sulphate solution, i.e. ranging from 50 to 65%. The purification study revealed the presence of multiple forms of protease(s) in cytosolic and membrane‐bound extracts of H. contortus.     

Comparative In Vitro Activity of 16 Antimicrobial Agents Against Actinobacillus Pleuropneumoniae

Sixteen antimicrobial agents were tested for their activity against 68 isolates of Actinobacillus pleuropneumoniae by determining the minimum inhibitory concentrations (MICs). Ceftiofur and the fluoroquinolones danofloxacin and enrofloxacin were the most active compounds, with a MIC for 90% of the isolates (MIC₉₀) of 0.05 µg/ml. The MIC₉₀ values of benzylpenicillin, amoxicillin and aspoxicillin were 0.78 units/ml, 0.39 µg/ml and 0.05 µg/ml, respectively. Three isolates (4.4%) were resistant to penicillins, but aspoxicillin was as active as ceftiofur against the susceptible isolates, with MICs of 0.05 µg/ml for all isolates. Resistance to oxytetracycline, chloramphenicol and thiamphenicol occurred in 22 (32.4%), 14 (20.6%) and 15 (22.1%) of the isolates, respectively. Doxycycline was more active than oxytetracycline, with a MIC₉₀ of 1.56 µg/ml as against 25 µg/ml. Florfenicol was not only as active as thiamphenicol, with a MIC for 50% of the isolates (MIC₅₀) of 0.39 µg/ml, but also active against thiamphenicol-resistant isolates. All the isolates were susceptible to florfenicol. All the isolates were also susceptible to gentamicin, spectinomycin, tilmicosin, colistin and tiamulin. Of these, spectinomycin was the least active, with a MIC₅₀ of 25 µg/ml, followed by tiamulin, with a MIC₅₀ of 6.25 µg/ml. Of the 68 isolates tested, 49 (72.0%) were of serotype 2; 14 (20.5%) were of serotype 1; 2 each (3.0$) were of serotypes 5 and 6; and one was of serotype 7. Of the isolates, 23 (33.8%) were resistant to one or more of the major antibiotics. Antibiotic resistance was found only infrequently among serotype 2, with 5 (10.2%) of 49 isolates being resistant to chloramphenicol and/or oxytetracycline, while it occurred in 18 (94.7%) of the 19 isolates of other serotypes.