Sequential response of milk leukocytes, albumin, immunoglobulins, monovalent ions, citrate, and lactose in cows given infusions of Escherichia coli endotoxin into the mammary gland

Changes in concentrations of both the cellular and the humoral components of milk are known to occur during mastitis. This study was conducted to determine temporal changes in the concentrations of leukocytes, albumin, immunoglobulins (Ig), monovalent ions, lactose, and citrate in milk during the initial phases of simulated mastitis. Ten cows whose udders were pathogen free and had milk leukocyte counts of less than 0.5 X 10(6)/ml were used. Two dosages of Escherichia coli endotoxin were administered to simulate various degrees of mastitis. Two quarters in each cow were infused with the endotoxin and the other 2 served as controls. Quarter milk samples were collected frequently before and after infusion. Within 2 hours after infusion of a 100-micrograms dose of endotoxin, clinical mastitis was observed in most of the infused quarters. Leukocytes, albumin, IgG1, and conductivity showed significant increases. Values before infusion and at postinfusion (PI) hour 2 were as follows: leukocytes, 0.33 and 3.65 X 10(6)/ml, respectively; albumin, 0.38 and 4.49 mg/ml; IgG1, 0.34 and 0.79 mg/ml; and conductivity, 6.0 and 6.9 mmho. Average of the peak values and their average relative time of appearance after infusion were as follows: leukocytes, 28.82 X 10(6)/ml at 16 hours; albumin, 9.37 mg/ml at 4 hours; IgG1, 1.35 mg/ml at 4 hours; and conductivity, 95.5 mmho at 10 hours. The IgG1 values tended to remain high in the presence of rapidly declining albumin concentrations, indicating the possibility of an active, rather than a passive, transfer of IgG1 from the circulation. The response to the 10-micrograms dose of endotoxin ranged from subclinical to clinically mild mastitis with lesser cellular and humoral responses.     

Intensification of milk somatic cell response to intramammary device

Treatments consisting of copper-impregnated polyethylene intramammary device (PIMD-Cu), PIMD-Cu which had been abraded to ensure exposure of surface copper (APIMD-Cu), PIMD which had been abraded in an identical manner (APIMD), and an untreated control were established in mammary quarters of 2 cows. Quarters selected were bacteria free and had milk somatic cell counts (MSCC) in strippings of less than 240 X 10(3) ml. Milk somatic cell counts were determined from strippings immediately after milking and 6 hours later. Cows were monitored for 4 weeks after devices were inserted. Milk samples (250 ml) were collected and analyzed for free fatty acids, a measure of hydrolytic rancidity, at 14 days. The devices were removed from mammary quarters after 4 weeks and examined with a scanning electron microscope. Surfaces of the devices were assessed for plaque buildup and presence of leukocytes. Immediately after and 6 hours later, geometric mean MSCC for APIMD, APIMD-Cu, PIMD-Cu, and control quarters average 946/1,556, 1,479/2,882, 512/1,148, and 161/190 X 10(3) ml, respectively. There was no difference in free fatty acid values of samples from treated and control quarters. Plaque formation was observed over the entire surface of APIMD. Numerous leukocytes were found associated with plaque and appeared to initiate development of plaque. Smaller amounts of plaque were found on APIMD-Cu, and minimal amounts were found on PIMD-Cu. Results indicate that modification of PIMD by abrading or addition of copper will increase MSCC to concentrations (greater than 900 X 10(3) ml) that should be protective against establishment of infection by mastitis pathogens.     

Secretion of angiogenic activity and progesterone by ovine luteal cell types in vitro

In the first experiment, minced luteal tissues from cyclic ewes (n = 5) were incubated for 6 h. Media conditioned by these luteal tissue explants stimulated proliferation and migration of endothelial cells. In a second experiment, corpora lutea (CL) from superovulated ewes (n = 12) were dissociated (two ewes/dispersion) and separated into three fractions: a non-elutriated fraction containing a mixed population of luteal cells, a fraction enriched with small steroidogenic luteal cells, and a fraction containing primarily large steroidogenic luteal cells. Fractions (2 X 10(5) viable steroidogenic luteal cells per milliliter of medium) were incubated with LH in doses of 0, .1, 1, 10, and 100 ng/ml for 7 d. Conditioned media were collected on d 1, 3, 5, and 7 of incubation. Across all days of incubation, media from small luteal cells stimulated proliferation of endothelial cells. Media from large luteal cell incubations, however, secreted an endothelial mitogen only on d 7 of culture. Mixed luteal cell cultures secreted mitogenic activity on d 3, 5, and 7 of incubation, but not on d 1. Luteinizing hormone did not influence release of mitogenic activity by any luteal cell fraction. Across all days of incubation, media from large luteal cells contained more progesterone than those from small luteal cells (528 +/- 137 vs 48 +/- 16 ng/ml with no LH). Mixed (non-elutriated) and small luteal cells increased progesterone secretion in response to LH, and this response was maintained during long-term culture. Large luteal cells did not increase progesterone secretion in response to LH. Steroidogenic activity of all cell types decreased as incubation time progressed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine costimulator. I. Production kinetics,partial purification,and quantification in serum-free Iscove's medium

Bovine peripheral blood leukocytes were examined for blast transformation in response to T-cell lectins in serum-containing RPMI 1640 medium and serum-free Iscove's medium. Phytohemagglutinin-induced blastogenesis was significantly greater in Iscove's medium than in RPMI containing ten percent fetal calf serum. Concanavalin A-induced blast transformation was equivalent in both media. However, the kinetics of lectin response and the quantity of lectin required for optimum blastogenesis was considerably different in the two culture media. Concanavalin A-induced blast transformation of bovine thymocytes in Iscove's medium revealed that at a concentration of 10⁶ cells/ml, inconsequential blastogenesis ensued; but at 10⁷ cells/ml blast transformation was significant and dose-dependent. Therefore, conditioned media from concanavalin A-stimulated bovine peripheral blood leukocytes, prepared in serum-free Iscove's medium, were assayed for costimulator activity using bovine thymocytes at 10⁶ cells/ml in Iscove's medium as indicator cells. Both optimum lectin requirements and cell concentrations for production of costimulator activity were found. Conditioned medium, generated with the total exclusion of serum and with optimal costimulator activity, was fractionated via gel exclusion chromatography. A quantitative assay was described, and results indicated that bovine costimulator had an approximate molecular weight of 20,000 daltons.     

Optimization and kinetics of in vitro stimulation of carp (Cyprinus carpio L.) leukocytes

The optimization of a leukocyte stimulation microassay with carp (Cyprinus carpio L.) leukocytes is described. Leukocytes were isolated from the thymus, anterior kidney, spleen, mid-kidney and peripheral blood.Leukocyte cultures were stimulated with PHA-P, LPS (Escherichia coli 055: B5) PWM, ConA and PPD from Mycobacterium fortuitum. The optimum incubation temperature for leukocyte cultures differed depending on the mitogen used. The optimum incubation period was 3.5 days for leukocyte cultures derived from lymphoid organs and 4.5 days for peripheral blood lymphocyte cultures. Leukocytes from various organ sources showed similar reactivity patterns to stimulation in vitro by different mitogens. The results of these mitogen stimulations did not present sufficient arguments in favour of compartmentation.     

The effect of Pasteurella haemolytica cytotoxin on bovine polymorphonuclear leukocytes can be attenuated by beta-adrenoceptor antagonists

It was investigated whether beta-adrenoceptor antagonists could disturb the interaction between cytotoxin preparations isolated from Pasteurella haemolytica and bovine polymorphonuclear leukocytes (PMNs). The toxicity of the cytotoxin preparation was evaluated by measuring the chemiluminescence response and the viability of the cells after incubation with the cytotoxin. No effect on cell viability was detected when PMNs were incubated with 63 micrograms cytotoxin per ml while the chemiluminescence response was diminished by approximately 30%. The beta-adrenoceptor antagonists alprenolol (10(-5) M) and propranolol (5 X 10(-7) - 5 X 10(-6) M) were able to attenuate this effect of cytotoxin on the chemiluminescence response of PMNs. It seemed unlikely that propranolol and alprenolol diminished the effect of cytotoxin on the chemiluminescence response of PMNs by their beta-adrenoceptor blocking potency because other beta-adrenoceptor antagonists used were without effect. Also, the membrane stabilizing characteristics of the beta-adrenoceptor antagonists used were probably not responsible for the diminished interaction between PMNs and the cytotoxin. Whether beta-adrenoceptor antagonists could be used in vivo to prevent or treat P. haemolytica infections in bovines remains to be examined.     

Development of a migration inhibitory factor assay under agarose of bovine mononuclear leukocytes, using an antigen of Brucella abortus

A study was conducted to develop a migration inhibitory factor assay under agarose of bovine mononuclear leukocytes, with an antigen of Brucella abortus. Different concentrations of mononuclear leukocytes were prepared by the Ficoll-Hypaque technique from the blood of nonvaccinated calves and from calves previously vaccinated with strain 19. Concentrations of 0.5, 1, 2, 3, 4, and 5 x 10(6) leukocytes were suspended in RPMI-1640 medium and various dilutions (20, 10, 1, and 0.1 microgram) of B abortus-soluble antigen, dispensed in triplicate wells cut in 1% agarose containing minimal essential medium and 10% bovine fetal serum. These agarose plates were incubated for 4-, 8-, 12-, 16-, 20-, and 24-hour periods and then were fixed; leukocytes were stained with Wright's stain. Migration distances were measured, and statistical analyses of the data revealed a concentration of 2 x 10(6) cells/well and an antigen concentration of 10 microgram/well. An incubation period of 20 hours was optimal for the assay.     

Treatment of horses with chronic diarrhea: immunologic status.

All chronically diarrheal horses given (orally) 2 series of treatments with normal horse serum recovered in 2 to 4 weeks. However, mild diarrhea sometimes persisted several months in the group of horses with severe diarrhea. Weight gains were approximately 35% in horses with severe diarrhea and approximately 10% in horses with mild diarrhea. Serum specimens from 12 diarrheal and 20 normal horses were examined for immunoglobulins by single radial immunodiffusion technique. Concentration of immunoglobulin A in serum of diarrheal horses was approximately 50% lower than that in serum of normal horses. By contrast, there was more immunoglobulin G in serum of diarrheal horses than in serum of normal horses. Phytohemagglutinin (PHA-M) responsiveness of blood lymphocytes showed transient suppression during the stage of severe diarrhea. The regaining of PHA-M responsiveness of lymphocytes was observed simultaneously with the recovery process. However, the responsiveness of lymphocytes in recovered horses remained markedly lower than that in normal horses. Allergic reactions in diarrheal and normal horses were studied by observing dermal response to injections of saline extracts from some of the horse feeds. A delayed hypersensitivity reaction to streptokinase-streptodornase and PHA-M was also studied. Allergic reactions to these extracts were not induced in either diarrheal or normal horses; however, inflammatory response to the extracts was approximately 50% greater in normal than in diarrheal horses. Response to intradermal injection of either streptokinase-streptodornase or PHA-M was significantly greater in normal horses than in diarrheal horses.     

In vitro stimulation of bovine peripheral blood lymphocytes: suppression of phytomitogen and specific antigen lymphocyte responses by aflatoxin.

The effects of aflatoxin B1 on responses of the peripheral blood lymphocytes (PBL) of 2 normal animals to phytohemagglutinin, concanavalin-A, and pokeweed mitogen and of the PBL of 2 Mycobacterium bovis-infected animals to phytohemagglutinin and purified protein derivative of M bovis (PPD) were studied. Aflatoxin concentrations of greater than or equal to 10 microgram/ml significantly suppressed the lymphocyte response of normal animals to the phytomitogens. Lymphocyte response of M bovis-infected animals to specific antigen PPD was significantly suppressed at aflatoxin concentration of 0.5 microgram/ml. Fifty- to 100-fold higher concentrations of aflatoxin were required to produce 50% suppression of lymphocyte response to phytomitogens, as compared with that produced to PPD.     

Quantitative urinalysis in kittens from four to thirty weeks after birth.

To evaluate renal function and obtain reference values for measurements of urinary excretion of various substances, quantitative urinalysis was performed in healthy, growing kittens from 4 to 30 weeks after birth. Endogenous creatinine clearance, 24-hour urine protein excretion, and urine protein-to-creatinine ratio were determined. Additionally, fractional excretion to creatinine clearance was calculated for calcium, inorganic phosphorus, sodium, potassium, and chloride. Mean +/- SD endogenous creatinine clearance values (range, 3.80 +/- 0.48 to 4.74 +/- 0.61 ml/min/kg) were significantly (P less than 0.0001) higher in kittens 9 to 19 weeks old, compared with younger (range, 1.39 +/- 0.85 to 3.59 +/- 0.86 ml/min/kg) and older kittens (range, 2.69 +/- 0.40 to 3.46 +/- 0.37 ml/min/kg). Mean values for all kittens for 24-hour urine protein excretion (range, 2.54 +/- 1.81 mg/kg at 4 weeks to 11.39 +/- 7.61 mg/kg at 14 weeks) and for urine protein-to-creatinine ratio (range, 0.14 +/- 0.03 to 0.34 +/- 0.18) varied from week to week of age. The urine protein-to-creatinine ratio in kittens greater than or equal to 9 weeks old correlated well (R2 = 0.861) with 24-hour urine protein excretion. Urinary fractional excretion of calcium, inorganic phosphorus, sodium, potassium, and chloride in kittens varied among age groups, being significantly (P less than 0.01) different for potassium and calcium in young kittens (4 to 6 weeks) and older kittens (greater than or equal to 7 weeks).     

Influence of cortisol and different steroidogenic pathways on estrogen synthesis by the bovine placenta

The influence of cortisol on estrogen synthesis by the bovine placenta and the importance of the delta 4 and delta 5 pathway for estrogen production were investigated. For experiment 1, portions of fetal villi (200 mg) were incubated for 48 hours with 0, 10, 100, and 1,000 ng of cortisol/ml with [3H]androstenedione (3H-A) or [3H]pregnenolone (3H-P5). Villi were also incubated for 4, 28, and 52 hours with or without cortisol (500 ng/ml) and with 3H-A or 3H-P5 (experiment 2). The conversion of various [3H]steroid metabolites such as A, P5, 17 alpha-OH-pregnenolone (17 alpha-OH-P5), progesterone (P4), 17 alpha-OH-P4, cholesterol (chol), and chol plus lipoprotein (500 micrograms/ml) into estrogen was measured during a 4-hour incubation (experiment 3). In experiment 1, cortisol increased conversion of 3H-A and 3H-P5 into estrogen by 3 to 41% and 7 to 34%, respectively, in a dose-dependent manner (P less than 0.05). In experiment 2, times of incubation did not influence conversion of 3H-A into estrogen, which, however, was increased significantly (P less than 0.05) over all times of incubation by administration of 500 ng of cortisol/ml. Conversion of 3H-P5 into estrogen increased over time of incubation and was stimulated by cortisol (P less than 0.05). However, there was no interaction between cortisol treatment and time of incubation. In experiment 3, conversion of 3H-A, 3H-P5, and 3H-17 alpha-OH-P5 into estrogen was greater than the conversion of the other precursors tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetics of rifampin given as a single oral dose in foals

Six foals from 6 to 8 weeks of age were given a single oral dose of rifampin at a dosage of 10 mg/kg of body weight. Serum rifampin concentrations were measured serially during a 24-hour period. The mean peak serum rifampin concentration was 6.7 micrograms/ml at 4 hours after treatment. The concentration decreased slowly, and at 24 hours the mean value was 2.7 micrograms/ml. The elimination half-life was 17.5 hours, and the elimination rate constant was 0.04/hr.     

The early events of infection with Theileria parva in cattle: infectivity for cattle of leukocytes incubated in vitro with sporozoites

Leukocytes were isolated from bovine blood and, after short periods of incubation in vitro with sporozoites of Theileria parva, were washed thoroughly, and their infectivity tested in autologous and allogeneic hosts. Using a standard inoculum of 10(6) viable cells, it was found that, after incubation in vitro for either 1 or 24 h, the cells initiated lethal infections in autologous cattle, but failed to infect allogeneic animals. Autologous and allogeneic erythrocytes and mouse lymphocytes similarly incubated with sporozoites failed to infect cattle. The supernatant from bovine lymphocyte suspensions incubated with sporozoites for 1 h produced lethal infections whereas after 24 h of incubation the supernatant was non-infective. All cattle which did not develop detectable infection were fully susceptible to subsequent challenge with a stabilate of sporozoites. By inoculating cattle with graded doses of autologous blood leukocytes which had been incubated for 24 h with sporozoites, it was found that as few as 2 X 10(3) cells gave rise to infection. The results indicate that this approach can be used to evaluate different cell populations as targets for infection and transformation by sporozoites of T. parva.     

Kinetics of in vitro bovine lymphocyte immunostimulation with a Brucella abortus antigen.

A Brucella abortus-soluble antigen was investigated, using in vitro assay of lymphocyte immunostimulation, to determine which concentration of this antigen and which period of incubation of the lymphocyte cultures would induce maximum specific lymphocyte immunostimulation as an additional method for further study of B abortus infection in cattle. Soluble antigen was prepared from autoclaved cells of B abortus strain 1119-3. Peripheral blood lymphocytes were obtained from cattle infected with B abortus and from healthy control cattle not infected with B abortus. The lymphocytes were prepared by the Ficoll-Hypaque density gradient technique, suspended in RPMI 1640 medium (1.5 X 10(6)/ml), cultured with several dilutions of soluble antigen, and incubated. Prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine and, after harvesting, assayed for [3H]thymidine incorporation in DNA by a liquid scintillation spectrometer. Maximum specific immunostimulation of lymphocytes from B abortus-infected cattle was induced in this assay system with 6 days' incubation and 22 microgram of protein/ml/1.5 X 10(6) lymphocytes, using protein content to express concentration of soluble antigen in this system.     

Measurement of phagocytosis of 32P-labeled Staphylococcus aureus by bovine leukocytes: lysostaphin digestion and inhibitory effect of cream.

A procedure to measure phagocytosis by blood and milk neutrophils was developed. One milliliter of heat-killed 32P-labeled Staphylococcus aureus ([32P]SA) (180-200 X 10(6) CFU), 1 ml of phosphate-buffered saline solution (PBSS), and 2 ml of serum, whole milk, skimmed milk, whey, or PBSS were incubated in duplicate for 60 minutes at 37 C. Isolated blood or milk nuetrophils (polymorphonuclear leukocytes (PMN), 25 X 10(6) cells/ml; 1 ml) were added and incubated at 37 C for 30 minutes. Unphagocytosed [32P]SA organisms were lysed by incubation with 5 ml of lysostaphin (10 U) at 37 C for 30 minutes, and the PMN and phagocytosed T2P]SA were removed by centrifugation. Radioactivity of the supernatant was determined in a scintillation spectrometer and was used in estimate the percentage of [32P]SA phogocytosed. With this procedure, 25 assays in duplicate could be conducted each day with an expected coefficient of variation between duplicates of 5.6%. Blood PMN phagocytosed 80, 44, 74, 72, and 11% of the [32P]SA when incubated in serum, whole milk, skimmed milk, whey, and PBSS, respectively. Mik PMN phagocytosed 78, 44, 72, 74, and 22%, respectively. The addition of cream to either skimmed milk or serum reduced phagocytosis of [32P]SA by both blood and milk PMN. The inhibitory effect of cream was verified by the microscopic observation that PMN containing large quantities of ingested fat contained fewer S aureus. Seemingly, PMN upon entering the alveoli of the mammary gland become less efficiently phagocytic for bacteria, because of the presence of milk fat globules. This phef intramammary infection by invading mastitic pathogens.     

Effects of the ingestion of whole colostrum or cell-free colostrum on the capacity of leukocytes in newborn calves to stimulate or respond in one-way mixed leukocyte cultures

OBJECTIVE: To evaluate effects of colostral cells on the ability of neonatal leukocytes to respond in a mixed leukocyte response (MLR) as a means of evaluating specific immune responsiveness. ANIMALS: 10 Holstein calves, their respective dams, and 10 unrelated adult Holstein cows. PROCEDURE: Soon after birth, their calves were fed maternal whole colostrum or colostrum after cells were removed by centrifugation. Responses for leukocytes obtained from calves during the first 5 weeks after birth, their dams, and unrelated cows were measured by use of 1-way MLR as an indicator of immune development. An internal control treatment, proliferation of lymphocytes stimulated with Staphylococcus enterotoxin B (SEB), was also measured. RESULTS: Transfer of colostral leukocytes had a significant effect on the MLR and SEB-induced response in calves. Calves receiving whole colostrum had enhanced responses to maternal and unrelated leukocytes 24 hours after ingestion of colostrum. These responses decreased quickly, indicating direct modulation of the neonatal immune response. Calves receiving whole colostrum effectively stimulated the MLR by 24 hours after ingestion of colostrum. In contrast, calves receiving acellular colostrum did not effectively stimulate the MLR until 2 to 3 weeks after birth. CONCLUSIONS AND CLINICAL RELEVANCE: Ingestion of maternal colostral leukocytes immediately after birth stimulates development of the neonatal immune system. These maternal leukocytes enhance development of antigen-presenting capacity as indicated by their ability to stimulate the MLR and SEB response. The influence of ingested maternal cells on neonatal immunity was also indicated by a reduction in reactivity of neonatal cells to maternal alloantigens.     

THE DURATION OF THE RESPONSE OF CATTLE TO INOCULATION WITH ATYPICAL MYCOBACTERIA

SUMMARY Each of 4 strains of atypical mycobacteria was inoculated into 2 cattle and the responses of the cattle were studied over the following 52 weeks. Each strain was injected subcutaneously into one animal and into a mesenteric lymph node of another. Within 7 days palpable lesions were produced at the sites of subcutaneous inoculation in response to all the strains. After intervals varying from 3 to 26 weeks, lesions due to 3 of the strains were no longer palpable. The lesion produced in response to the fourth strain, a non-agglutinable serotype of Mycobacterium intracellulare, was still palpable at necropsy, 52 weeks post-inoculation (PI). Of the 8 cattle inoculated with mycobacteria, the latter was the only animal that had a lesion with features consistent with a mycobacterial infection and from which mycobacteria were isolated. The inoculated cattle and 4 uninoculated control cattle were turberculin tested on 8 occasions during the post-inoculation period. Bovine purified protein derivative (PPD), avian PPD and PPD tuberculins prepared from each of the atypical mycobacteria were used. In inoculated cattle, sensitivity to both avian and bovine PPD was short lived, significant levels not persisting in any animal beyond 16 weeks PI. From the results of intradermal tests on the control cattle, a 95% confidence interval for their response to any of the 6 tuberculins used, was found to be ±1.36mm. On this basis all inoculated cattle developed sensitivity to the homologous tuberculin. The animal with mycobacterial granuloma at the subcutaneous inoculation site at necropsy had never developed significant levels of sensitivity to bovine PPD, had not shown significant levels of avian sensitivity after week 16 PI nor had it shown homologous sensitivity after week 22 PI. In all animals the level of sensitivity to bovine PPD decreased between successive tests. This fact could be used to clarify the status of a reactor if non-specific bovine sensitivity was suspected. Alternatively, the comparative intradermal tuberculin test using both bovine and avian PPD may be employed.     

RESPONSE OF CATTLE TO INOCULATION WITH ATYPICAL MYCOBACTERIA OF BOVINE ORIGIN

SUMMARY Ten strains of atypical mycobacteria originally isolated from cattle were inoculated into cattle. Each strain was injected subcutaneously into one animal and into a mesenteric lymph node of another. Four weeks and 10 weeks after inoculation the cattle were tuberculin tested with bovine PPD, avian PPD and the appropriate homologous PPD. Three strains produced a significant level of sensitivity to bovine PPD at the 4-week test but by the 10-week test no animal gave a significant response. The sensitivity to all tuberculins was less at the 10-week test than at the 4-week test. At both tests the response to avian PPD was equal to or exceeded that to bovine PPD. Of 4 strains originally from cattle sensitive to mammalian tuberculin only 2 produced sensitivity of bovine PPD in this experiment. Cultural isolation of mycobacteria from necropsy material was correlated neither with sensitivity to bovine PPD nor with the presence of lesions.     

基于结核菌素与CFP10／ESAT6的牛IFN-γ检测法在牛结核病诊断中的比较研究

本研究通过比较三种刺激物（牛结核菌素、禽结核菌素和牛型结核菌特异性抗原CFP10／ESAT6对结核菌素皮内变态反应阳性牛的IFN-γ刺激反应,探讨IFN-γ检测法在我国牛结核病诊断中的应用前景。无菌采集22头结核菌素皮内变态反应阳性牛的血液．肝素抗凝。每1mL全血与1mL RPMI1640完全培养基混合均匀,并加入0.1mL（20μg）PHA（阳性对照孔）、0．1mL（20μg PHA）CFP10／ESAT6融合蛋白、0．1mL（2000U）＆结核菌素（PPD／B）、0．1mL（2500u）禽结核菌素（PPD／A）或等量PBS（无刺激阴性对照）,37℃培养过夜。次日用夹心ELISA法检测各刺激组0．1mL培养上清的IFN-γ,以OD630表示IFN-γ浓度。结果,特异性抗原CFP10／ESAT6刺激组与牛结核菌素（PPD／B）刺激组的IFN-γ反应具有良好的相关性．相关系数为0．84。但CFP10／ESAT6刺激组IFN-γ浓度与牛和禽PPD的比较反应（以两刺激组IFN-γ浓度差值表示）间无相关性,相关系数为-0．11。分析禽PPD组的IFN-γ反应,发现实验牛中有少数牛对禽PPD有反应。以OD630=0．17为阳性反应切割值,牛PPD检出阳性牛21头,CFP10／ESAT6检出20头,牛和禽PPD比较反应（以OD值差值表示）检出14头,禽PPD阳性反应3头。扣除禽PPD阳性反应牛后,牛和禽PPD比较反应与牛PPD刺激组的相关系数增至0．54。结果表明,牛PPD的IFN-γ释放反应检测灵敏度最高。当出现牛型结核菌与环境分枝杆菌混合感染时．应用牛和禽PPD比较反应检测牛结核的准确度低,混合感染牛被误判为结核阴性牛。而基于CFP10／ESAT6的IFN-γ释放反应不受环境分枝杆菌的影响,检测具有良好的特异性与敏感性。     

Effect of timing of blood collection on serum phenobarbital concentrations in dogs with epilepsy

OBJECTIVE: To determine whether there are therapeutically relevant changes in serum phenobarbital concentrations throughout a daily dosing interval in epileptic dogs receiving phenobarbital for > or = 3 weeks. DESIGN: Prospective study. ANIMALS: 33 epileptic dogs receiving phenobarbital. PROCEDURE: Serum phenobarbital concentrations were measured at 0 hour (trough), 3 hours, and 6 hours after oral administration of phenobarbital in epileptic dogs that had received phenobarbital twice daily for a minimum of 3 weeks. For each dog, trough, 3-hour, and 6-hour serum phenobarbital concentrations were evaluated to determine whether they were within the same therapeutic category (lower, middle, or upper end of the therapeutic range of 15 to 45 micrograms/ml), or whether there was a > 30% change in serum concentrations throughout the day. RESULTS: Ninety-one percent (30/33) of dogs had trough, 3-hour, and 6-hour serum phenobarbital concentrations in the same therapeutic category. Only 9% (3/33) of dogs had trough, 3-hour, and 6-hour serum concentrations in different therapeutic categories with a > 30% change in concentrations throughout the day. Significant differences were not detected among mean serum phenobarbital concentrations when comparing the trough, 3-hour, and 6-hour samples for all dogs. CONCLUSIONS AND CLINICAL RELEVANCE: There is no therapeutically relevant change in serum phenobarbital concentrations throughout a daily dosing interval in most epileptic dogs. Therefore, timing is not important when collecting blood samples to measure serum phenobarbital concentrations in most epileptic dogs treated long-term with phenobarbital.