三种抗原对家畜日本血吸虫病诊断价值的比较

以日本血吸虫基因重组抗原LHD-Sj23／pGEX、可溶性虫卵抗原（soluble egg antigen，SEA）及SEA经Sephadex G-200柱层析分离后得到的第一峰SEAl作为诊断抗原，应用ELISA检测人工感染日本血吸虫绵羊血清及自然感染日本血吸虫水牛血清。结果显示，三种抗原用于检测人工感染日本血吸虫绵羊血清的阳性符合率分剐为88．79％、98．15％、100％，阴性符合率均为100％；自然感染日本血吸虫水牛血清的阳性符合率分别为80％、80％、84％，阴性符合率分别为88．9％、93．3％、91．1％；三种抗原与伊氏锥虫病牛血清无交叉反应；检测感染日本血吸虫不同时间绵羊血清，发现感染6周后均被检出针对这三种抗原的特异性抗体，且SEA及SEAl在感染4周后即可检出特异性抗体。三种抗原的诊断效果差异不显著。     

应用LHD-Sj23和SjCL基因重组抗原诊断水牛日本血吸虫病的研究

目的：验证日本血吸虫基因重组抗原检测水牛日本血吸虫病的效果。方法：以日本血吸虫基因重组抗原LHD-Sj23／pGEX和SjCL／pET-28a( )作为抗原，应用ELISA法检测水牛日本血吸虫病。结果：感染日本血吸虫病阳性符合率分别为93．3％(42／45)和95．6％(43／45)，阴性符合率分别为93．3％(42／45)和80％(36／45)。和以血吸虫成虫抗原(SWAP)和虫卵(SEA)抗原作为诊断抗原获得的结果差异不明显。同时将上述两种基因重组抗原混合作为诊断抗原，检测水牛血吸虫病，结果阳性符合率为88．9％(40／45)，阴性符合率为33．3％(15／45)。结论：由于基因重组抗原制备简便、价廉，有利于诊断技术的标准化。因此，基因重组抗原在血吸虫病诊断领域有着良好的研究和应用前景。     

东方田鼠抗日本血吸虫抗体水平检测与分析

东方田鼠具有抗日本血吸虫病的特性，观察东方田鼠感染日本血吸虫尾蚴后的特异性抗体水平变化，可为阐明其抗病机制提供依据。本试验用间接ELISA方法检测东方田鼠抗成虫可溶性抗原（SWAP）和几种日本血吸虫基因重组抗原（Sj28-GST、LHD-SjTP1、C-SjParamyosion）的特异性抗体水平动态变化，并检测东方田鼠感染日本血吸虫尾蚴前、后的IgG3亚类特异性抗体水平。结果东方田鼠在攻击日本血吸虫尾蚴后，抗SWAP的特异性抗体水平有所升高，而抗几体基因重组抗原的特异性抗体水平低，没有显著变化。东方田鼠正常血清中抗SWAP和IgG3亚类抗体的OD值是牛血清白蛋白（BSA）对照的31倍。结果表明，在东方田鼠血清中有较高水平的SWAP的IgG3亚类抗体，值得进一步深入探讨。     

可视化核酸恒温扩增技术在日本血吸虫病筛查和诊断中的应用

日本血吸虫病的筛查和诊断在血吸虫病的防治中处于重要的地位,而核酸诊断是其潜在的优良检测方法。拟建立一套完整的"可视化日本血吸虫核酸恒温扩增检测试剂",用于基层血防单位对日本血吸虫病的筛查和诊断等工作。用腹壁拔毛贴片法给新西兰大白兔感染日本血吸虫,用多种核酸提取方法抽提其血清或血浆中的基因组DNA,并用"可视化日本血吸虫核酸恒温扩增检测试剂"对其进行检测。结果表明,该研究所设计的检测方法可以方便地检测到100条日本血吸虫尾蚴感染的新西兰大白兔,说明可视化核酸恒温扩增技术在日本血吸虫病的筛查和诊断中具有潜在的应用可能性。     

东方田鼠感染日本血吸虫前后血常规和血清生化指标的动态变化

东方田鼠具有天然抗日本血吸虫病特征,为了探索东方田鼠抗日本血吸虫机制,本研究分析了东方田鼠和小鼠感染日本血吸虫尾蚴前后血液细胞以及生化指标的动态变化。东方田鼠和BALB/c鼠定量感染日本血吸虫尾蚴,于感染后第0、1、3、5、8、12、16、21、28、35、42 d分别采集抗凝血和血清,进行血常规和血清生化分析。血常规检测结果表明,东方田鼠感染后第3~16 d,WBC数量显著升高(Neu数量显著升高),而小鼠血常规检测结果正常;血清生化结果显示,东方田鼠感染日本血吸虫后ALB、HDL-C显著升高,小鼠各指标变化不明显。本研究结果显示东方田鼠天然免疫细胞Neu和血清ALB可能与其抗日本血吸虫特性相关,对补体杀伤日本血吸虫机制研究具有重要的指导意义。     

IgM-ELISA法用于日本血吸虫病早期诊断初探

目的 验证血清特异性IgM抗体检测对急性日本血吸虫病的早期诊断价值。方法 采用IgM-ELISA方法检测感染血吸虫的小鼠和急性血吸虫病人血清中的特异性IgM抗体水平，与IgG—ELISA相比较，并与急性血吸虫病人的流行病学调查资料相比对。结果 感染4周时小鼠血清中特异性IgM抗体水平显著升高，5周时阳性检出率达100％，比IgG-ELISA提早2-3周。检测急性血吸虫病人血清阳性率达100％，IgG—ELISA为91．4％。急血病人接触疫水后5周，特异性IgM抗体水平明显高于IgG。7周时两者水平相近，8周时IgG抗体水平超过IgM。结论 IgM—ELISA具有早期诊断急性血吸虫病的价值.     

湖北省江陵县猫日本血吸虫感染情况调查

以饱和食盐水漂浮虫卵法、顶管毛蚴孵化法检测猫日本血吸虫的感染情况。结果显示,饱和食盐水漂浮虫卵法检测阳性率为7.82%;粪便毛蚴孵化法检测阳性率为6.80%;含5-9个毛蚴杯子数占总杯数的17.35%。结果提示,血吸虫病的老疫区江陵县猫轻度感染日本血吸虫。     

日本血吸虫Sj32KD抗原基因的克隆、表达及诊断应用

目的开展日本血吸虫中国大陆株Sj32KD抗原基因研究,探讨其作为诊断抗原的潜力。方法应用PCR方法克隆Sj32KD抗原基因,并在大肠杆菌系统中进行表达,利用H is亲和层析方法纯化融合蛋白,并应用重组的Sj32KD抗原检测羊日本血吸虫病。结果克隆了ORF为1 272bP的日本血吸虫中国大陆株Sj32KD抗原基因,成功构建表达质粒Sj32-pET28 a,并在BL21(DE3)菌株中获得了分子量为47KD的特异性表达产物。应用Sj32KD重组抗原应用于检测羊日本血吸虫病,结果阴性血清的特异性为85.71%,阳性血清的敏感性为95.45%。结论成功克隆表达了日本血吸虫Sj32KD抗原,原核表达融合蛋白有一定的诊断应用潜力。     

日本血吸虫病疫苗及免疫增强技术研究进展

血吸虫病是由血吸虫感染引起的一种危害严重、流行广泛的人兽共患寄生虫病。目前,对血吸虫病的防治以吡喹酮化疗为主,但会出现重复感染,防治实践表明在我国单靠药物治疗不能消灭血吸虫病。疫苗是预防和控制血吸虫感染的重要手段,本文对日本血吸虫病多价疫苗、免疫佐剂及疫苗递送技术的研究现状、存在的问题及其展望进行了概述。     

湖北省江陵县湖滩地区牛日本血吸虫病流行特征的研究

选择湖北省江陵县5个行政村的水牛和黄牛，用血清学诊断法、饱和盐水漂浮虫卵法、顶管毛蚴孵化法检测日本血吸虫感染情况，并采用抽样法对调查村所属的湖滩钉螺分布情况进行了检查。结果，牛日本血吸虫病血清抗体检查阳性率为17．92％，饱和盐水漂浮虫卵法检查阳性率为19．46%，粪便毛蚴孵化法检查阳性率为16．04%，5个村湖滩钉螺的平均密度为0．445只／m^2。结果显示，该县日本血吸虫病流行严重。     

The significance of leptospiral titres associated with bovine abortion

To investigate relationships between serological titres to 2 serovars, pomona (L. pomona) and hardjo (L. hardjo), of Leptospira interrogans and abortions, log linear and logit models were fitted to herd and individual cow data from cattle serologically negative for brucellosis. Serological titres to both serovars were significantly related to abortions in individual cows, with L. pomona having a stronger relationship than L. hardjo. L. hardjo was not significant when herd data were analysed. Differences between dairy and beef cattle in the serological titres found to both L. pomona and L. hardjo were detected when data sets of all cattle or cattle with no history of abortion were analysed. The beef/dairy differences may be due to different management practices and/or to different geographical distributions of both serovars and populations of beef and dairy cattle. If there are no cattle in a herd with a reciprocal titre of 3000 or greater for L. pomona, it is unlikely that L. pomona is associated with the abortion problem. There was no specific L. hardjo titre which separated high and low probabilities that the serum came from a cow or herd with an abortion history.     

The assessment of blood copper status in cattle: a comparison of measurements of caeruloplasmin and elemental copper in serum and plasma

AIM: To evaluate the effect of test, either copper (Cu) concentration or caeruloplasmin (CP) activity, and sample type, either serum or plasma, on the diagnosis of blood Cu status in cattle. METHODS: Paired serum and heparinised plasma samples taken from 125 cattle in 13 herds were tested for Cu concentration and CP activity. The individual results for serum Cu concentration and serum and plasma CP activities were compared with the plasma Cu concentration results, as were their diagnostic values as determined by reference ranges, i.e. 'marginal', 'adequate', 'excess'. RESULTS: The overall mean serum Cu concentration was 2.92 micromol/L lower than the mean plasma Cu concentration; however, there was significant variability between individual samples, and the 95% limits of agreement ranged from 0.44 micromol/L more to 6.28 micromol/L less. The relationship between CP activity and plasma Cu concentration was less variable; the 95% prediction interval for plasma Cu concentration from CP activity was +/- 2.8 micromol/L, and was unaffected by whether CP activity was measured in plasma or serum. Using the threshold currently recommended for 'marginal' status of <8.0 micromol/L for serum Cu concentration identified a significantly different population of cattle than a threshold of <9.0 micromol/L for plasma samples. Altering the threshold to <7.0 micromol/L for serum Cu concentration produced better agreement. For CP activity, a threshold of 15 IU/L for both serum and plasma identified the same population as a threshold of <9 micromol/L for plasma Cu concentration. CONCLUSIONS: Serum Cu concentration is not a suitable substitute for plasma Cu concentration for the detection of 'marginal' blood Cu status in cattle as the individual variability in the apparent loss of Cu during clotting is too great. In this study, CP activity, in both serum and plasma, was found to be a suitable substitute for the detection of 'marginal' blood Cu status. CLINICAL RELEVANCE: The use of serum Cu concentration rather than plasma Cu concentration in the diagnosis of Cu responsive disease in cattle needs to be re-evaluated as does the way in which individual sample results are used in such tests.     

Determinants and Utility of the Anion Gap in Predicting Hyperlactatemia in Cattle

The objectives of this study were to investigate the determinants of the anion gap (AG) in cattle and to evaluate the utility of AG in detecting hyperlactatemia in sick neonatal calves and adult cattle. The AG was calculated as AG = ([Na+] + [K+]) - ([Cl^-] + [HCO₃]), with all values in mEq/L. The AG of healthy neonatal calves (n = 16) was 29.6 ± 6.2 mEq/L (mean ± SD), and the blood L-lactate concentration ranged from 0.5 to 1.2 mM/L. The AG was significantly (P > .05) correlated with serum phosphate (r = .66) and creatinine (r = .51) concentrations. The AG of neonatal calves with experimentally induced diarrhea (n = 16) was 28.6 ± 5.6 mEq/L, and the blood L-lactate concentration ranged from 1.1 to 2.9 mM/L. The AG was significantly correlated with blood L-lactate concentration (r = .67), serum phosphate concentration (r = .63), creatinine concentration (r = .76), and blood pH (r = -.61). The AG of adult cattle with abomasal volvulus (n = 41) was 20.5 ± 7.8 mEq/L, and the blood L-lactate concentration ranged from 0.6 to 15.6 mM/L. The AG was significantly correlated with blood L-lactate concentration (r = .60), serum phosphate concentration (r = .71), creatinine concentration (r = .65), albumin concentration (r = .47), total protein concentration (r = .54), blood pyruvate concentration (r = .67), and blood pH (r = -.41) but not plasma β-OH butyrate concentration. The results indicate that the AG in cattle is only moderately correlated with blood L-lactate concentration and is similarly correlated with serum phosphate and creatinine concentrations in neonatal calves and adult cattle, as well as with serum albumin and total protein concentrations in adult cattle. Anion gap determination is of limited usefulness in predicting blood L-lactate concentration in sick cattle, whereas the correlation between AG and serum creatinine concentration in sick cattle suggests that an increased AG should alert the clinician to the potential presence of uremic anions.     

An evaluation of the effect of clotting on the recovery of copper from caprine blood

In the goat, diagnosis of copper (Cu) deficiency is often based on measurement of Cu in serum or plasma. Previous research in cattle and sheep has shown that these values are not interchangeable, but data for goats have not been published. Paired serum and heparinised plasma samples taken from 119 goats in eight herds were tested for Cu concentration. Plasma and serum Cu were significantly correlated (r=0.95). On average serum Cu was 3.5 μmol/L lower than plasma Cu, but this difference was related to Cu status (r=0.45). Mean serum Cu concentration was 83% of plasma Cu, with the 95% limits of agreement ranging from 66% to 100%. Similar to cattle and sheep, individual variability in Cu loss during clotting is too great for serum Cu to be used as a measure of Cu status in goats.     

Minimal herd sample size for determination of blood copper status of cattle

Copper is required by cattle for synthesis of numerous proteins and enzymes. Copper deficiency in cattle results in a variety of signs ranging from weight loss to diarrhea. In the fall of 1984 and 1985, blood samples were collected from 22 cattle herds near Gunnison, Colo. Approximately one third of the herds were classified as copper deficient (ie, mean serum copper concentration less than 0.6 mg/L). The inherent variability of serum copper concentrations within a herd mandates the determination of the minimal number of cattle to be tested to properly assess the blood copper status of a herd. Coefficients of variation for serum copper concentration were used to calculate a minimal sample size, with a 95% confidence interval for each herd. Minimal sample size ranged from 3 to 55 cattle/herd (ie, 1 to 22% of the herd); this finding suggested that the usual procedure of testing 10% of the herd may be inappropriate.     

Enzyme-linked immunosorbent assay for differentiation of the antibody response of cattle naturally infected with Brucella abortus or vaccinated with strain 19

Purified O chain of Brucella abortus was passively attached to polystyrene to differentiate antibody responses of cattle vaccinated with B abortus strain 19 from those of naturally infected cattle. In the indirect assay, using O polysaccharide as antigen, a single serum dilution was used and mouse monoclonal antibody to bovine L chain conjugated with horseradish peroxidase was the detection reagent. Measurable antibody was not found in sera of vaccinated cattle, except for 3 sera from cattle that were persistently infected with strain 19. Sera from 25 cattle infected with pathogenic strains contained antibody on the basis of results of indirect enzyme immunoassay, using smooth lipopolysaccharide or O chain as antigens, or results of competitive enzyme immunoassay, using the O-chain antigen. Results in sera from calves with experimentally induced Yersinia enterocolitica serotype 0:9 infection or inoculated with a low dose of B abortus strain 2308 were comparable with those in sera of cattle that were vaccinated with strain 19. The data correlated with those from competitive enzyme immunoassay, using one serum dilution and horseradish peroxidase-conjugated mouse monoclonal antibody to smooth lipopolysaccharide. On the basis of results of the indirect enzyme immunoassay, all sera (except those samples obtained before inoculation) contained antibody to smooth lipopolysaccharide.     

Diagnosis of nitrate toxicosis in cattle, using biological fluids and a rapid ion chromatographic method

An ion chromatographic method was used to simultaneously determine nitrate and nitrite ions in biological samples. Ultrafiltration was used to produce a protein-free filtrate. Chloride interferences were eliminated by precipitation as the silver salt. Detection limits and average recoveries were 0.5 mg/L and 102% for nitrate and 0.2 mg/L and 78% for nitrite, respectively. Nitrate concentration was 2.1 +/- 1.8 mg/L and 4.9 +/- 0.8 mg/L in serum and ocular fluid of healthy cattle, respectively; nitrite was not detected. A severe case of nitrate poisoning in cattle was described and used to study the concentrations of nitrate and nitrite in samples obtained under natural conditions. Nitrate concentration of acutely poisoned cattle was 35% lower in ocular fluid at 158.1 +/- 51.4 mg/L, than in serum at 256.3 +/- 113.4 mg/L. Nitrite was not detected, because of the long processing time (greater than 3 hours) required for samples obtained in the field. A gradual decrease in ocular fluid nitrate of 29.4% at 24 hours, 25.9% at 36 hours, 51.6% at 48 hours, and 73.2% at 60 hours was observed; however, concentrations remained diagnostically significant (73.2 mg/L) 60 hours after death. Twenty-four hours after poisoning, the serum nitrate concentration of severely ill (52.7 +/- 51.9 mg/L) and moderately affected (12.4 +/- 5.7 mg/L) cattle that survived was indicative of the severity of clinical signs previously observed. Nitrate in serum and ocular fluid was stable in samples stored for 24 hours at 23 C, 1 week at 4 C, and 1 month at -20 C.     

Preliminary report: immunodiagnosis of pre-type II ostertagiasis

Ostertagia ostertagi soluble antigens were prepared by gel electrophoresis and electrophoretic transfer onto nitrocellulose for enzyme-linked immunosorbent assays with serum probes. Serologic responses to L3-derived antigen of approximately 32 kDa may be unique and diagnostic for cattle harboring inhibited larvae, or pre-Type II ostertagiasis. Specificity was evaluated by comparing sera from pre-Type II cattle to sera from Type I, uninfected, Fascioloides magna infected, Fasciola hepatica infected or Cooperia oncophora infected cattle.     

The effect of copper deficiency on the peripheral blood cells of cattle

Six female cattle were given molybdenum (30 ppm) and sulphate (225 ppm) to induce experimental secondary copper deficiency. The total and differential leukocyte numbers and lymphocyte subpopulations were counted and the neutrophil activity was assessed by means of nitroblue tetrazolium reduction and phagocytosis of sheep erythrocytes. The serum caeruloplasmin activity and concentration were also determined. Copper deficiency was confirmed from decreased serum copper levels, the animals with values less than 5.9 µmol/L being considered deficient. Total leukocyte numbers were not affected by the copper deficiency. However, differential counts showed a marked increase in monocyte subpopulations, a significant decrease in B lymphocytes and reduced neutrophil activity. The serum caeruloplasmin activity was decreased about 50%, but the total serum protein concentration was less altered. We concluded that the effect of these changes on the animals' immune competence may contribute to a greater incidence of infectious diseases in copper-deficient cattle.     

Cerebrospinal fluid constituents collected at the atlanto-occipital site of xylazine hydrochloride sedated, healthy 8-week-old Holstein calves.

Cerebrospinal fluid (CSF) collected at the atlanto-occipital site and serum were obtained from 10 male, 8-week-old, Holstein calves after sedation with xylazine hydrochloride. Glucose, creatine kinase, alkaline phosphatase, urea nitrogen, creatinine, sodium, potassium, chloride, calcium, phosphorus, total protein, and albumin were determined in serum and CSF. Optical characteristics, specific gravity, total red blood cell and nucleated cell counts and differentials were also evaluated in the CSF. Additionally, CSF protein electrophoresis and immunoglobulin concentrations were determined. Then, albumin quotients (AQ) were derived. Erythrocytes were observed in 9 of 10 CSF samples. Total nucleated cell counts ranged from 0-10 cells x 10(6)/L with a mean of 3 cells x 10(6)/L. Differential nucleated cell count in the CSF consisted primarily of lymphocytes/small mononuclear cells (57%), fewer monocytes/ large mononuclear cells (38%), and scant neutrophils (4%) and eosinophils (0.05%). The concentration of sodium (134 to 139 mEq/L) was similar to that of serum, but the concentration of potassium (2.8 to 3 mEq/L) was lower than that of serum. Creatine kinase activity (0 to 4 U/L) of CSF was markedly lower than serum activity. The CSF glucose concentration was approximately 80% of the serum value. Cerebrospinal fluid total protein concentration determined by electrophoresis ranged from 110 to 330 mg/L with a mean of 159 mg/L. Cerebrospinal fluid albumin ranged from 48 to 209 mg/L with a mean of 86 mg/L. In all CSF samples, radial immunodiffusion of unaltered CSF and concentrated CSF (four-fold concentration) revealed quantities undetectable by the present techniques in which the lowest standard values for IgG1, IgG, and IgM determinations was 70 mg/L and IgG2 was 30 mg/L. The albumin quotient ranged from 0.15 to 0.65 with a mean of 0.25. Based on the results of this study, CSF may be collected at the atlanto-occipital site safely and efficiently in calves, and reported values for CSF from adult cattle may not be suitable for evaluation of CSF collected from immature cattle.