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1.
Biochem Soc Trans ; 35(Pt 2): 311-3, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17371267

ABSTRACT

ODC (ornithine decarboxylase) is a central regulator of cellular polyamine synthesis. ODC is a highly regulated enzyme stimulated by a variety of growth-promoting stimuli. ODC overexpression leads to cellular transformation. Cellular ODC levels are determined at transcriptional and translational levels and by regulation of its degradation. Here we review the mechanism of ODC degradation with particular emphasis on AzI (antizyme inhibitor), an ODC homologous protein that appears as a central regulator of ODC stability, cellular polyamine homoeostasis and cellular proliferation.


Subject(s)
Cell Division/physiology , Enzyme Inhibitors/metabolism , Ornithine Decarboxylase/metabolism , Polyamines/metabolism , Animals , Kinetics , Mammals , Ornithine Decarboxylase Inhibitors , Proteins/metabolism
2.
Oncogene ; 25(37): 5163-72, 2006 Aug 24.
Article in English | MEDLINE | ID: mdl-16568078

ABSTRACT

Antizyme inhibitor (AzI) is a homolog of ornithine decarboxylase (ODC), a key enzyme of polyamine synthesis. Antizyme inhibitor retains no enzymatic activity, but exhibits high affinity to antizyme (Az), a negative regulator of polyamine homeostasis. As polyamines are involved in maintaining cellular proliferation, and since AzI may negate Az functions, we have investigated the role of AzI in regulating cell growth. We show here that overexpression of AzI in NIH3T3 cells increased growth rate, enabled growth in low serum, and permitted anchorage-independent growth in soft agar, while reduction of AzI levels by AzI siRNA reduced cellular proliferation. Moreover, AzI overproducing cells gave rise to tumors when injected into nude mice. AzI overexpression resulted in elevation of ODC activity and of polyamine uptake. These effects of AzI are a result of its ability to neutralize Az, as overexpression of an AzI mutant with reduced Az binding failed to alter cellular polyamine metabolism and growth properties. We also demonstrate upregulation of AzI in Ras transformed cells, suggesting its relevance to some naturally occurring transformations. Finally, increased uptake activity rendered AzI overproducing and Ras-transformed cells more sensitive to toxic polyamine analogs. Our results therefore imply that AzI has a central and meaningful role in modulation of polyamine homeostasis, and in regulating cellular proliferation and transformation properties.


Subject(s)
Cell Division/physiology , Proteins/genetics , 3T3 Cells , Animals , Base Sequence , Cell Line , Cell Transformation, Neoplastic , DNA Primers , Fibroblasts/cytology , Fibroblasts/physiology , Mice , Ornithine Decarboxylase/genetics , Ornithine Decarboxylase/metabolism , Proteins/physiology , Reverse Transcriptase Polymerase Chain Reaction , Transfection
3.
Vet Q ; 22(3): 123-30, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10952440

ABSTRACT

Brucellosis, caused by bacteria of the genus Brucella, is a contagious disease that causes economic loss to owners of domestic animals due to loss of progeny and milk yield. Because cattle, sheep, goats, and to a lesser extent pigs are considered to be the source of human brucellosis, serological tests have been used to screen domestic animals for antibodies against Brucella. Although the serological tests helped to eradicate brucellosis in many countries, serological tests are not always adequate to detect latent carriers of Brucella. Therefore, the use of the skin delayed-type hypersensitivity (SDTH) test, which is independent of circulating antibodies, might improve the diagnosis of brucellosis. In the literature, however, there are conflicting reports as to the value of the SDTH test for the diagnosis of brucellosis. Some studies consider the test unreliable, whereas others advocate its use because it detects brucellosis earlier than serological tests. The objectives of this study were therefore to assess the characteristics of the SDTH test, to select a Brucella strain that will yield a suitable brucellin for use in the field, and to determine whether the use of serological tests in combination with the SDTH test improves the detection of brucellosis. The results of this study clearly show that the SDTH test detects latent carriers of Brucella and confirms brucellosis in cattle with ambiguous serological test results. Brucellins prepared from smooth or mucoid strains of Brucella are better suited for use in the field than brucellins prepared from rough strains because they detect brucellosis in cattle with acute as well as chronic infection. The SDTH test is highly specific (99.3% specificity), and repeated testing of naive cattle or cattle infected with microorganisms that serologically cross-react with Brucella does not sensitize cattle to subsequent SDTH tests. However, it is possible that some naive cattle may serologically react to the injection of brucellin. The effect of these serological reactions on the sero-diagnosis of brucellosis is limited, because cattle may only now and then react serologically either with the serum agglutination test (SAT) or the complement fixation test (CFT). Nevertheless, cattle infected with microorganisms that serologically cross-react with Brucella may test seropositive for brucellosis 4 to 7 weeks after injection of brucellin, depending on the cross-reacting microorganism. The value of the SDTH test for the diagnosis of brucellosis was demonstrated after an outbreak of brucellosis. When the SDTH test was used in combination with SAT and CFT at diagnostic threshold > or =2 mm or > or =1 mm (increase in skinfold thickness), respectively, 39/44 (88%) or 42/44 (95%) of the infected cattle were detected compared with only 27/44 (61%) when SAT and CFT were used. When cattle in areas of low prevalence or in areas free from brucellosis are tested with the SDTH test an increase > or =2 mm in skinfold thickness should be considered indicative of infection. When the control and eradication of brucellosis is based on test-and-slaughter, an increase of > or =1 mm in skinfold thickness should be considered indicative of infection. Repeated serological testing complemented with the SDTH test in this programme will shorten the quarantine (movement control) period of a suspect herd, limiting the financial loss incurred during outbreaks of the disease. Consequently, since the SDTH test usually does not interfere with the serological diagnosis and can safely be used to establish the infection status of cattle in a suspect herd, it is opportune to consider adding the SDTH test to the procedure currently used to diagnose brucellosis in individual animals.


Subject(s)
Brucella/isolation & purification , Brucellosis, Bovine/diagnosis , Hypersensitivity, Delayed/veterinary , Skin Tests/veterinary , Animals , Antibodies, Bacterial/analysis , Antigens, Bacterial/immunology , Brucella/immunology , Brucellosis, Bovine/epidemiology , Brucellosis, Bovine/prevention & control , Cattle , Prevalence , Reproducibility of Results , Serologic Tests/veterinary
4.
Vet J ; 157(1): 61-7, 1999 Jan.
Article in English | MEDLINE | ID: mdl-10030130

ABSTRACT

Eight-hundred-and-ninety-six cattle belonging to herds officially designated Brucella-free, and 190 cattle belonging to infected herds were tested with the skin delayed-type hypersensitivity (SDTH) test, using brucellin (273) prepared from a mucoid strain of Brucella abortus. An increase in skinfold thickness > or = 2 mm was considered a positive SDTH test. The serum agglutination test, complement fixation test and bacteriological examination were used to confirm SDTH test results. Results show that 6 of the 896 uninfected cattle tested positive in the SDTH test, indicating a 99.3% specificity. Of the 44 cattle that tested serologically or bacteriologically positive, 33 tested positive in the SDTH test, indicating a 75% sensitivity. The value of the SDTH test was demonstrated by its ability to detect infection earlier than serological tests, and by confirming infection in cattle with ambiguous serological test results. An increase in skinfold thickness > or = 1 mm in cattle in suspected herds should not be ignored, as it may indicate specific sensitization. We recommend the use of the SDTH test in combination with serological tests for early diagnosis of brucellosis in cattle.


Subject(s)
Antigens, Bacterial/immunology , Brucellosis/veterinary , Cattle Diseases/diagnosis , Hypersensitivity, Delayed/veterinary , Skin Tests/veterinary , Animals , Antigens, Bacterial/administration & dosage , Brucella/immunology , Brucellosis/diagnosis , Cattle
5.
Vet Microbiol ; 62(4): 313-20, 1998 Aug 15.
Article in English | MEDLINE | ID: mdl-9791877

ABSTRACT

A study was conducted to determine the repeatability of a procedure used to prepare brucellin from a mucoid strain of Brucella abortus, and to determine the biological activity of those brucellins. The brucellins were standardized to contain 1 mg protein/ml, and their potency was estimated according to the European pharmacopoeia norm for tuberculin. Estimation of the potency was done in cattle that have been sensitized with living or killed brucellae. A brucellin that effectively detected acute and chronic brucellosis in cattle with experimentally induced brucellosis was used as reference brucellin. Results show that five of the nine batches of brucellin equalled the potency of the reference brucellin. Three brucellins had 79-88% potency of the reference brucellin and one had only 59%. Since the potency of various brucellins may vary it is suggested to estimate the potency of the brucellins according to the European pharmacopoeia norm for tuberculin.


Subject(s)
Antigens, Bacterial/immunology , Brucella abortus/immunology , Allergens , Animals , Antigens, Bacterial/isolation & purification , Antigens, Bacterial/pharmacology , Cattle , Europe , Immunization , Pharmacopoeias as Topic , Skin/immunology , Skin Tests/veterinary
6.
Vet Q ; 20(3): 81-8, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9684294

ABSTRACT

This review covers some epidemiological aspects that allow Brucella to survive, spread, and maintain itself in the environment. Because the success of maintaining Brucella-free herds is determined by the efficiency of the serological tests to detect a single infected animal the limitations of the traditional serological tests are emphasized. Serological tests cannot differentiate between cattle infected with Brucella and cattle infected with microorganisms that serologically cross-react with B. abortus antigen. These cattle and cattle with 'natural' antibodies jeopardize the Brucella-free status of a herd. Likewise, infected cattle with serologically inconclusive test results or which elude detection are also a hazard to Brucella-free herds. Since cattle that elude detection with serological tests and the presence of non-specific serum antibodies in healthy cattle have long been recognized as problems, it is opportune to reconsider the procedures currently used to diagnose brucellosis in individual animals. Use of the skin delayed-type hypersensitivity test in addition to serological tests will significantly improve the diagnosis of brucellosis. This will limit the financial loss incurred by outbreaks of brucellosis.


Subject(s)
Brucella abortus/pathogenicity , Brucellosis, Bovine/epidemiology , Animals , Animals, Domestic , Antibodies, Bacterial , Brucellosis, Bovine/diagnosis , Cattle , Cross Reactions , Diagnosis, Differential , Disease Transmission, Infectious/veterinary , Hypersensitivity, Delayed/veterinary , Serologic Tests/veterinary
7.
Vet Microbiol ; 51(1-2): 85-93, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8828125

ABSTRACT

An antigen prepared from a mucoid strain of B. abortus was repeatedly injected intradermally into cattle to determine whether it sensitizes cattle so that they test false positive with the skin delayed-type hypersensitivity (SDTH) test. Cattle (n = 14) that were experimentally inoculated. with microorganisms that serologically cross-react with B. abortus, and uninfected cattle (n = 15) were tested with the antigen 2 to 5 times over a period of 16 to 17 weeks. An increase in skinfold thickness of > or = 2.0 mm on the injection site was considered a reaction elicited by the antigen. The sera from the cattle were tested with the serum agglutination test, complement fixation test, and an enzyme-linked immunosorbent assay for antibodies against B. abortus. Results showed that none of the animals had an increase in skinfold thickness of > or = 2.0 mm on the injection site of the antigen, which is considered a positive reaction. However, cattle experimentally inoculated with microorganisms other than B. abortus tested seropositive for brucellosis after one SDHT test only. We conclude that the B. abortus antigen inoculated intradermally does not sensitize cattle after repeated inculations, and therefore does not interfere with the subsequent use of the SDTH test in the diagnosis of brucellosis.


Subject(s)
Antigens, Bacterial/administration & dosage , Brucella abortus/immunology , Brucellosis, Bovine/diagnosis , Hypersensitivity, Delayed/veterinary , Animals , Antibodies, Bacterial/blood , Brucellosis, Bovine/immunology , Brucellosis, Bovine/microbiology , Cattle , False Positive Reactions , Hypersensitivity, Delayed/etiology , Injections, Intradermal/veterinary , Skin Tests/veterinary
8.
Vet Res Commun ; 20(2): 141-51, 1996.
Article in English | MEDLINE | ID: mdl-8711894

ABSTRACT

The potency of Brucella allergens prepared from a smooth Brucella abortus strain S-99, mucoid strain Leewarden, rough strain 45/20, and rough Brucella melitensis strain B-115 was assessed. The potency of these allergens was compared with that of a standard allergen prepared from smooth Brucella abortus S-99 that efficiently detected bovine brucellosis in other studies. Eight cattle experimentally inoculated with Brucella abortus 544 were tested with the allergens 4 and 10 weeks after infection, and again 8 months after infection. All the allergens effectively detected infection but there was a clear distinction in the mean skin reactions 48 and 72 h after injection of the allergens. The skin reactions provoked by the allergens prepared from smooth or mucoid strains of Brucella were most pronounced 48 h after injection. Skin reactions provoked by allergens prepared from rough strains of Brucella were strongest 72 h after injection. Allergens prepared from smooth or mucoid Brucella strains were more potent in detecting brucellosis than those prepared from rough strains of Brucella.


Subject(s)
Allergens , Brucella abortus/immunology , Brucella melitensis/immunology , Brucellosis, Bovine/diagnosis , Brucellosis, Bovine/immunology , Acute Disease , Allergens/immunology , Animals , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Antigens, Bacterial/immunology , Brucellosis, Bovine/pathology , Cattle , Chronic Disease , Hypersensitivity, Delayed/immunology , Hypersensitivity, Delayed/pathology , Hypersensitivity, Delayed/veterinary , Skin/immunology , Skin/pathology , Skin/physiopathology , Skin Tests/standards , Skin Tests/veterinary
9.
Vet Microbiol ; 48(1-2): 174-8, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8701573

ABSTRACT

A study was conducted to determine whether an allergen that has been prepared from a mucoid strain of Brucella abortus triggers a serum antibody response that interferes with the interpretation of serologic tests results. Fifteen cattle seronegative for Brucella antigen were tested with the SDTH test several times. Blood samples were collected weekly and tested with the serum agglutination test and complement fixation test. Results show that some cattle tested seronegative after each of the SDTH tests while other cattle tested weakly positive with the serum agglutination test or the complement fixation test. All seropositive cattle tested seronegative 4-7 weeks after the last SDTH test indicating an antibody response of a transient nature. We conclude that serologic tests results indicating infection are reliable when recorded four weeks after a single SDTH test. If cattle are tested with the SDTH test several times an interval of seven weeks should be observed after the last test to ensure a reliable interpretation of the serologic test results.


Subject(s)
Allergens/immunology , Antibodies, Bacterial/blood , Antigens, Bacterial/immunology , Brucella abortus/immunology , Agglutination Tests , Allergens/administration & dosage , Animals , Antigens, Bacterial/administration & dosage , Cattle , Complement Fixation Tests , Female , Intradermal Tests/veterinary , Time Factors
10.
Zentralbl Veterinarmed B ; 42(1): 19-27, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7483897

ABSTRACT

A study was conducted to evaluate the biological activity of Brucella allergens extracted with hydrochloride or trichloroacetic acid. Smooth and mucoid Brucella abortus cells and the medium in which brucellae were propagated were used to prepare the allergens. The biological activity of the allergens was estimated in guinea-pigs sensitized with Brucella abortus strain 544. The guinea-pigs were intradermally injected with several allergen dilutions. The dilutions were coded and randomized for site of injection so that none of the dilution was injected twice on the same site. Variance analysis using incomplete Latin square was used for the statistical calculation of the results. The calculated biological activity of the allergens was compared with the biological activity of a 'standard' allergen that has proved effective in detecting cattle brucellosis. The skin erythema diameter was best when recorded 32 h after allergen injection. Statistical analysis of the skin erythema diameters showed a great variation in biological activity (12-105%) between the allergens. Only the allergen extracted from the medium in which a mucoid Brucella strain was propagated was as potent as the standard. The use of the incomplete Latin square for variance analysis resulted in the estimation of the biological activity of nine batches of allergen in only 27 guinea-pigs.


Subject(s)
Allergens , Brucella abortus/immunology , Brucellosis, Bovine/diagnosis , Allergens/isolation & purification , Animals , Biological Assay/veterinary , Cattle , Guinea Pigs , Hypersensitivity, Delayed/veterinary , Intradermal Tests/veterinary , Random Allocation
11.
Eur J Biochem ; 226(2): 547-54, 1994 Dec 01.
Article in English | MEDLINE | ID: mdl-8001569

ABSTRACT

Recent studies have provided convincing evidence to add to a number of earlier observations suggesting that the rapid intracellular degradation of mammalian ornithine decarboxylase (ODC) is further accelerated by the action of ornithine decarboxylase antizyme (ODC-Az), a polyamine-induced protein. However, the mechanism whereby ODC-Az exerts its effect in this proteolytic process is mostly unknown. Here, by using reticulocyte-lysate-based synthesis and degradation systems, we demonstrate that interaction of ODC-Az with ODC results in two related outcomes: (a) ODC is inactivated as a result of its monomerization, and (b) ODC degradation is dramatically accelerated. While ODC inactivation requires the integrity of the ODC-Az binding site of ODC and the ODC binding site of ODC-Az, acceleration in ODC degradation also requires the previously characterized carboxyl-terminal destabilizing segment of ODC and a specific segment of ODC-Az that may be functionally distinct from that required for ODC binding. Interestingly, an active ODC variant with a mutant ODC-Az binding site is stable under basal degradation conditions. This, together with the ability of anti-(ODC-Az) antibody to specifically inhibit the basal degradation of ODC in the lysate, suggests that ODC-Az is an essential general mediator of ODC degradation. Based on these observations, we propose a model for the degradation of ODC which always require interaction with antizyme.


Subject(s)
Ornithine Decarboxylase/metabolism , Polyamines/pharmacology , Proteins/pharmacology , Reticulocytes/enzymology , Amino Acid Sequence , Animals , Binding Sites , Macromolecular Substances , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Ornithine Decarboxylase/chemistry , Ornithine Decarboxylase Inhibitors , Point Mutation , Proteins/chemistry , Proteins/metabolism , Structure-Activity Relationship
12.
Zentralbl Veterinarmed B ; 40(8): 582-8, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8122447

ABSTRACT

A study was conducted to determine possible nonspecific skin delayed-type hypersensitivity (SDTH) test reactions in cattle tested with a Brucella allergen. Cattle (n = 14) experimentally inoculated with microorganisms known serologically to cross-react with Brucella and cattle (n = 549) from Brucella free herds were tested serologically and with the SDTH test. The increase in skinfold thickness at the injection site of the allergen was measured to the nearest mm with calipers 48 hours after injection. The results show that none of the SDTH test reactions in cattle experimentally inoculated with microorganisms other than Brucella exceeded 2.0 mm. This indicates that an increase in skinfold thickness > or = 2.0 mm can be considered a positive SDTH test reaction. When this norm was applied to cattle in Brucella free herds 11/549 (2%) cattle showed an increase > or = 2.0. It is concluded that infections with microorganisms other than Brucella are unlikely to cause sensitization that interferes with the SDTH test when used to detect brucellosis. Therefore, the SDTH test can be used to verify positive serologic tests results that might have been caused by microorganisms that serologically cross-react with Brucella.


Subject(s)
Allergens/immunology , Brucella/immunology , Brucellosis, Bovine/diagnosis , Hypersensitivity, Delayed/veterinary , Animals , Cattle , Sensitivity and Specificity , Skin Tests/veterinary
13.
Eur J Biochem ; 213(1): 205-10, 1993 Apr 01.
Article in English | MEDLINE | ID: mdl-8477695

ABSTRACT

Eukaryotic cells have been shown to contain two high-molecular-mass proteases of 700 kDa and 1400 kDa (20S and 26S proteases, respectively). It has been suggested that the 20S protease, also known as proteasome, may constitute the catalytic core of the 26S protease. While the role of the free 20S protease in intracellular protein degradation is unclear, the 26S protease is implicated in the degradation of ubiquinated proteins. We have recently demonstrated, that ornithine decarboxylase (ODC), one of the most labile proteins in mammalian cells, is degraded via an ATP-dependent but ubiquitin-independent proteolytic pathway. Here we extend these observations by demonstrating that in reticulocyte lysate ODC degradation is inhibited by antibodies raised against the C9 subunit of rat proteasome. Partial fractionation of the lysate demonstrated preferential degradation of ODC in the fraction of the lysate proteins that are precipitated by 38% ammonium sulfate. Since it was demonstrated that the 26S protease precipitates at this concentration of ammonium sulfate while the 20S proteasome remains soluble, our results suggest that the 26S protease is the one degrading ODC.


Subject(s)
Cysteine Endopeptidases/metabolism , Multienzyme Complexes/metabolism , Ornithine Decarboxylase/metabolism , Adenosine Triphosphate/metabolism , Ammonium Sulfate/metabolism , Animals , Complement C9/immunology , Immune Sera , Mice , Proteasome Endopeptidase Complex , Rabbits
14.
Biochem J ; 277 ( Pt 3): 683-5, 1991 Aug 01.
Article in English | MEDLINE | ID: mdl-1872804

ABSTRACT

One of the most interesting characteristics of ornithine decarboxylase (ODC) is its extremely short half-life. In a recent study we have demonstrated that deletion of a C-terminal segment converts ODC into a stable protein. In the present study we have extended this observation by testing the degradation of an ODC heterodimer composed of one rapidly degraded wild-type subunit and one stable mutant subunit. Our study was motivated by the possibility of trans-recognition of stable subunits due to their association with labile subunits. Our results demonstrate that such an association did not confer lability upon the stable subunits, not did it stabilize the short-lived subunits.


Subject(s)
Ornithine Decarboxylase/metabolism , Reticulocytes/metabolism , Animals , Cell-Free System , Cloning, Molecular , In Vitro Techniques , Molecular Structure , Rabbits , Structure-Activity Relationship
15.
Eur J Biochem ; 196(3): 647-51, 1991 Mar 28.
Article in English | MEDLINE | ID: mdl-2013288

ABSTRACT

Mouse ornithine decarboxylase is a 461-amino-acid protein that is extremely labile. A set of contiguous in-frame deletions were introduced into its C-terminal hydrophilic region. The resulting mutant proteins were expressed in cos monkey cells using an expression vector based on simian virus 40 (SV40) or by in vitro translation in reticulocyte lysate. The degradation of wild-type and mutant proteins was determined in transfected cos cells and in a degradation system based on reticulocyte lysate. Deletion mutants lacking segments of the C-terminus (amino acids 423-461, 423-435, 436-449 and 449-461) were converted into stable proteins in both experimental systems. The mutant lacking amino acids 295-309 was significantly stabilized in transfected cos cells, but was rapidly degraded in reticulocyte-lysate-based degradation mix. Our results suggest that the carboxyl-terminal region encompassing amino acids 423-461 and perhaps also amino acids 295-309 may constitute a signal recognized by the proteolytic machinery that degrades ornithine decarboxylase.


Subject(s)
Ornithine Decarboxylase/metabolism , Reticulocytes/metabolism , Animals , Cells, Cultured , Mutation , Protein Conformation , Transfection
16.
Zentralbl Veterinarmed B ; 37(10): 753-9, 1990 Dec.
Article in English | MEDLINE | ID: mdl-2127977

ABSTRACT

Individual milk samples and artificially constructed tank milk samples from cows with naturally occurring brucellosis were examined by the enzyme-linked immunosorbent assay (ELISA) using sonicated B. abortus S-99 antigen, and mouse monoclonal anti-bovine IgM, IgA, and IgG1 conjugates. ELISA results were compared with the results of the milk ring test using either 1 ml milk (MRT-1) or 8 ml milk (MRT-8). The ELISA using mouse monoclonal anti-bovine IgG1 conjugate was sensitive and specific. In testing individual milk samples and constructed tank milk samples containing milk with low titers in the MRT-1 the ELISA was superior to the MRT-1, and MRT-8. In testing other milk samples, the ELISA was as sensitive or slightly less sensitive than the MRT-8. From a total of 5,910 milk samples collected from cows free from brucellosis, only 24 (0.4%) samples tested positive in the ELISA. All 500 tank milk samples collected from farms negative for brucellosis tested negative in the ELISA. We concluded that the ELISA is a good substitute for the MRT-1 to detect antibodies against Brucella in milk from individual cows. When tank milk is tested for antibodies against Brucella, however, both the MRT-8 and the ELISA should be used.


Subject(s)
Antibodies, Bacterial/analysis , Antibodies, Monoclonal , Brucella abortus/immunology , Enzyme-Linked Immunosorbent Assay , Milk/immunology , Animals , Cattle , Predictive Value of Tests
17.
Vet Q ; 12(4): 231-7, 1990 Oct.
Article in English | MEDLINE | ID: mdl-2125383

ABSTRACT

Calves (n = 2) born to dams with experimentally induced brucellosis, and calves (n = 4) born to dams with naturally occurring infection were examined by the delayed-type hypersensitivity (DTH) test for possible B. abortus infection. The results were compared with the serum agglutination test, complement fixation test, and Coombs test. Five calves were nursed by their dams for 8-10 weeks after birth. One calf was separated from its dam and fed artificial milk. Three to five months after birth, four calves tested seropositive in the serologic tests. Antibodies were detected in one calf as early as 1 week after birth. The calf fed on artificial milk was seronegative 4-5 weeks after birth. All calves reacted to the DTH test antigen from week 12 until the end of the experiment, even though serologic tests were negative. We conclude that the DTH test is a valuable technique for diagnosing Brucella in calves born to infected dams.


Subject(s)
Brucellosis, Bovine/diagnosis , Hypersensitivity, Delayed , Agglutination Tests/veterinary , Animals , Antibodies, Bacterial/analysis , Brucella abortus/immunology , Brucellosis, Bovine/transmission , Cattle , Complement Fixation Tests/veterinary , Coombs Test/veterinary , Female , Maternal-Fetal Exchange , Pregnancy
18.
Vet Microbiol ; 22(2-3): 241-8, 1990 Apr.
Article in English | MEDLINE | ID: mdl-2353446

ABSTRACT

A field study was conducted to evaluate the delayed-type hypersensitivity (DTH) test in diagnosing brucellosis in cattle, in particular the diagnosis of infection in individual cows. A total of 93 cows that were negative, suspect, or positive to the serum agglutination test (SAT), complement fixation test (CFT), or the milk ring test (MRT) were subjected to the DTH test. The cows were then slaughtered and the supramammary lymph nodes were collected for bacteriologic examination. In 989 cows the DTH test, MRT and serologic tests were negative. When the DTH test results were compared with bacteriologic results, 12 of the 93 cows with CFT titres greater than 1:200 tested negative in the DTH test while bacteriologic results were positive. The sensitivity of the DTH test (calculated on the remaining 81 cows) was 100%; the specificity was 83%. The sensitivity of the DTH test (calculated on 93 cows) was 81%; the specificity was 83%. The sensitivity and specificity of the DTH test correlated well with those of the CFT (86-83%). We conclude that the DTH test is very sensitive, and specific enough to diagnose brucellosis in individual cows. The DTH test should be used in combination with serologic tests in the diagnosis of brucellosis.


Subject(s)
Brucellosis, Bovine/diagnosis , Hypersensitivity, Delayed , Agglutination Tests , Animals , Cattle , Complement Fixation Tests , Evaluation Studies as Topic , Female , Predictive Value of Tests
19.
Vet Microbiol ; 21(3): 255-62, 1990 Jan.
Article in English | MEDLINE | ID: mdl-2305547

ABSTRACT

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Leptospira interrogans serovar hardjo (hardjo) infection in cattle was compared with the microscopic agglutination test (MAT). Glutardialdehyde was used in the ELISA to couple sonicated hardjo antigen to the microtiter plate. Mouse monoclonal anti-bovine IgG1 coupled to peroxidase was used as conjugate. Sera from calves experimentally inoculated with hardjo reacted positively in the MAT as early as 10 days after inoculation; these sera did not react positively in the ELISA until 25 days after the first inoculation. Positive and negative field sera from 704 adult cattle on 90 farms were examined by the MAT and the ELISA; a 90% correlation between the two tests was demonstrated. Eighty-six sera from calves inoculated with four Leptospira serogroups other than hardjo and 227 field sera from adult cattle with naturally occurring leptospirosis other than hardjo were examined by the ELISA. Fewer than 1% of these heterologous sera reacted with hardjo antigen in the ELISA. We concluded that the ELISA described in this report is an advantageous alternative to the MAT for diagnosing leptospirosis.


Subject(s)
Cattle Diseases/diagnosis , Enzyme-Linked Immunosorbent Assay , Leptospirosis/veterinary , Agglutination Tests , Animals , Antibodies, Bacterial/analysis , Antibody Specificity , Antigens, Bacterial/immunology , Cattle , Leptospira/immunology , Leptospirosis/diagnosis , Predictive Value of Tests
20.
Eur J Biochem ; 185(2): 469-74, 1989 Nov 06.
Article in English | MEDLINE | ID: mdl-2555193

ABSTRACT

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines in mammalian cells is characterized by an extremely short half-life. In the present study, ODC degradation was investigated in 653-1 mouse myeloma cells that overproduce ODC and in ts85 cells that are thermosensitive for conjunction of ubiquitin to target proteins. Addition of 2-deoxyglucose and dinitrophenol (agents that efficiently deplete cellular ATP) to the growth medium of these cells inhibited ODC degradation. In contrast, chloroquine and leupeptin, inhibitors of intralysosomal proteolysis, did not affect ODC degradation. Shifting ts85 cells to 42 degrees C (a non-permissive temperature that inhibited conjugation of ubiquitin to target proteins) did not prevent ODC degradation. The ATP-dependent degradation of ODC in 653-1 cells was inhibited substantially by N alpha-tosyl-L-lysine chloromethane (TosPheMeCl), iodoacetamide and o-phenanthroline. These results suggest that ODC degradation occurs via a non-lysosomal. ATP-requiring and ubiquitin-independent cellular proteolytic mechanism, and that serine proteases and enzymes containing sulphydryl groups and metalloenzyme(s) may be involved in this process.


Subject(s)
Adenosine Triphosphate/physiology , Ornithine Decarboxylase/metabolism , Ubiquitins/physiology , Adenosine Triphosphate/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Animals , Cell Line , Electrophoresis, Polyacrylamide Gel , Mammals/physiology , Polyamines/pharmacology , Tumor Cells, Cultured
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