Neuraminidase production by Erysipelothrix rhusiopathiae

In order to characterise neuraminidase activity by Erysipelothrix, 85 isolates of Erysipelothrix spp. from a variety of sources including human clinical, marine and terrestrial animals, and the environment were investigated for neuraminidase production. Neuraminidase activity was detected by a peanut lectin haemagglutination method. The effects of media, incubation conditions and pH on the production and activity of neuraminidase were also investigated. Enzyme activity was detected only in the supernatants of the isolates of Erysipelothrix rhusiopathiae which had been incubated in cooked meat broth and Todd Hewitt broth supplemented with horse serum after 16 and 36 h incubation at 37 degrees C. The maximum titres were reached at 40 h in cooked meat broth and 56 h in Todd Hewitt broth supplemented with horse serum. All 58 isolates and the type strain (ATCC 19414) of E. rhusiopathiae produced detectable neuraminidase activity with titres between 10 and 320. The optimal pH for the enzyme activity varied among the isolates with a pH between 6.0 and 7.0 covering the highest enzyme activity of the most. There was no statistically significant difference in the level of neuraminidase activity between isolates from different sources (p > 0.05). Neuraminidase activity was not detected in the non-pathogenic Erysipelothrix spp. such as E. tonsillarum. Neuraminidase was detected only in E. rhusiopathiae suggesting its possible role as a virulence factor. Enzyme production and activity were medium and pH dependent. The peanut lectin haemagglutination assay is a simple, rapid and sensitive method and is particularly useful for the analysis of multiple samples.     

不同培养基及诱导物对捕食性真菌Duddingtonia flagrans发酵液中蛋白质的产生及杀虫作用的影响

为研究捕食性真菌Duddingtonia flagrans发酵液中蛋白质的产生及杀虫作用,使用不同营养成分组成的液体培养基及诱导物,对Duddingtonia flagrans进行培养和诱导,测定其发酵液中的蛋白质含量、蛋白酶活性、磷酸酶活性和杀虫作用,进而确定Duddingtonia flagrans产生胞外蛋白质和杀虫作用最适的培养基与诱导物。结果显示,不同培养基及诱导物对Duddingtonia flagrans的代谢过程会产生明显影响,导致其分泌的蛋白质种类、浓度以及蛋白酶和磷酸酶的活性显著不同;Duddingtonia flagrans发酵液不仅具有杀线虫活性,而且有杀蝇蛆作用。上述结果为进一步研究捕食线虫性真菌的杀虫机理提供了重要参考依据。     

A large potentiation effect of serum on the in vitro potency of tulathromycin against Mannheimia haemolytica and Pasteurella multocida

The antimicrobial properties of tulathromycin were investigated for M. haemolytica and P. multocida. Three in vitro indices of antimicrobial activity, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time‐kill curves, were established for six isolates of each organism. Each index was measured in two growth media: Mueller–Hinton broth (MHB) and calf serum. It was shown that MICs and MBCs were markedly lower in serum than in MHB. MHB:serum ratios for MIC were 47:1 (M. haemolytica) and 53:1 (P. multocida). For both serum and MHB, adjustment of pH led to greater potency at alkaline compared to acid pH. Tulathromycin MIC was influenced by size of inoculum count, being 4.0‐ to 7.7‐fold greater for high compared to low initial counts. It was concluded that for the purpose of determining dosages for therapeutic use, pharmacodynamic data for tulathromycin should be derived in biological fluids such as serum. It is hypothesized that in vitro measurement of MIC in broth, conducted according to internationally recommended standards, may be misleading as a basis for estimating the in vivo potency of tulathromycin.     

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.     

Survival of Salmonella strains differing in their biofilm-formation capability upon exposure to hydrochloric and acetic acid and to high salt

Acidic and osmotic treatments are part of hurdle systems to control pathogens such as Salmonella in food. In the current study, Salmonella enterica isolates previously shown to differ in their ability to form biofilms were grown in diluted tryptic soy broth (TSB) (1:5 dilution in distilled water) and subsequently exposed to phosphate-buffered saline (PBS) adjusted to pH 3.0 with HCl, PBS adjusted to pH 3.9 with acetic acid or rice vinegar diluted 1:15 with distilled water (pH 3.9). Cells grown in diluted TSB were also exposed to distilled water, pH 7.6, containing 5 M NaCl. No differences in survival upon exposure to PBS adjusted to pH 3.0 with HCl or distilled water containing high salt were observed between the isolates; however, exposure to acetic acid and rice vinegar resulted in lower survival levels of isolates previously shown to be poor biofilm formers. The numbers (log(10) cfu/ml) of surviving cells after exposure for 36 hr to acetic acid and rice vinegar were 4.43 ± 0.24 vs. 2.27 ± 0.87 (P<0.05) and 5.19 ± 0.12 vs. 2.33 ± 0.93 (P<0.05) for isolates with a high vs. low biofilm-forming ability. The survival data could be fitted with the Weibull model. The data suggest that the ability of Salmonella strains to survive in the presence of acetic acid and rice vinegar parallels their ability to form biofilms. Thus, Salmonella with a high biofilm-formation capability might be more difficult to kill with acetic acid found in foods or cleaning solutions.     

Determination of minimum inhibitory and minimum bactericidal concentrations of tiamulin against field isolates of Actinobacillus pleuropneumoniae

Tiamulin activity was measured against 19 UK field isolates of Actinobacillus pleuropneumoniae collected between 2003 and 2009 and the type strain ATCC 27090 as a control, with the intention of comparing broth with serum as growth media. Broth microdilution MIC/MBC tests were performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) guideline M31-A3, in 'Veterinary Fastidious Medium' (VFM) (supplemented Mueller-Hinton broth at pH 7.3) and in 100% swine serum. For improved precision, a modified, overlapping doubling-dilution series was used (tiamulin concentration range 0.3-72 μg/ml). The MBC was reported as the lowest concentration producing a 99.9% reduction in bacterial density in the sub-cultured well contents, relative to the starting inoculum. The mean MBC/MIC ratio for tiamulin against A. pleuropneumoniae in VFM was low (1.74:1), even though tiamulin is classed as a bacteriostatic drug. Only three of the 19 isolates and the reference strain grew in 100% serum and their MICs were higher than those determined in VFM. It is postulated that this difference was due to differences in pH of the matrices or binding of tiamulin to serum proteins or a combination of both factors.     

B .methylotrophicus SWU6菌株产纤维素酶发酵条件的优化?

前期从自然界土壤中分离筛选到一株纤维素酶产生菌 Bacillus methylotrophicus SWU6菌株,为提高该菌株发酵产酶能力,本研究采用3,5二硝基水杨酸显色法(DNS)对该菌株产酶所需碳源、氮源、温度、pH 值、装瓶量、接种量等发酵条件进行优化,并比较优化前后等量发酵上清液中 CMCase 酶活大小。结果表明：SWU6菌株产纤维素酶的最适碳源为淀粉,最适氮源为牛肉膏,最适培养温度为50℃,培养基最适初始 pH 值为5.0,最适装瓶量为20%,最适接种量为2%;在最优发酵产酶条件下,SWU6菌株发酵上清液中的 CMCase 酶活达到454.69 U/mL,明显高于优化前利用基础培养基发酵所产的 CMCase 酶活,且酶活提高约3倍。通过发酵条件优化后,SWU6菌株单位体积发酵液显示出了更强的纤维素酶活性。     

Cultivation and Maintenance of Sphaerophorus necrophorus Part I: An Anaerobic Medium for Aerobic Incubation

Out of 185 media formulated and studied for the growth of Sphaerophorus necrophorus, ten were selected for intensive investigation. Their ability to promote growth was compared photometrically. Medium 156 made up of Brewer's thioglycollate broth, yeast extract, L-cystine, and ascorbic acid, was found to yield a very heavy growth. The viability of S. necrophorus was maintained indefinitely by weekly subcultures. When not subcultured, all isolates remained viable for fifteen weeks and some isolates for 30 weeks at 37°C.

Twenty-nine isolates were tested. No variation in maximum growth was noticed.

Medium 156 is an optimum medium for S. necrophorus. It can be incubated aerobically without interfering with maximum growth. The medium does not deteriorate when stored for at least 12 weeks at room temperature.

     

Studies on kinetic properties of non-specific phosphomonoesterases in the sheep nematode, Bunostomum trigonocephalum Rudolphi, 1808

The maximum activity (Vmax) of acid phosphomonoesterase (E.C.3.1.3.2.) at pH 5.5 and 37 degrees C was found to be 2.68 +/- 0.25 and 3.85 +/- 0.24 mu moles phenol mg protein-1 min-1 in male and female Bunostomum trigonocephalum, respectively. The Vmax of alkaline phosphomonoesterase (E.C.3.1.3.1) at pH 10.0 and 37 degrees C was 0.75 +/- 0.04 and 1.15 +/- 0.05 mu moles phenol mg protein-1 min-1 in male and female B. trigonocephalum, respectively. The Michaelis constant (Km) values were 10.25 mM and 11.76 mM for acid and 8.69 mM and 9.09 mM for alkaline phosphomonoesterase in male and female worms, respectively. Enzymal activities were optimum at 7.0 and 9.0% enzyme concentrations, at incubation periods of 60 and 20 min and at temperatures of 50 and 45 degrees C for acid and alkaline phosphomonoesterases, respectively. Dialysis in distilled water decreased the activity of both enzymes, while only acid phosphomonoesterase activity increased in citrate buffer (pH 5.5) and alkaline phosphomonoesterase activity in carbonate buffer (pH 10.0).     

Comparison of growth rates of Tritrichomonas foetus isolates from various geographic regions using three different culture media

The growth rates of 16 isolates of Tritrichomonas foetus from three distinct geographic regions were investigated in modified Diamond's medium, liver infusion broth medium and a commercially available culture kit. While some differences in growth characteristics were detected for different isolates and in the three different media, all isolates grew. Trichomonads reached peak concentrations from an initial concentration of 10(4) trichomonads/ml on Days 2, 3 and 4 in modified Diamond's medium, on Days 2-6 (excluding CAPTF102) in the commercial culture kit and on Days 2-7 in liver infusion broth medium. Viable parasites were detectable for longer periods in liver infusion broth medium and the commercial culture kit than in Diamond's medium. Peak concentrations for isolates tended to be higher in modified Diamond's medium than in liver infusion broth medium or the commercial culture kit. Results show that these three media are suitable for the growth of all 16 T. foetus isolates from three continents and suggest that these media could be used effectively throughout the world.     

Purification and some of the properties of alkaline phosphatase in guinea fowls (Numida meleagris galeata)

1. Alkaline phosphatase activity in the plasma of different strains of guinea fowls showed considerable variation both within and between sexes as well as within and between strains. 2. The enzymes from different strains of wild guinea fowls had different mobilities on disc polyacrylamide electrophoresis but each was characterised by a single band. 3. When the enzyme was purified 163-fold from the plasma of a domesticated grey breasted strain, both ion-exchange chromatography and gel-filtration purification steps yielded a single band of enzyme. 4. The purified enzyme had a molecular weight of 79,400 +/- 3,000 and was stable up to 60 degrees C at the optimum pH of 9.6. 5. Evidence is provided that guinea fowl alkaline phosphatase is a metalloenzyme.     

Results of histochemical and biochemical studies of uterus of gilts during the estrous cycle]

Alkaline phosphatase activity as well as lipid, polysaccharide, glycogen, and acid mucopolysaccharide levels in the uteri of 79 gilts were histochemically tested during the first three sexual cycles. Intra-cyclic alkaline phosphatase activity exhibited clearly pronounced variations in the surface epithelium, with maximum values reached in metoestrus, and moderate variations in the endometrial stroma, with maximum values reached in dioestrus. Cycle-dependent variations were recordable also from the glycogen and lipid levels in the surface and glandular epithelia and from the acid mucopolysaccharide levels in the endometrial stroma. The activity of alkaline phosphatase as well as the levels of glycogen and acid mucopolysaccharides during the first cycle still were lower than those in the second and third. Biochemical examinations of the endometrium revealed significant cycle-dependent variations in the activities of alkaline phosphatase, inorganic pyrophosphatase, and succinate-dehydrogenase as well as in the concentrations of soluble protein and glycogen, with maximum values being recordable on the tenth day of cycle. No significant intracyclic variations were recordable from the activities of acid phosphatase as well as of aspartate alanine-amino transferase.     

Alkaline phosphatase activity in goblet cells in the small intestine of piglets experimentally infected with the coccidium Isospora suis

Alkaline phosphatase activity (EC. 3.1.3.1.) in goblet cells was investigated in the small intestine of 16 gnotobiotic piglets infected one day after delivery (DAD) by different rates of oocysts of Isospora suis coccidia. At a high infection rate of I. suis (750,000) the goblet cells were found to be highly positive to alkaline phosphatase on day 3 to day 4 after infection (DAI). In piglets infected by a low infection rate of I. suis oocysts (100,000) the activity of alkaline phosphatase activity in goblet cells was proved on days 4 to 10 after infection. In the first group of piglets, the positive goblet cells prevailed in the middle region of jejunum, with the peak on 4th DAI. It the second group of piglets a marked increase in the alkaline phosphatase activity was recorded in the goblet cells in the posterior part of jejunum on days 4 to 5 after infection and on 10th DAI. No alkaline phosphatase activity in the goblet cells was demonstrated in the control gnotobiotic piglets at the age of two to seven days.     

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.

     

Examination of the origin of increased equine serum alkaline phosphatase concentrations

Serum alkaline phosphatase activity was found to be increased in 32.6% of equine samples analyzed at the Ontario Veterinary College over an 18 month period. An attempt was made using sensitivity to L-phenylalanine and heat to identify the origin of increased serum alkaline phosphatase isoenzymes present in 44 clinical cases. No difference in sensitivity to either procedure was observed for serum alkaline phosphatase from groups of foals and horses representing different clinical problems. Alkaline phosphatase of osseous tissue origin appeared to be the major source of activity for each group of animals reported.     

The enzymatic profile of urine and plasma in bovine urinary bladder cancer (enzootic bovine haematuria)

The enzymatic profile of urine and plasma in field cases of bovine bladder cancer was studied. Urinary lactate dehydrogenase activity was significantly altered along with the isoenzyme pattern. Activity of alkaline phosphatase and -glucuronidase was decreased in the affected animals. No significant changes were observed in acid phosphatase, or arylsulphatase A and B activity. In plasma, lactate dehydrogenase activity was elevated without any change in the isoenzyme pattern. No significant changes were observed in the other plasma enzymes studied or in the sialic acid concentration.     

Effect of anthelmintics on phosphatases in Ascaridia galli.

In vitro addition of the drugs tetramisole (TMS) and levamisole (LMS) caused an inhibition of the specific activities of acid phosphatase and Mg(++)-dependent adenosine triphosphatase. The inhibition was non-competitive in nature. No significant inhibition was caused by TMS in the activity of glucose-6-phosphatase, but LMS inhibited the enzyme in a non-competitive manner. The activity of alkaline phosphatase was, however, increased in the presence of both TMS and LMS.     

Cytometric and cytochemical study of peritoneal and alveolar macrophages from Yersinia pseudotuberculosis infected ground squirrels (Citellus citellus).

The enzymic activity (succinate dehydrogenase, acid and alkaline phosphatase) of alveolar and peritoneal macrophages as well as leucocytic reaction of ground squirrels infected with Yersinia pseudotuberculosis (I and III serovar) have been investigated in dynamics from the 1st up to the 30th day. The animals infected with III serovar survived only to the 7th day, while those infected with I serovar survived up to the 30th day after inoculation. A massive influx of leucocytes having peak values (100-fold increase) on the 3rd day after infection has been found in the peritoneal cavity of the animals infected with I serovar. Moderate leucocytosis in the blood, and insignificant fluctuations in alveolar macrophage number have been established too. An earlier and higher activation of succinate dehydrogenase in alveolar and peritoneal macrophages from animals infected with III serovar in comparison with those infected with I serovar was observed. No differences in alkaline phosphatase activity of alveolar and peritoneal macrophages have been found between the animals infected with I and III serovar. A correlation has been found between the number of leucocytes and changes in the enzymatic activity of the macrophages. A metabolic transformation was demonstrated typical for different macrophages (peritoneal and alveolar), in the course of this experimental intraperitoneal infection. Obviously, more virulent serovar III of Y. pseudotuberculosis fails to attract leucocytes to the peritoneal cavity sufficiently quickly, so it overcome the local protective mechanisms with consequent systemic cytochemical changes. On the other hand the virulent serovar I attracts leucocytes to the peritoneum and is presumably destroyed by them.     

牛源性停乳链球菌最佳培养基的筛选及影响因素研究

为了提高奶牛乳房炎多联苗的免疫效果,对停乳链球菌进行了最佳培养基的筛选及生长过程中影响因素的研究。结果表明,停乳链球菌在改良肉汤中生长缓慢,不适宜其生长,但在优化培养基和THB培养基中长势良好,尤其是在优化培养基中,停乳链球菌生长延迟期大大缩短,培养4 h即进入对数生长期,约12 h达到生长高峰期,生长周期比改良肉汤缩短近约50%,比THB缩短约2 h,且生长高峰期的细菌数量比THB和改良肉汤亦明显提高。培养过程中影响因素试验结果表明,溶氧量对停乳链球菌生长无影响;在优化培养基中,葡萄糖较适宜的浓度为10 g/L;在对数生长期内补加NaOH调节pH值,能明显提高细菌的产量,并能延长其对数生长期。