Dot immunobinding assay for the detection of bluetongue virus antibodies in sheep experimentally inoculated with bluetongue virus type 1.

Dot immunobinding assay (DIA) was evaluated for the detection of bluetongue virus (BTV) antibodies in sheep experimentally inoculated with BTV 1. Serum samples collected on 14, 21, 28, 43 and 60 day post infection (dpi) were positive for precipitating antibodies by the agar gel precipitation test (AGPT) while antibodies could be detected as early as 7 dpi by DIA and ELISA. Virus neutralizing antibodies were detected first at 14 dpi. The sensitivity of the four tests was compared on the same serum samples collected at different intervals. The results indicated that DIA was more sensitive than AGPT and the serum neutralization test and as sensitive as ELISA. Thus due to sensitivity simplicity and economy, DIA could replace AGPT for diagnosis and serological survey for BTV infection in animals.

Dot immunobinding assay in comparison with enzyme-linked immunosorbent assay for the detection of bluetongue virus antibodies in sheep.

A total of 384 sheep serum samples collected from two organised sheep farms was tested by dot immunobinding assay (DIA) and indirect enzyme-linked immunosorbent assay (I-ELISA) for the presence of bluetongue virus (BTV) antibodies. The results of both these assays were compared to find a sensitive, specific, rapid, easily performed and economical test for the diagnosis of bluetongue disease. DIA detected BTV antibodies in 210 samples (54.94%) and I-ELISA detected 157 positive samples (40.88%). Competitive ELISA (C-ELISA) was performed to check the discrepancies in I-ELISA and DIA. On the basis of these tests the overall agreement, relative specificity and sensitivity between ELISA and DIA were 75%, 87.6% and 100%, respectively. DIA was found to be a rapid, sensitive, easily performed and economical test as compared to ELISA.

Mass screening and confirmation of codeine and morphine in urine by radioimmunoassay-GLC.

A rapid, sensitive, and specific procedure is described for the mass screening and confirmation of codeine and morphine in urine specimens. The method is sensitive to 0.5-mug/ml levels of both opiates in free and/or conjugate forms. The raw urine is screened directly by radiommunoassay, which is reactive to both free and glucuronide forms of codeine and morphine. Specimens that are screened positive are confirmed by GLC using a flame-ionization detector. The opiates are analyzed as their acetyl derivat-ves on two different columns, OV-25 and Poly-A 103. This multiple approach eliminates false positives caused by interfering substances or structurally similar compounds present in the urine.

Evaluation of phencyclidine by EMIT d.a.u. utilizing the ETS analyzer and a 25-ng/mL cutoff.

This study evaluates the usefulness of the EMIT d.a.u. phencyclidine assay using the Syva ETS instrument to reliably detect phencyclidine in urine specimens at 25 ng/mL and above. More than 1600 urine specimens were screened over a one-month period. Fifty three (53) specimens screened positive (25 of these were previously tested positive by EMIT and GC/MS at or above the 25-ng/ml level and resubmitted as unknowns). Of the 53 specimens, 52 were confirmed positive by GC/MS. Reanalysis by EMIT of the one sample that was confirmed negative by GC/MS yielded a negative result. The absorbance difference between a negative sample and a sample containing 25 ng/mL of phencyclidine averaged 53 absorbance units with a standard deviation of 7.7 units. The absorbance difference between a negative sample and a sample containing 75 ng/mL of phencyclidine averaged 105 absorbance units with a standard deviation of 6.8 units.

Identification of underivatized basic drugs in urine by capillary column gas chromatography.

A capillary column gas chromatographic (GC) method is described for detecting basic drugs of abuse and their metabolites in urine. One milliliter of urine, buffered with 400 microL Tris buffer (pH 9.5) containing 1.0 microgram/mL internal standard SKF 525A (proadifen hydrochloride), is extracted with 100 microL of 5% (v/v) isoamyl alcohol/hexane. The sample is vortexed for 30 sec and centrifuged for 10 min. The upper organic layer is directly injected into the GC, thus eliminating the need for an evaporation step. The GC is equipped with two DB-1701 fused silica capillary columns and nitrogen phosphorus detectors. The column oven is temperature programmed from 185 degrees to 265 degrees C at 8 degrees/min. Identification of the unknown drugs is accomplished by using retention time relative to that of a common internal standard.

Simultaneous identification of cocaine and benzoylecgonine using solid phase extraction and gas chromatography/mass spectrometry.

A procedure for the simultaneous detection and quantitation of cocaine and benzoylecgonine from urine is described. Using solid phase extraction, cocaine, benzoylecgonine, deuterated cocaine, and deuterated benzoylecgonine (2 internal standards) are extracted from urine. Benzoylecgonine and the deuterated benzoylecgonine are derivatized to their trimethylsilyl esters. The underivatized cocaine and derivatized benzoylecgonine are detected by electron impact mass spectrometry in the selected ion monitoring mode. Both cocaine and benzoylecgonine are detected at concentrations of 50 ng/mL. In addition, the procedure is simple, rapid, and suitable for a large number of specimens.

Simultaneous identification of amphetamine and methamphetamine using solid-phase extraction and gas chromatography/nitrogen phosphorous detection or gas chromatography/mass spectrometry.

A method for the simultaneous detection and quantitation of amphetamine and methamphetamine in urine is described. Using solid-phase extraction, amphetamine, methamphetamine, and n-propylamphetamine (internal standard) are extracted from urine samples. Drugs in their free form are identified using gas chromatography/nitrogen-phosphorous detection (GC/NPD), whereas their heptafluorobutyric anhydride derivatives are detected by gas chromatography/mass spectrometry (GC/MS) in the selected ion monitoring (SIM) mode. Limits of detection for both amphetamine and methamphetamine are approximately 35 ng/mL. The procedure is simple, rapid, and suitable for a large number of specimens (25 or more).

Evaluation of Emit II reagents on the Chem 1.

An evaluation study of Syva Emit II reagents using the Chem 1 was performed for the following drugs: barbiturates, benzodiazepines, cannabinoids, benzoylecgonine, opiates, and phencyclidine. The Emit II reagents (100-mL bottles) were reconstituted to 70 mL and evaluated against the Emit d.a.u. reagents. A minimum of 446 samples were run for each drug. For all drugs tested, there were a total of 11 discordant results between the two reagents. The Emit II reagent was found to be correct on 8 of the 11 discordances after retesting by FPIA or GC/MS. The CV of within-run and day-to-day precision of the Emit II was 1.8% or less and 12.3% or less, respectively.

Bizarre lymphoid cells in serous effusion of a dog with mediastinal lymphoma.

An 8-year-old collie dog was examined because of dyspnoea. Thoracocentesis revealed a modified transudate containing a predominance of large (10 to 15 microns diameter) unusual-appearing mononuclear cells showing cloverleaf-shaped nuclei. These cells were categorized as lymphoid in origin on the basis of ultrastructural and cytochemical methods. Subsequent necropsy examination revealed lymphoma, localized primarily to the cranial mediastinum, but also infiltrating tissues surrounding the thyroid, parathyroid and adrenal glands.

Ultrastructural and biochemical observations on antineutrophil antibody- and complement-induced immuno-injury to equine neutrophils.

Antibody-induced damage to neutrophils was studied to elucidate processes associated with destruction of neutrophils in immune-mediated neutropenias. Cytomorphological changes and release of certain cellular constituents were determined for neutrophils treated with an antineutrophil serum in the presence or absence of rabbit complement. Neutrophils exposed to the antineutrophil serum alone showed endocytotic vacuoles and degranulation. In contrast, neutrophils exposed to the antineutrophil serum and complement showed marked morphologic changes. The plasma membrane developed numerous vesicles, villous processes and minute areas of bilayer discontinuity. Highly damaged cells exhibited cellular and nuclear swellings, disruption of cytoplasmic integrity and disordered distribution of lysosomal granules. Cytoplasmic constituents (K+ and lactate dehydrogenase) were released extracellularly from neutrophils exposed to the antineutrophil serum with or without complement. Cytological changes induced by the antineutrophil serum and complement were analogous to those reported for leucocytes exposed to the activated complement components C5b-9 (the membrane attack complex) and bacterial toxins. It was concluded that the cytological abnormalities observed were most probably associated with immune-mediated damage to the cell membrane, leading to leakage of cytoplasmic constituents like K+, colloidal osmotic swelling, and disruption of the cytoskeletal system.

Mechanisms of platelet destruction in immune-mediated thrombocytopenia: in vitro studies with canine platelets exposed to heterologous and isologous antiplatelet antibodies.

Normal canine platelets coated with heterologous or isologous antiplatelet antibody were interacted with viable canine neutrophils in vitro. Platelet phagocytosis was assessed by detecting nitroblue tetrazolium (NBT) reduction, measuring uptake of opsonised 51Cr-labelled platelets, and electron microscopy. The NBT reduction and uptake of 51Cr-labelled opsonised platelets were markedly increased. Electron microscopy revealed phagocytosis of antibody-coated platelets and their degradation intracellularly. Exposure of canine platelets to rabbit anti-canine platelet antibody in vitro produced morphological changes in platelets and caused serotonin release. Serotonin was not released in the absence of antiplatelet antibody or in the presence of normal rabbit gamma-globulin. Morphological changes in the platelets included disappearance of alpha and dense granules and exaggeration of the open canalicular system. These observations indicate that circulating platelets may be vulnerable to an antiplatelet antibody and that antibody-mediated phagocytosis of platelets is an important mechanism in the pathogenesis of immune-mediated thrombocytopenia.

Fusiform erythrocytes resembling sickle cells in angora goats: light and electron microscopic observations.

Fresh blood from nine mature non-pregnant angora goats was found to contain a varying proportion of spindle-shaped, fusiform, triangular, pear-shaped, and other bizarre forms of erythrocytes in addition to normal discoid biconcave erythrocytes. The number of spindled and fusiform erythrocytes varied from 2 to 66 per cent, with two goats having such cells in excess of 50 per cent. Other aberrant forms of erythrocytes ranged from 3 to 54 per cent. Although the aberrant erythrocytes seemed to be present intravascularly, all goats studied were clinically normal and exhibited no other haematological abnormality. Light and electron microscopic observations indicated that polymerisation of haemoglobin in the form of longitudinal tubular fibres was responsible for conferring the fusiform and spindle shapes to erythrocytes, a phenomenon akin to that seen in sickle-shaped human and deer erythrocytes.

Leucocytic changes in cows given intravenous injections of Escherichia coli endotoxin.

Single intravenous injections of Escherichia coli endotoxin into normal cows produced dose dependent leucocytic and febrile responses. Injections of 5 to 20 microgram endotoxin induced a neutrophilia between 4 and 8 h, whereas 50 to 500 microgram produced a severe neutropenia for 4 to 6 h followed by a gradual increase in neutrophils along with slight to moderate left shift. Higher doses caused progressively severe lymphopenia and greater increase in fever than the lower doses. It was concluded that blood leucocytic changes during the early part of endotoxin-induced mastitis are partly related to systemic absorption of endotoxin, and that the size of the bone marrow neutrophil reserve in the cow could be estimated by injecting 5 to 10 microgram of endotoxin intravenously.

Experimental immunologic thrombocytopenia in dogs: a study of thrombocytopenia and megakaryocytopoiesis.

Immunologic thrombocytopenia was induced in dogs by intravenous or intraperitoneal injection of rabbit anti-canine platelet serum (APS) or by intraperitoneal injection of the IgG fraction of the APS. In contrast, dogs injected with normal rabbit serum (NRS) or the IgM fraction of the APS did not develop thrombocytopenia. Marrow samples obtained at 24-96 h after inoculation of the IgG fraction contained megakaryocytes giving a positive reaction with flurescein isothiocyanate or horseradish peroxidase-conjugated anti-rabbit globulin. In contrast, negative results were obtained with megakaryocytes in marrow samples collected before injection of the IgG fraction or after injection of the IgM fraction or NRS. The attachment of anti-platelet antibody to the surface of megakaryocytes was associated with appearance of morphologic abnormalities, particularly in mature megakaryocytes. Simultaneously, the number of immature megakaryocytes and acetylcholine esterase (AChE)-positive small cells (presumably megakaryocyte precursors) increased considerably. These changes coincided with the development of thrombocytopenia, and subsided as recovery ensued.

Fusiform erythrocytes resembling sickle cells in angora goats: observations on osmotic and mechanical fragilities and reversal of cell shape during anaemia.

Osmotic and mechanical fragilities of erythrocytes were determined for seven goats having 3.4-71 per cent fusiform erythrocytes. The osmotic fragility was related to the erythrocyte shape in that the osmotic resistance was considerably higher for bloods containing more than 26 per cent fusiform erythrocytes. A decrease in the proportion of fusiform erythrocytes in the same goats was related to an increase in the osmotic fragility. Anaemia was induced in two goats by removal of 200-400 ml of blood at three or four day intervals for eight weeks. Red cell values decreased by 28-43 per cent within three weeks, but further bleeding produced either no or less (0-21 per cent) reductions in these values. Slight reticulocytosis was seen during the anaemic phase and there was a concomitant increase in the mean corpuscular volume and mean corpuscular haemoglobin values. Reticulocytosis diminished before the start of recovery from anaemia and disappeared during the recovery phase. The most significant finding was the change in the erythrocyte morphology during production of and recovery from anaemia. The development of anaemia was associated with a gradual reduction in the proportion of fusiform erythrocytes or discoid cells and simultaneous increase in the proportion of erythrocytes exhibiting distinct poikilocytosis. Recovery from the anaemia was rapid (within five weeks), but reversal of the erythrocyte shape took several months. Severe blood loss anaemia in the goat is known to induce synthesis of haemoglobin C, and in these anaemic goats formation of a new haemoglobin, most likely haemoglobin C, was demonstrated by electrophoretic and column chromatographic analyses. It was concluded that the formation of haemoglobin C was responsible for the morphological changes in the erythrocytes.

Haematological studies on normal lactating Indian water buffaloes.

Haematological studies were conducted on 50 clinically normal lactating Murrah buffaloes in India. The range and mean (with one standard deviation), respectively, for the parameters examined were: red blood cells, 5.07 to 8.27, 6.54 +/- 0.77 million per microliter; haemoglobin, 9 to 13.5, 11.1 +/- 0.96 g/dl; packed cell volume, 0.26 to 0.34, 0.31 +/- 0.02; mean corpuscular volume, 40.6 to 55.2, 48.2 +/- 4.6 fl; mean corpuscular haemoglobin concentration, 30.9 to 38.5, 35.2 +/- 2.34 g/dl; mean corpuscular haemoglobin, 13.5 to 20.5, 17.10 +/- 1.85 pg; icterus index, 2 to 5, 2 +/- 1.25 units; erythrocyte sedimentation rate, 17 to 69, 53 +/- 12.3 mm at one hour; plasma protein, 6 to 9, 7.8 +/- 0.7 g/dl; fibrinogen, 0.2 to 0.8, 0.37 +/- 0.2 g/dl; reticulocytes, 0 per cent; white blood cells, 6250 to 13,050, 9676 +/- 1789 microliters; band cells, 0 to 1, 0.2 +/- 0.34 or 0 to 106, 18 +/- 40/microliters; neutrophils, 13 to 54, 32.9 +/- 8.74 per cent or 1285 to 6893, 3257 +/- 1262/microliters; lymphocytes, 26 to 75, 52.7 +/- 12.0 per cent or 2554 to 9637, 5065 +/- 1595/microliters; monocytes 1 to 11.5, 5.9 +/- 2.63 per cent or 63 to 1349, 584 +/- 301/microliters; eosinophils, 2 to 14.0, 6.9 +/- 4.64 per cent or 170 to 1471, 592 +/- 452/microliters and basophils, 0 to .5, 1.4 +/- 1.02 per cent or 0 to 326, 131 +/- 98 microliters. Normal species characteristics evident from this investigation included average size of the erythrocytes similar to that in cattle, low icterus index, conspicuous erythrocyte sedimentation rate, absence of reticulocytes and predominance of lymphocytes over neutrophils.

Detection of equine antiplatelet and antineutrophil antibodies by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was standardised and applied for the detection of antiplatelet and antineutrophil antibodies using a heterologous system consisting of equine platelets or neutrophils and antisera raised in rabbits. The standardised technique consisted of using Immulon type 3 plate, 1 per cent gelatine as a blocking solution, poly-L-lysine buffer as a coating solution, unfixed antigen, 90 microliters test serum, horseradish peroxidase conjugated antibody and o-phenylenediamine dihydrochloride as a substrate. The number of unfixed platelets or neutrophils required for optimum detection of antibodies was 250,000 per well. Unfixed cellular antigens were as good as their extracts and superior to paraformaldehyde-fixed antigens in detecting specific antibodies. Microtitre plates coated with platelet or neutrophil antigens could be stored at 4 degrees and -70 degrees C for four to five weeks without significant loss of antigenicity. The ELISA was very sensitive in that antiplatelet antibody was detected up to a titre of 1:204,800 and antineutrophil antibody to a titre of 1:51,200. Some cross-reactivity (1:1600) was detected in antiplatelet and antineutrophil sera for neutrophil and platelet antigens, respectively. Platelet-associated antibody was also detected in extracts from platelets pretreated with 1:2 and 1:8 dilutions of antiplatelet serum. Standardised ELISA detected antiplatelet antibodies in nine and antineutrophil antibodies in three of 100 isologous equine blood typing sera.

Haematological changes in buffalo calves inoculated with Escherichia coli endotoxin and corticosteroids.

Haematological studies were conducted on 10 clinically normal water buffalo calves to determine leucocytic responses to Escherichia coli endotoxin, prednisolone and dexamethasone. Intravenous injection of 10 micrograms endotoxin induced minimal decreases in leucocyte numbers, whereas 20, 50 and 100 micrograms produced a marked leucopenia within one hour. Moderate to marked leucopenia, neutropenia and lymphopenia persisted for three to 14 hours. Significant rebound neutrophilia was evident at six to eight hours after inoculation in calves given only 10 and 20 micrograms. Intramuscular injection of prednisolone (100 mg) and dexamethasone (5 mg) produced increases in total leucocyte counts and neutrophil numbers within two hours. Moderate to marked leucocytosis and neutrophilia persisted for eight to 24 hours. Lymphocyte response was unlike that in other species in that lymphopenia was not a consistent feature of the corticosteroid response. A transient monocytosis was seen following administration of prednisolone but not of dexamethasone, while eosinopenia and basopenia developed in both cases. In conclusion, endotoxin and corticosteroid induced changes in total and differential leucocyte counts in water buffalo were largely similar to those seen in cattle.

Bluetongue virus infection in India: a review.

The history and epizootiology of bluetongue (BT) in India are reviewed. BT has become endemic in India. The first outbreak of BT in sheep and goats in the country was recorded in 1964 in Maharashtra State. Since then, several outbreaks of BT have been reported in sheep. Exotic sheep are more susceptible than indigenous and cross-bred sheep. A serological survey has indicated the presence of bluetongue virus (BTV) antibodies in cattle and buffalo in several states in India. However, clinical BT has not been observed in cattle or buffalo to date. Of the 24 known serotypes of BTV, 18 have been reported in India. Although BTV has been isolated from Culicoides midges, the particular species responsible for transmission has not yet been identified.

Intravascular leukostasis and systemic aspergillosis in a horse with subleukemic acute myelomonocytic leukemia.

Leukemia is a neoplastic disease of one or more of the cell types of the hemopoietic system and is rarely diagnosed in the horse. This report describes a case of subleukemic acute myelomonocytic leukemia in an 11-year-old gelding. Preliminary cytological diagnosis was supported by two types of laboratory investigations. Cytochemical characterization of blood and bone marrow neoplastic cells was consistent with a myelomonocytic origin. Neoplastic blast cells in peripheral blood were labeled by monoclonal antibodies specific for cell surface molecules of horse granulocytes, but they were not labeled by antibodies to T- or B-lymphocytes or macrophages. Treatment was attempted but was unsuccessful. At necropsy, intravascular leukostasis was present in all tissues examined. Fungal hyphae were also found in lung interstitium and colonic submucosa, suggesting the presence of a systemic mycosis. Nucleated cells were isolated from peripheral blood and cultured in vitro; they survived for up to 2 weeks and had evidence of cell division that was not sustained. Frozen-thawed cells stored in liquid nitrogen were also successfully cultured in vitro, but no permanent cell lines could be established.