Comparison of serological tests and faecal culture for the detection of Mycobacterium avium subsp. paratuberculosis infection in cattle and analysis of the antigens involved.

Three hundred and forty-one sera from cattle in Western Australia and 106 sera from Mycobacterium paratuberculosis faecal culture positive cattle were used to evaluate the performance of two absorbed enzyme-linked immunosorbent assays (ELISA) (one locally produced, the other a commercial test) and a complement fixation test (CFT) for the detection of Johne's disease in cattle. The diagnostic sensitivity (47.2%) of the local ELISA was significantly higher than that of the commercial ELISA (31.1%), and significantly higher than that for the complement fixation test (17.9%) and immunoblot (20.8%). Diagnostic specificity for the two ELISAs was 99.7% and 97.9% and similar for CFT and immunoblot (97.1% and 97.7%, respectively). The diagnostic sensitivity rose for both ELISAs and the CFT as the number of M. paratuberculosis isolated from the faeces increased. The ELISA antigen was characterised by polyacrylamide gel electrophoresis and electrophoretic immunoblotting and was found to consist mostly of a carbohydrate-type macromolecule of 32-42 kDa. This macromolecule was identified as lipoarabinomannan (LAM) by using a LAM-specific monoclonal antibody in immunoblots and purified LAM in absorption experiments. By applying more complex antigen preparations in immunoblots, serum antibodies against proteins of 47, 37, 30, 24 and 21 kDa, and against the 32-42 kDa carbohydrate component were frequently found in infected cattle, and of these the 47 kDa protein and the 32-42 kDa antigen were immuno-dominant. Pre-absorption of the sera with M. phlei sonicate indicated that the protein antigens contributed markedly to non-specific serological cross-reactions, while the 32-42 kDa non-protein macromolecule appeared to be specific.

Development and initial evaluation of an indirect enzyme-linked immunosorbent assay for the detection of Leptospira interrogans serovar hardjo antibodies in bovine sera.

Outer sheath antigen from Leptospira interrogans serovar hardjo type hardjoprajitno and acetic acid extracted antigens from serovar hardjo types hardjoprajitno and hardjobovis were evaluated in an immunoassay for ability to detect hyperimmune rabbit serum to serovar hardjo. The degree of cross-reactivity with hyperimmune rabbit sera to L. interrogans serovars pomona, copenhageni, grippotyphosa, canicola and sejroe, and Leptospira biflexa serovar patoc was also measured for each antigen. All of the antigens reacted with the antiserum to L. interrogans serovar hardjo. The outer sheath antigen however, also showed wide cross-reactivity with the antisera to all of the serovars of L. interrogans tested and with the antiserum to L. biflexa serovar patoc. The acetic acid extracted antigen from either type hardjoprajitno, or type hardjobovis, showed a high degree of specificity for serovar hardjo antiserum. The hardjobovis acetic acid extracted antigen was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting, and was incorporated into an indirect ELISA for detection of anti-serovar hardjo antibodies in bovine serum. This ELISA showed a relative specificity of 100% with 156 bovine sera which were negative at a dilution of 1:100 in the microscopic agglutination test (MAT) for L. interrogans serovars hardjo, pomona, sejroe, icterohaemorrhagiae, copenhageni, canicola, and grippotyphosa. The relative sensitivity of this assay with 192 bovine sera which had serovar hardjo MAT titres of > or = 100 was 95.3% (95% confidence limit = 2.99%). The degree of cross-reactivity with 289 bovine sera which had serovar pomona MAT titres of > or = 100 (with no detectable serovar hardjo MAT titres) was approximately 1.0%. This assay was: easily standardized, scored objectively, repeatable, semi-automated and used a non-hazardous antigen that can be routinely prepared in gram amounts.

A closed system for the filtration of Mycobacterium bovis liquid cultures using disposable capsule filters.

A filtration system was designed to sterilize large volumes of Mycobacterium bovis BCG Tokyo culture safely, needed to purify protein antigens for immunodiagnosis of bovine tuberculosis. A closed system consists of culture bottles connected to three disposable filter capsules of decreasing pore size in series: a depth prefilter over a 1.2 microns filter; 0.8 micron prefilter over a 0.45 micron filter; and a 0.2 micron sterile filter. Low air pressure (3 psi) forces liquid from below the bacillary pellicle. The system features a stainless steel clamp to hold rubber stoppers on the culture bottles, pleated filters to exclude bacillary clumps, a quick disconnector to minimize aerosols, and a closed system with plastic disposable filters that can be autoclaved as a unit without dismantling.

Competitive and indirect enzyme-linked immunosorbent assays for Mycobacterium bovis infections based on MPB70 and lipoarabinomannan antigens.

A competitive enzyme-linked immunosorbent assay (C-ELISA) using M. bovis BCG Tokyo culture filtrate as antigen and anti-MPB70 4C3/17 monoclonal antibody was developed for use in multiple animal species. An analysis of the C-ELISA data for cattle and bison serum panels revealed specificities of 68% to 85% and sensitivities of 85% to 89%. Receiver operator characteristics (ROC) of this data revealed areas of 81% to 92% for C-ELISA and demonstrated that C-ELISA as well as the indirect ELISA protocols, MPB70-ELISA and LAM-ELISA, discriminate M. bovis infected animals from non-infected animals for these particular panels. The kappa statistic values for agreement beyond chance between C-ELISA and MPB70-ELISA were determined after ELISA cutoffs were adjusted to minimize false positives. There were poor to excellent agreements between C-ELISA and MPB70-ELISA in all species tested (Bovidae, Cervidae, and Camelidae) that were consistently higher than the kappa statistic between C-ELISA and LAM-ELISA. The humoral response to one antigen and little or no response to the other in many animals argued for a parallel interpretation of C-ELISA and LAM-ELISA to increase sensitivity.

Purification of Mycobacterium bovis BCG Tokyo antigens by chromatofocusing, lectin-affinity chromatography, and hydrophobic interaction chromatography.

A combination of chromatofocusing, lectin-affinity chromatography, and hydrophobic interaction chromatography resulted in a simple purification of protein antigens of Mycobacterium bovis BCG Tokyo culture filtrate. Identification was established on the basis of chromatographic separation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis determination of molecular weights, and N-terminal amino acid determination. Chromatofocusing on PBE 94 accomplished the separation of BCG85B from other BCG85 complex antigens and partial separation of MPB64 and MPB70 antigens. Subsequently, MPB64 and MPB70 were completely separated on a high-performance liquid chromatography TSK Phenyl 5PW hydrophobic interaction chromatography column. This column also separated BCG85B from a 17-kDa protein with an N-terminal amino acid sequence of A-V-P-I-T-G-K-L-G-S-E-L-T-M-T-D-( )-V-G-Q, which is similar to the sequence of MPT63. Concanavalin A-Sepharose-affinity chromatography separated MPB64 from a 43- and 47-kDa doublet with an amino acid sequence of D-P-E-P-A-P-P-V-P-P-V-P-A-( )-A-A-S-P, which is similar to the sequence of MPT32 and which appears to be glycosylated.

Modification of the Mycobacterium bovis extracellular protein MPB70 with fluorescein for rapid detection of specific serum antibodies by fluorescence polarization.

The principle of fluorescence polarization described by Perrin (F. Perrin, J. Phys. Radium 7:390-401, 1926) was applied to the development of a novel assay that used fluorescein-labeled Mycobacterium bovis secretory protein MPB70 for rapid detection of anti-MPB70 antibodies in selected sera from three M. bovis-infected species (elk, Ilama, and bison). Labeling of purified MPB70 with fluorescein isothiocyanate resulted in the incorporation of 0.96 +/- 0.08 (mean +/- standard deviation; n = 3) fluorescein group per MPB70 molecule. The labeled protein fluoresced strongly with an emission maximum at 518 nm when excited with light of a wavelength near 493 nm, and its immunoreactivity with anti-MPB70 monoclonal antibody 4C3/17 was not altered by modification with fluorescein. The fluorescence polarization assay protocol was optimized for analysis of serum samples by incorporating into the assay buffer 0.05% lithium dodecyl sulfate, which prevents the occurrence of some nonspecific interactions. Sera from M. bovis-infected animals, selected on the basis of exhibiting the presence of anti-MPB70 antibodies, as detected by enzyme-linked immunosorbent assay (ELISA), reacted with fluorescein-labeled MPB70, resulting in an increase in polarization of up to 330 milli-polarization units, in contrast to the values for noninfected sera (167 to 178 mP), which were close to that obtained in the absence of specific antibodies (164.7 +/- 3.3 mP; n = 6). These results demonstrated the feasibility of using fluorescein-labeled MPB70 to detect anti-MPB70 antibodies by fluorescence polarization and suggested that the assay described here can be an alternative to ELISA or other antibody assay systems. The advantages of this original methodology and its general applicability to the diagnosis of infectious diseases are discussed.

Field tests of an acephate baiting system designed for eradicating undesirable honey bees (Hymenoptera: Apidae).

Field evaluations were made of a baiting system designed for use by regulatory agencies in suppressing populations of undesirable feral honey bees, Apis mellifera L. (e.g., bees posing hazards [especially Africanized bees] and colonies infested with parasitic mites). Bees from feral or simulated feral (hived) colonies were lured with honey and Nasonov pheromone components to feeders dispensing sucrose-honey syrup. After 1-3 wk of passive training to feeders, colonies were treated during active foraging by replacing untreated syrup with syrup containing 500 ppm (mg/liter) acephate (Orthene 75 S). In four trials using hived colonies on Grant Terre Island, LA., 21 of 29 colonies foraged actively enough at baits to be treated, and 20 of the 22 treated were destroyed. In the lower Rio Grande Valley of Texas (two trials at each of two trials), treatments killed 11 of 16 colonies (6 of 10 hived; 50 of 6 feral). Overall results showed that all 11 colonies that collected greater than 25 mg acephate died, whereas 3 of 10 colonies receiving less than 25 mg survived. Delivering adequate doses required a minimum of approximately 100 bees per target colony simultaneously collecting treated syrup. The system destroyed target colonies located up to nearly 700 m away from baits. Major factors limiting efficacy were conditions inhibiting foraging at baits (e.g., competing natural nectar sources and temperatures and winds that restricted bee flight).

Mycobacterium paratuberculosis antigen D: characterization and evidence that it is a bacterioferritin.

By using a combination of agarose and polyacrylamide gel electrophoresis, Mycobacterium paratuberculosis antigen D was resolved from a crude sonicated preparation of the organism and characterized as a component with a molecular mass of approximately 400,000 Da. While this component was composed mainly of protein, with unusually high proportions of glutamic acid and leucine, it was resistant to digestion with a number of proteolytic enzymes. Structural detail revealed by electron microscopy, amino acid sequence data, and the demonstration of a Soret band in its absorption spectrum indicated that antigen D was similar to an Escherichia coli bacterioferritin.

Chromatographic purification and characterization of antigens A and D from Mycobacterium paratuberculosis and their use in enzyme-linked immunosorbent assays for diagnosis of paratuberculosis in sheep.

The protein antigens A and D were purified from culture filtrates and sonic extracts of laboratory strains of Mycobacterium paratuberculosis by salt precipitation and chromatography. The characterization of antigen A is shown here, and both antigens were evaluated along with lipoarabinomannan antigen in indirect enzyme-linked immunosorbent assays (ELISA) for the serodiagnosis of ovine paratuberculosis. After anion-exchange (DEAE-5PW) and hydrophobic (phenyl-5PW) chromatography using high-performance liquid chromatography, antigen A showed a prominant band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 31 kDa with small amounts of low-molecular-mass proteins but with no evidence of antigen D. A single precipitin arc was evident with purified antigen A in crossed immunoelectrophoresis. The determination of the N-terminal amino acid sequence showed a high degree of homology between the 31-kDa component of antigen A and antigens of the BCG85 complex of Mycobacterium bovis BCG, a total of 24 of 26 residues being identical to those of BCG85C. A prominant SDS-PAGE band at 400 kDa and a single crossed-immunoelectrophoresis arc was also evident for antigen D after gel filtration (Sephacryl S-200), anion-exchange (DEAE-Sephacel), and concanavalin A-Sepharose affinity chromatography. By ELISA, purified antigen A detected antibody in the sera of 18 of 22 paratuberculosis-infected sheep (82% sensitivity), whereas the purified antigen D detected antibody in all 22 infected animals (100% sensitivity). Combined ELISA results showed increased specificity with some loss in sensitivity.

An evaluation of selected screening tests for bovine paratuberculosis.

The objective of this study was to evaluate the performance of the lipoarabinomannan antigen enzyme-linked immunosorbent assay (LAM-ELISA), carbohydrate antigen complement fixation (CH-CFT), and protein D antigen agar gel immunodiffusion (D-AGID) tests for bovine paratuberculosis, relative to histopathology, and to culture and isolation of Mycobacterium paratuberculosis from tissues and feces. Samples for test evaluation were collected from four sources including blood and tissues from 400 cull cows at three abattoirs in Ontario, blood and feces from a paratuberculosis survey of cattle from 120 dairy farms in Ontario, a serum bank containing samples from cattle from Ontario and Québec, and a bank of sera from cattle from Pennsylvania and the northeastern United States. The data were analyzed using receiver operator characteristic curves, estimates of relative sensitivity and specificity, and kappa statistics of agreement between tests. The LAM-ELISA performed significantly better than both the CH-CFT and the D-AGID tests. The LAM-ELISA was better at predicting fecal shedding status than tissue infection. However, the LAM-ELISA also had limitations. When interpreted as positive or negative (+/-), at a critical optical density of 0.675, its sensitivity and specificity relative to bacteriology were 49% and 87% respectively. Although the serological tests examined in this study provided some information, they did not predict well the infection status of individual animals.

Serodiagnosis of ovine paratuberculosis, using lipoarabinomannan in an enzyme-linked immunosorbent assay.

The use of lipoarabinomannan (LAM; obtained from Mycobacterium paratuberculosis) in and ELISA (LAM-ELISA) to test 75 sheep sera from a paratuberculosis-infected flock resulted in an approximate threefold increase in sensitivity (from 23.5% to 70.6%), compared with the use of Annau's polysaccharide in a complement fixation test (P-CFT). Even after manipulation of the LAM-ELISA cut-off value to produce a specificity of 100% to match that of the P-CFT, the sensitivity still was approximately twofold greater than that of the P-CFT. Anti-bovine monoclonal antiglobulin-enzyme conjugates matched commercially available anti-ovine polyclonal antiglobulin-enzyme conjugates with respect to sensitivity and specificity. False-positive results were found to be less frequent after combining 2 serodiagnostic tests, LAM-ELISA and D antigenagar gel immunodiffusion, resulting in an increase in specificity from 88.1% to 95.2%. The repeatability of true seropositive and seronegative results was found to be 89.5% and 91.1%, respectively, for sera obtained less than or equal to 1 month prior to slaughter and 91.7% and 95.5%, respectively, for reanalysis of sera obtained at the time of slaughter.

A comparison of the bovine leukemia and bovine syncytial virus status in utero-tubal cells recovered from fluids used to flush the uterus and oviducts of BLV-infected, superovulated cattle.

Twenty-four cell lines were established from uterine-oviductal flush fluid (UOFF) cells from 20 bovine leukosis virus (BLV)-infected embryo-donor cows and 4 BLV-free control cows harvested by the Ficoll-gradient technique. Similar epithelial-like and fibroblast-like cells were observed in the primary cultures of UOFF from both groups. BLV-antigens were not detected with direct immunofluorescence test in any of the cell-lines from the 20 positive BLV-cows but a positive reaction was observed with the competitive radioimmunoassay in one cell line only. Bovine syncytial virus (BSV) was detected (multinucleated cells) in five of the 20 cows, bovine virus diarrhea (BVD) in six and infectious bovine rhino-tracheitis (IBR) in one (by D-IF). Some of the cell lines had antigens to one (3/20) two (2/20) or three (1/20) different viruses as demonstrated by D-IF. There were no antigens to BLV, BSV or IBRV demonstrated in the BLV-free cows by both immunofluorescence test and radioimmunoassay. Permissiveness to the growth of BLV in the cell lines of bovine utero-tubal (BUT) origin was demonstrated by inoculating three of the 20 cell lines from BLV-infected cows and three cell lines from the 4 BLV-free cow by BLV suspensions. All six cell lines permitted the growth of BLV as shown by syncytia, and positive reactions with the immunofluorescence test but only three out of six lines were BLV-positive by radioimmunoassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical changes in progressive muscular dystrophy. XIV. Skeletal muscle myosin mRNA translatability in dystrophic mice.

Variations in the content and translatability of the poly(A)+ RNA and mRNA molecules coding for myosin (M) were studied in the hind leg muscles of genetically dystrophic mice. The poly(A)+ RNA content of total skeletal muscle failed to increase normally during progression of the disease. M mRNA, isolated from dystrophic normally during progression of the disease. M mRNA, isolated from dystrophic murine muscle poly(A)+ RNA, was mostly found to be associated with the 26S RNA species. The translation of M mRNA in an in vitro heterologous wheat germ system was lower at 8 and 16 weeks in the dystrophic group as compared with the controls. Analysis of the translation products via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and densitometric autoradiographic tracing demonstrated the gradual disappearance of a protein band corresponding to M, the major component of skeletal muscle. cDNA was synthesized, using M mRNA that was isolated and purified from normal and dystrophic mouse muscle as a template. Total radioactivity was measured in some cDNA fractions produced from normal and dystrophic mouse muscle, while other fractions were utilized for separation and sizing of cDNA by disc gel electrophoresis. The cDNA from normal muscle was hybridized with M mRNA from normal and 16-week-old dystrophic mouse muscles. The cDNA probe, hybridization experiments, and studies involving the content and synthesis of M mRNA suggest that murine muscular dystrophy elicited a shorter species of mRNA or shorter sequences of the same species of mRNA coding for M. Not all poly(A)+ mRNA sequences coding for M, found in control mice, were present in their dystrophic counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoarabinomannan and lipid-free arabinomannan antigens of Mycobacterium paratuberculosis.

Lipoarabinomannan (LAM) and lipid-free arabinomannan (AM) were prepared from Mycobacterium paratuberculosis. Purification of LAM was done by ultracentrifugation of the phenol-water-extracted crude polysaccharide, followed by affinity and anion exchange chromatography. AM was purified from the supernatant of the ultracentrifuged polysaccharide or from alkaline-extracted material by gel filtration and anion exchange chromatography. Chemical analysis revealed arabinose and mannose in LAM (1.4:1) and AM (3.5:1) and the presence of palmitic, stearic, and tuberculostearic acids for a total of 7.8% lipid in LAM. Traces of phosphorus were found in the AMs, particularly LAM (0.05%). Nuclear magnetic resonance confirmed the presence of alpha-arabinosyl residues and the acylated nature of LAM. LAM exhibited lipid-dependent aggregation, as indicated by a Triton-induced decrease in molecular weight. By using bovine sera, LAM was found to be active in the complement fixation test, whereas AM was inactive and inhibited this activity. Thus, the presence of AM in crude polysaccharide could explain the variable complement fixation results. Triton-dissociated LAM exhibited a precipitin (Cl) in common with that of AM, confirming shared determinants. LAM in its lipid-dependent aggregated form, however, exhibited a second precipitin (C2), which may be due to the disparity in antigen size or a novel epitope. The lipid content of LAM rendered it 100 times more effective for coating plates in the enzyme immunoassay than lipid-free AM.

Ectopic mineralization and nutritional hyperparathyroidism in boars.

The influence of a high phosphorus (1.5%) and high calcium (2.2%) diet on ectopic mineralization in boars was examined over a four month period. The high phosphorus diet caused metastatic mineralization in the left atrial endocardium in 84% of animals and in the pulmonary and diaphragmatic pleura in 21 and 58% of animals, respectively. Mineralization, that apparently commenced as deposits of extraneous calcium and progressed by metastatic deposition, occurred also in the lamina propria mucosae of the respiratory airways and in the lamina muscularis mucosae of fundic stomach. Hyperplasia of osteoclasts and microfractures in costochondral junction, morphological features of activated parathyroid cells and a significant drop in serum phosphorus during the fourth month in boars fed high phosphorus, suggest that nutritional hyperparathyroidism was experimentally induced. No systemic bone disease developed. Feeding a high calcium diet resulted in 20% incidence of discrete lesions in the left atrial endocardium and no pleural involvement. Also, lesions in respiratory airways and fundic stomach were mild. The fact that high phosphorus but not high calcium increased the incidence and extent of ectopic mineralization suggests that hyperparathyroidism under normocalcemic conditions might be involved in the pathogenesis of these lesions.

High pressure liquid chromatographic determination of nitrofurazone and furazolidone in chicken and pork tissues.

A high pressure liquid chromatographic (HPLC) procedure is presented for the determination of nitrofurazone and furazolidone in chicken and pork tissues in the 2-40 ppb range. Muscle, liver, and kidney are homogenized with cold methanol and water (50 + 50). Following methanol evaporation, the nitrofurans are partitioned into ethyl acetate and cleaned up on an alumina column. After elution with 20% methanol in ethyl acetate and evaporation to dryness, residues are determined by HPLC, using a reverse phase analytical column. Overall average recoveries for nitrofurazone and furazolidone were 65.7 and 73.5%, respectively. Average relative standard deviations of 11.9% (nitrofurazone) and 9.5% (furazolidone) at the 2 ppb level were achieved.

Copper toxicosis in lambs fed milk replacer.

Thirty-four hysterectomy derived, crossbred lambs were fed a commercial, lamb milk replacer, containing added copper from birth. Twenty-five lambs died, four were killed and five survived. At necropsy, generalized icterus, enlarged kidneys and enlarged or small livers were found.CLINICAL PATHOLOGY FINDINGS INCLUDED: responsive hemolytic anemia and occasional spherocytosis, hemoglobinemia, hemoglobinuria and urinary casts, leukocytosis with neutrophilia, a liver copper content range of 38-584 ppm; a kidney copper content 6-86 ppm, and serum aspartate amino-transferase level range of 150-302 I.U./L. Histopathologically there was either periacinar hepatic necrosis or widespread portal fibrosis and nephrosis.The role of age and stress in the development of toxicosis is discussed.

Focal mineralization and nonspecific granulomatous inflammation of respiratory mucous membranes in pigs.

Discrete mineralized foci and granulomatous inflammation occurred in the lamina propria mucosae of respiratory mucous membranes of adult pigs. Lesions were present in clinically healthy pigs of both sexes, including castrated males, fed various pelleted or non-pelleted diets. They were mainly in longitudinally corrugated mucosae of the dorsal wall of the trachea. Identical mineralization and inflammation occurred in the nasal cavity and, in decreasing frequency and intensity, in the thoracic trachea and bronchi. The lesions in respiratory mucous membranes occurred in pigs with and without mineralization in other organs. The distribution of lesions in the respiratory tract, and the higher frequency in pigs fed non-pelleted dusty feeds, suggest that focal mineralization was caused by inhaled particles of calcium salts.