猪蛔虫幼虫琼脂胶移行法的建立

本实验首次在国内建立起了线虫的琼脂胶移行法 (Agar- gel Migration Assay,AMA)。通过对影响幼虫活力的某些理化因素 ,如 :琼脂胶的温度、每孔加入的琼脂胶和幼虫混合液的体积、琼脂胶的浓度及幼虫在琼脂胶内移行的时间等研究发现 :加胶的温度和加胶的量对幼虫的移出率有较大的影响。幼虫的移出率随加胶温度的升高而降低 ,当温度达到 70℃时 ,则几乎无虫体从琼脂胶内移出 (移出率仅为 0 .5 % ) ;幼虫的移出率随加入琼脂胶量的增多而减小 ,当每孔加入 10 0 0μl时 ,幼虫的移出率为 16 .6 8% ,当加入 4 0 0μl时幼虫的移出率则能达到2 5 .17%。琼脂胶的浓度对幼虫移出的影响并不明显 ,采用 SAS软件 (for Windows V6 .12 )分析发现 1.2 %、1.0 %、0 .8%、0 .4 %的结果间无显著差异 (P>0 .0 5 ) ,但琼脂胶的浓度能影响胶液凝固的时间和凝固后的韧性。实验表明 :最适宜的加胶量为 4 0 0μl /孔 ,最适宜的加胶温度为 5 3℃ (Agar- gel,sigm a) ,在琼脂胶中最佳培养移行时间为 2 4 h,琼脂胶液的最适实验浓度为 1.5 %琼脂胶     

A comparison of the efficacy of two ivermectin formulations against larval and adult Ascaris suum and Oesophagostomum dentatum in experimentally infected pigs

A study was conducted to evaluate and compare the efficacy of two injectable formulations of ivermectin (IVM-1 and IVM-2) at a dose rate of 0.3 mg/kg bodyweight versus placebo in the treatment and control of larval and adult stages of Ascaris suum and Oesophagostomum spp. in experimentally infected pigs. Seventy helminth free pigs were allocated on a liveweight basis to 7 groups each comprising 10 pigs (A-G). Group A served as an untreated control group. Groups B and C were used to investigate the efficacy of both formulations against adult stages of A. suum and Oesophagostomum spp., Groups D and E for efficacy against larval stages of A. suum and Groups F and G for efficacy against larval stages of Oesophagostomum spp. Pigs of groups A, B, C, D and E were infected on Day-0 with 1000 infective A. suum eggs each. Infective larvae of Oesophagostomum spp. (10,000/pig) were given on Day-0 to pigs of Groups F and G and on Day-21 to pigs of Groups A, B and C. Treatment was given to pigs of Group A (saline as placebo) on Day-7 and -28, IVM-1 to pigs of Group F on Day-7, pigs of Group D on Day-14 and pigs of Group B on Day-49. IVM-2 was given to pigs of Group G on Day-7, Group E on Day-28 and Group C on Day-49. Pigs of Groups F and G were sacrificed on Day-28, pigs of Groups A, D and E on Day-49 and pigs of Groups B and C on Day-56. Post mortem worm counts showed the following efficacies: (IVM-1) against larval A. suum 100%, against adult A. suum 94.4%, against larval Oesophagostomum spp. 52.0% and against adult Oesophagostomum spp. 83.0%. (IVM-2) against larval A. suum 100%, against adult A. suum 90.3%, against larval Oesophagostomum spp. 94.0% and against adult Oesophagostomum spp. 94.7%.     

Optimization of the under-agarose assay of porcine neutrophil migration

Important procedural factors in the under-agarose assay for porcine neutrophil migration were identified, and optimal conditions were established. Three factors were tested: the concentration of zymosan-activated serum inoculated into the outer well; the number of neutrophils inoculated into the center well; and the time of incubation of the agarose plates. All factors had a significant (P less than 0.0001, 0.0001, and 0.01, respectively) effect on the chemotactic index of porcine neutrophils. The optimal combination of these 3 factors was undiluted zymosan-activated serum as the chemoattractant, 8 X 10(5) neutrophils inoculated into the center well, and 5 hours of incubation. The assay was validated, using standard conditions, and the data were used to predict the number of pigs and/or repetitive assays needed to identify differences among experimental groups.     

Use of a soluble tetrazolium/formazan assay for chicken cells.

We evaluated a soluble tetrazolium/formazan assay using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl ]-2H- tetrazolium hydroxide (XTT) for chicken cell growth. Fifty microliter of solution containing 1 mg/ml of XTT and 0.025 mM phenazine methosulfate was added to the cells in a well of 96-well microplate. After 4 hr incubation at 37 degrees C, the absorbance was measured at 490 nm. Under this condition, absorbances were well correlated with cell number of Marek's disease tumor cells and chicken embryo fibroblasts. Proliferation of chicken lymphocytes stimulated with mitogens was also effectively measured. The formazan of XTT is water-soluble and can be quantitated in culture medium without the necessity for extraction with organic solvents. Thus XTT assay is simple and useful for the quantity assay with chicken cells.     

Use of the D-dimer assay for diagnosing thromboembolic disease in the dog

Although the exact incidence of pulmonary thromboembolism (PTE) in small animals is unknown, it is thought that PTE is a substantial, under-diagnosed complication. The difficulty in diagnosing PTE in small animals is confounded by its subtle symptomatic presentation and a lack of clinical suspicion, coexisting disease states, and lack of noninvasive tests that are sensitive and specific for the diagnosis of PTE. Although numerous laboratory markers of coagulation have been studied, only the D-dimer assay has shown clinical utility in detecting early embolism in humans. This paper examines the use of D-dimer assays and other clinical modalities in the diagnostic approach to thromboembolic disease in small animals.     

Characteristics of a flubendazole resistant isolate of Oesophagostomum dentatum from Germany.

Two controlled tests were performed to investigate the benzimidazole resistance of a nodular worm isolate "GIBZ" from a pig breeding farm in Germany. In Trial I, groups of five pigs, artificially infected with Oesophagostomum larvae isolated from that farm were treated with flubendazole at a single dose of 5 mg kg(-1) bodyweight (BW) or remained untreated. In Trial II, three groups of three pigs each infected with larvae after a further laboratory passage of this isolate were treated with flubendazole either at a single dose of 5 mg kg(-1) BW or at a divided dose of 1.5 mg kg(-1) BW daily for 5 consecutive days, or with fenbendazole at a single dose of 5 mg kg(-1) BW, the fourth infected group remained untreated. The respective doses of anthelmintics were mixed with a small amount of feed and administered to individual pigs in both trials. Fecal egg counts before and after treatment and post-mortem worm burdens 7 days after (last) treatment were examined to assess the anthelmintic efficacies. Only infections with Oesophagostomum dentatum were found in both trials. In Trial I, the mean worm count reduction by flubendazole was 30% as compared to the untreated controls. In Trial II, flubendazole administered at a single or divided dose reduced the mean worm burden by 0 and 85%, respectively, whereas fenbendazole was 100% effective. These results establish resistance to flubendazole in the isolate "GIBZ" of O. dentatum. The failure to reveal side resistance to fenbendazole may be explained by that the currently recommended dose rate of this compound is supra-optimal for porcine nodular worms.     

Long-term cryopreservation, infectivity and characterisation of Oesophagostomum dentatum

The successful cryopreservation for 6 years of infective larvae of a pure strain of Oesophagostomum dentatum is described. Changes in the characteristics of the isolate in comparison with the original strain could not be demonstrated.     

Use of a polymerase chain reaction assay for detection of Haemobartonella canis in a dog.

A polymerase chain reaction (PCR) originally developed for detection of Haemobartonella felis in cats was successfully used for detection of H canis in an 8-year-old spayed Great Dane. The dog had been splenectomized and was undergoing immunosuppressive chemotherapy at the time of diagnosis. Sequence analysis of the 16S ribosomal RNA gene revealed that the Haemobartonella spp infecting this dog was 97% homologous to the sequence previously reported for the Ohio strain of H felis. Clinical and hematologic abnormalities as well as identification of the organisms by use of light and electron microscopy supported the diagnosis of H canis. The PCR assay used for detection of H felis may be useful for the detection of H canis in dogs prior to blood donation, splenectomy, or treatment with immunosuppressive drugs.     

Use of a fiberoptic accessory for retrieval of adult heartworms in a dog with postcaval syndrome

Use of a spiral fiberoptic basket for the removal of adult heartworms from the vena cava and right atrium of a dog with postcaval syndrome is described. Complications reported with the use of alligator forceps were not encountered with the basket.     

An interferon-gamma assay for diagnosis of Corynebacterium pseudotuberculosis infection in adult sheep from a research flock

The optimal method of control of caseous lymphadenitis of sheep caused by Corynebacterium pseudotuberculosis is eradication of infection by identification and removal of infected carrier animals. Current serological approaches to identification of infected sheep are generally hampered by low sensitivity and specificity of available tests. The objective of this study was to develop a whole blood assay for detection of C. pseudotuberculosis-infected sheep, based on detection of IFN-gamma response to whole cell C. pseudotuberculosis antigens, and to determine the reliability of the assay. A commercially available bovine interferon-gamma assay enzyme-linked immunosorbent assay was used and the test optimised using experimentally infected sheep. The assay was also tested on known CLA-negative sheep. Setting a IFN-gamma optical density cut-off at 0.100 as positive under the conditions used, the test detected C. pseudotuberculosis experimentally infected sheep over a 450-day period with a reliability of 95.7%. It identified known non-infected sheep with a reliability of 95.5%. Repeated vaccination of three uninfected sheep with a commercially available bacterin-toxoid vaccine did not interfere with the assay. The IFN-gamma response of sheep whole blood to C. pseudotuberculosis antigens offers promise for use in a test-and-removal approach to eradication of CLA in sheep.     

Vertical migratory behavior of the infective third-stage larvae of Oesophagostomum dentatum

The vertical migratory behavior of third-stage infective larvae (L3i) of Oesophagostomum dentatum was investigated using upright truncated agarose cones and equivalent conical depressions in agarose. Geotactic response varied with the age of the infective larvae. Four-day-old L3i showed no preference for the sloping surfaces of either indented or upright cones, while the 8-day-old L3i showed a positive geotactic reaction, migrating down the sloping surface of the depressions.     

Use of a new and rapid milk progesterone assay to monitor reproductive activity in the cow

A new and rapid enzyme-amplified immunoassay (AELIA) has been developed for the measurement of progesterone in milk. The AELIA system is a non-isotopic method that gives results within 35 minutes. Milk progesterone concentrations measured in 10 cows sampled daily at various stages of the reproductive cycle were very similar to those recorded by a validated radioimmunoassay. The results show that the speed and sensitivity of the AELIA system would make it possible to diagnose pregnancy rapidly at about 24 days after insemination, to predict the onset of behavioural oestrus from decreasing progesterone values during the third week after a preceding oestrus, and to obtain a daily record of milk progesterone levels in animals treated for infertility of ovarian origin.     

Use of a time-resolved immunofluorometric assay for determination of canine C-reactive protein concentrations in whole blood

OBJECTIVE: To develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for measurement of C-reactive protein (CRP) in canine whole blood. ANIMALS: 12 healthy dogs and 35 dogs with inflammatory processes. PROCEDURE: CRP was isolated from acute-phase serum by affinity chromatography and used as a standard for calibration. Analytic and functional limit of detection and intra-assay and interassay precision were calculated. Accuracy was evaluated by recovery assays and by comparison with results of a commercial ELISA. Correlation between CRP concentrations in whole blood and corresponding plasma fractions was tested by use of TR-IFMA. Stability of blood samples at 4 degrees C was assessed during a 1-month period, and effects of anticoagulants were evaluated. Measurements of CRP in blood samples from 12 healthy dogs were compared with those of 35 dogs with inflammatory diseases. RESULTS: Analytic and functional limits of detection were 0.53 and 3.26 microg/mL, respectively. Intra-assay and interassay coefficients of variation varied between 2.1% to 8.9% and 8.0% to 12.3%, respectively. Mean recoveries of added CRP were 104% and 114%. Measurements of CRP by use of TR-IFMA and ELISA were highly correlated (R2 = 0.97). Measurements of CRP in whole blood and in corresponding plasma fractions by use of TR-IFMA were also highly correlated (R2 = 0.97). Neither storage nor use of anticoagulants disturbed measurement of CRP concentrations in whole blood. Concentrations of CRP in whole blood of dogs with inflammation were significantly higher than in healthy dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Determination of CRP concentrations in whole blood may provide a diagnostic test for inflammation in dogs.     

Use of an enzyme-linked immunosorbent assay in a bovine brucellosis eradication program

An enzyme-linked immunosorbent assay (ELISA) was developed and was compared with the complement fixation test (CFT) in a bovine brucellosis eradication program. The ELISA detected significantly more reactors than the CFT in both strain 19 vaccinated infected herds (1.79% versus 1.14%) and non-vaccinated infected herds (4.2% versus 3.59%) but not in either vaccinated or non-vaccinated brucella-free herds. The specificity for both tests in brucella-free herds was greater than 0.998. The specificity and sensitivity of the ELISA were compared with those of 3 other tests (the Rose Bengal test; the indirect haemolysis test [IHLT] and the CFT) on serum from 151 animals cultured at slaughter. The calculated specificity of the ELISA in this infected group was lower than for both the CFT and the IHLT (0.58 versus 0.67 versus 0.75). The sensitivity however was much greater (1.0 versus 0.73 versus 0.71). The value of the ELISA when used in an eradication program is discussed.     

Use of a feline cell line in the syncytia infectivity assay for the detection of bovine leukemia virus infection in cattle

This report describes a modified syncytia infectivity assay (SIA) for the direct detection of bovine leukemia virus (BLV) in blood lymphocytes of cattle, using transformed feline (CC81) cells as the indicator system. The data show that the syncytia present in cultures of CC81 cells inoculated with BLV-infected cells are specific and arise through a mechanism similar to that responsible for the phenomenon of "late" polykaryocytosis described in other virus systems. The susceptibility of the CC81 cells to the syncytia-inducing effect of BLV-infected cells is comparable with that of early passages of bovine embryonic spleen cells, which were previously used as the indicator system in the SIA. Unlike the bovine embryonic spleen cells, CC81 cells retain their susceptibility to syncytia induction for long periods of cultivation. Furthermore, the syncytia induced in the CC81 cultures are larger and easier to identify. Thus, the CC81 cells can be used advantageously as the indicator system when the SIA is applied to the detection of BLV-infected lymphocytes. The results of the SIA for the detection of infective BLV agreed closely with those of the radioimmunoassay for the detection of BLV antibodies in randomly examined cattle. On the other hand, many cattle in early stages of infection were positive in the radioimmunoassay several months before they reacted in the SIA. The detection of BLV in blood lymphocytes provides a useful method for the diagnosis of BLV infection in cattle when serologic tests cannot be used, eg, calves that may have passively acquired maternal antibodies and cattle given BLV vaccines.     

Effects of Oesophagostomum dentatum and dietary carbohydrates on morphology of the large intestine of pigs

The effects of Oesophagostomum dentatum infection and dietary carbohydrates on the morphology and epithelial cell proliferation in the gastrointestinal tract of pigs were investigated experimentally. Thirty-two worm-free pigs (n=32) from a specific pathogen-free farm were randomly divided into four groups (A-D), of eight animals each. Pigs in groups A (control) and B (infected) were fed Diet 1, and pigs in groups C (control) and D (infected) were fed Diet 2. The two diets were formulated: Diet 1 (%) contained barley flour, oat husk meal plus soya bean meal (55:21:24) and Diet 2 (%) contained barley flour, inulin and sugar beet fibre (SBF) (80.1:7:12.9) plus soya bean meal (3:1) to contain carbohydrates from inulin and sugar beet fibre (SBF) that were readily fermentable in the large intestine. The two infected pig groups (16 pigs total) were inoculated with 6000 infective larvae of O. dentatum and all pigs, including the controls, were slaughtered 12 weeks p.i. The combination of O. dentatum infection and highly fermentable dietary carbohydrates affected the mucosal architecture, the epithelial cell proliferation and mucin secretion of the large intestine. Infection had a significant influence on the crypt volume, height and density, and on muscularis externa at the proximal and middle colon. The changes in the affected gut sections were proportional to the number of worms present. However, these parameters appeared unaffected by those diets alone. In pigs without infection non-digestible dietary carbohydrates significantly influenced the tissue weight of colon.