Recognition of Mycoplasma arthritidis membrane antigens by rats and rabbits: comparison by immunoblotting and radioimmunoprecipitation

Sera from rats convalescing from infection with Mycoplasma arthritidis were tested for their ability to react with M. arthritidis membrane antigens by immunoblotting and radioimmunoprecipitation. The absence of metabolism-inhibition (MI) antibody activity in these sera suggested that rats might fail to recognize those membrane antigens involved in eliciting MI antibodies therefore rabbit antisera, which are strongly MI positive for M. arthritidis, were used for comparison. Antigenic recognition patterns of M. arthritidis surface and membrane antigens were not identical for rats and rabbits. The most striking and reproducible difference was the failure of rats to produce IgG antibodies against a surface antigen migrating in the 47,000-50,000 molecular weight range on SDS-polyacrylamide gels. However, rats recognized at least 2 antigens which we had previously shown to be "MI antigens", therefore the inability to express MI antibodies probably cannot be explained by their inability to recognize M. arthritidis "MI antigens".     

Antigenic cross-reactions between Mycoplasma arthritidis and rat tissues

An antigenic relationship between Mycoplasma arthritidis and rat tissues could be demonstrated using the enzyme-immune assay and the agar gel double diffusion test. Cross-reactions were observed with joint homogenates as well as with muscle, lung, liver, kidney, spleen and brain tissue from rats. In addition, antibodies against joint homogenate were found in the sera of rats infected with M. arthritidis ISR 1 using the indirect hemagglutination test. The significance of common antigens between M. arthritidis and rat tissues for the pathogenesis of the M. arthritidis polyarthritis in rats is discussed.     

Vaccination of Lewis rats against Mycoplasma arthritidis-induced arthritis.

The nature of Mycoplasma arthritidis antigens responsible for eliciting protective immunity in rats was studied by inoculation of rats with mycoplasmal components that had been subjected to a variety of physical and chemical treatments. All inocula tested induced good protection against development of clinical illness, as assessed by changes in body weight and appearance of joint swelling and/or temporary hind limb paralysis. Although all preparations stimulated development in inoculated rats of high titer of antimycoplasmal antibodies measured by ELISA, the complement-fixation antibody response was poor and, in some cases, lacking altogether. This indicated that completion-fixation antibodies may not be involved in protecting rats against M arthritidis-induced illness. Protective antigens were stable to heat (100 C for 10 minutes), formalin, and denaturation by sodium dodecyl sulfate (SDS). Inoculation with membrane and soluble cytoplasmic fractions was protective, as was inoculation with 5 M arthritidis fractions separated according to molecular weight by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). For this latter experiment, rat antisera obtained after vaccination, but prior to challenge exposure, were tested by immunoblot analysis against electrophoretically separated M arthritidis membrane proteins. Interestingly, all antisera from these rats recognized antigens migrating far outside the molecular weight range of the cell fractions with which rats were inoculated. This indicated either that the protective antigens may be composed of numerous antigenically related subunits that separated by SDS-PAGE into a variety of molecular weight ranges or that a few major antigens may exist in several forms or phases within a given population of M arthritidis.     

Monoclonal antibodies to human antigens recognise feline myeloid cells

Immunological techniques have been used to study the expression of a series of cell surface antigens in cat haemopoietic tissues. Forty-two monoclonal antibodies raised against well-defined antigens of human origin were tested by indirect immunofluorescence on feline blood, bone marrow, spleen and thymus. Myeloid cells from all tissues reacted with antibodies to CD9, CD10 and CD18 antigens. No antibodies specific for T or B lymphocytes were found to react with cat lymphoid cells. Osteoclasts, isolated from juvenile bone marrow, were found to express the 23C6 human osteoclast-specific antigen. The potential use of such antibodies in experimental and diagnostic veterinary haematology are discussed.     

Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink.     

Immunohistochemical demonstration of chromogranin A in endocrine organs of the rat and horse by use of region-specific antibodies

Chromogranin A (CgA) is an acidic glycoprotein that is co-stored with hormones or neurotransmitters in granular components of endocrine cells and neurons, and released together with them in response to adequate stimulation. In addition to acting as a packaging protein, CgA functions as a precursor molecule that yields several bioactive peptides by proteolytic cleavage. The purpose of this study is to elucidate how different the processing of CgA is among endocrine tissues by immunostaining using multiple region-specific antisera, and to evaluate the availability of region-specific antisera. When various endocrine organs of rats were immunostained with four region-specific antisera against rat CgA (CgA 1-28, 94-130, 296-314, and 359-389), all amine/peptide-secreting endocrine tissues except the pineal body were stained positively. The adrenal medulla and gastric endocrine cells were equally intensely immunoreactive to all four antisera, while the other endocrine tissues, represented by pancreatic islets, showed different staining patterns depending on the antiserum. These results suggest that the processing of CgA differs from tissue to tissue. An antiserum against horse CgA 335-365, corresponding to rat CgA 359-389 which shows the highest concentration in the plasma and urine of the rat, again stained all endocrine tissues of the horse except the pineal body. Therefore, the anti-horse CgA 335-365 serum is useful for immunohistochemical survey of horse CgA, and may make possible the establishment of a CgA assay system for the measurement of CgA in the plasma, urine and saliva.     

Experimental induction of arthritis in LEW rats and antibody response to four Mycoplasma arthritidis strains

Four Mycoplasma arthritidis strains were examined for differences in virulence for LEW rats and elicitation of antibody responses in the immunoglobulin (Ig) M and G classes and in the four IgG subclasses. Two strains were highly arthritogenic and two were relatively avirulent. When the latter strains did induce arthritis, it was significantly less severe (P less than 0.05) and developed significantly later (P less than 0.001) than in rats injected with the two virulent strains, suggesting that the low-virulence organisms are able to persist asymptomatically in rats for several weeks. None of the M. arthritidis-injected rats developed metabolism-inhibiting (MI) antibodies at any time during the 6-week observation period. Responses to other M. arthritidis antigens from all four strains were measured by enzyme immunoassay (ELISA); they were similar qualitatively but differed quantitatively. Rats injected with the two avirulent strains showed significantly lower titers of IgM antibodies (P less than 0.01) throughout the 6-week observation period and significantly lower early titers of IgG antibodies (P less than 0.05) than rats injected with the two virulent strains. In addition, peak IgM antibody titers, IgM titers measured 1 and 6 weeks after injection and IgG antibody titers measured 1 week after injection all correlated significantly with peak arthritis scores (P less than 0.05). The IgG antibody response against all four strains appeared mostly in the IgG2a and IgG2b fractions, with very little in the IgG1 and IgG2c fractions. Using immunoblotting, the immunodominant antigens of the two virulent strains appeared very similar, but the avirulent strains differed slightly from each other and from the other two. This study indicates that immune responses of rats to virulent and avirulent strains are similar but not identical and that immunogenicity for LEW rats may be a strain-specific characteristic for M. arthritidis.     

The effect of exercise on the lactic dehydrogenase and creatine kinase isoenzyme composition of horse serum.

The distribution of lactic dehydrogenase, aldolase and creatine kinase in various horse tissues was determined. Using polyacrylamide gel electrophoresis the lactic dehydrogenase and creatine kinase isoenzyme composition of horse serum, taken before and after exercise, was studied. Horse tissue isoenzyme patterns were also obtained. By comparing tissue and serum patterns, skeletal muscle was found to be the tissue of origin of the increase in serum lactic dehydrogenase and creatine kinase observed after exercise.     

Inhibition of turkey herpesvirus replication by anti-cellular serum

Quail embryo fibroblasts were used to investigate how rabbit and chicken antisera against chicken erythrocytes carrying different B alleles of the major histocompatibility antigens affect the neutralization of herpesvirus of turkeys (HVT). Although the neutralizing activities of these antisera were rather low, the HVT propagated in chicken embryo fibroblasts (CEF) from certain genotypic embryos was neutralized more by the antisera to the corresponding erythrocytes. After absorption of these antisera with homologous erythrocytes, the neutralizing activity of the absorbed sera was reduced slightly. These results reveal that the virion antigens of HVT might be partially associated with the host cell antigens of the fibroblast infected with the virus. The virus grown in these cells might incorporate the host cell antigens, including histocompatibility antigens, into the virion envelope.     

Agammaglobulinemia in a horse.

Immunologic deficiency was suspected in an 18-month-old Standardbred horse with persistent fever, multifocal bacterial infection, and neutropenia with a large number of immature neutrophils. Serum protein electrophoresis revealed marked depression of the gamma-globulin fraction (0.2 g/100 ml). Immunologic testing and histologic examination of lymphoid tissues identified the immune deficit as agammaglobulinemia. Serum concentrations of immunoglobulin (Ig)G and IgG(T) were initially low and declined with time; IgM and IgA were not detectable. The horse failed to produce antibodies when inoculated with foreign antigens but had a positive cell-mediated skin reaction to intradermal phytolectin injection, and lymphocytes responded normally to in vitro stimulation by mitogens. Histologic examination of lymphoid tissues revealed absence of germinal centers and plasma cells.     

Antigenicity of a chemically induced neurogenic rat sarcoma (author's transl)

A neurogenic sarcoma was induced in the plexus brachialis of a male Long-Evans rat by administration of N-methyl-N-nitrosourea in the drinking water. The tumor was established in vitro and designated 76LE-NS-369. Cells from tissue culture grew as tumors when isografted in young rats. 76LE-NS-369 cells did not react with antiserum directed against gliaspecific S-100 protein. We used the cultured cells as target cells, and found specific antibodies in the sera of tumor-bearing and immunized rats by indirect fluorescent antibody stain and a complement-dependent antibody-mediated microcytotoxicity assay. In immunization experiments, incubation of tumor cells with Vibrio cholerae neuraminidase yielded higher antibody titers than an antigen preparation with untreated cells. Incubation with neuraminidase enhanced the sensitivity of tumor cells to antibody and complement in vitro, whereas trypsinized cells showed complete loss of reactivity with autologous antisera. The specificity of antisera was tested by absorption with tumor, lyophilized rat whole body and rat nerve tissues.     

Monoclonal and polyclonal antibodies for immunohistochemical detection of bovine parainfluenza type 3 virus in frozen and formalin-fixed paraffin-embedded tissues.

Accurate identification of bovine parainfluenza type 3 virus in bovine respiratory disease requires dependable, sensitive, and specific techniques for detection in affected animals. Immunohistochemical testing can be a rapid and reliable means of demonstration of virus in tissues from suspect cases; however, this procedure is dependent upon the quality of the antisera directed against the viral antigens. The production of rabbit polyclonal and murine monoclonal antibodies directed against bovine parainfluenza type 3 virus and techniques for their use in fresh-frozen and formalin-fixed paraffin-embedded tissues in immunofluorescence and immunoperoxidase-based immunohistochemical tests are described.     

Co-migrating and shared antigens of selectedPasteurella haemolytica untypable strains

Bacterial cell envelope preparations from eight untypable strains ofPasteurella haemolytica were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting with rabbit antisera prepared against the eight untypable strains (one untypable strain per rabbit) and with cattle antisera prepared againstP. haemolytica serotypes 1, 2, 5, 6, 9 and against one heterologous untypable strain. Numerous comigrating and shared antigens were recognized by the eight rabbit antisera and theP. haemolytica serotype cattle antisera. Comigrating antigens at 43 and 30 kilodaltons (kDa) were recognized by all eight rabbit antisera. Shared antigens, detected by all eight rabbit antisera when reacting againstP. haemolytica serotype 1, were recognized at 43, 32, 30, 20 and 15 kDa.     

Application of the peroxidase-antiperoxidase procedure to the localization of pituitary hormones and calcitonin in various domestic animals and human beings

Specific cell populations in the pituitary glands of the rat, cat, pig, and human being were positive for thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH). When reacted with prediluted rabbit anti-human TSH, LH, and FSH, antisera were not positive for the demonstration of these hormones in the horse, cow, or dog. Immunocytochemical staining was obtained in the horse, cow, and dog by the use of a primary antiserum against a specific beta-subunit of bovine TSH. The immunocytochemical staining of TSH, LH, FSH, adrenocorticotropic hormone, growth hormone, prolactin, and calcitonin was examined by the peroxidase-antiperoxidase method, using standard commercially available kits. All species examined had a strong positive reaction in specific pituitary cell populations for adrenocorticotropic hormone, growth hormone, and prolactin. Sections of normal thyroid gland tissue had positive staining of C cells containing calcitonin at the dilution of 1:100 of the primary antibody in the rat, horse, cow, dog, cat, pig, and human being.     

Characterization of monoclonal antibodies against ovine Sarcocystis spp. antigens by immunoblotting and immuno-electron microscopy

Six monoclonal antibodies were raised in mice against purified cytozoite extracts of Sarcocystis gigantea and S. tenella from sheep. Each monoclonal antibody was evaluated for specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy using homologous and heterologous antigenic preparations. All six monoclonal antibodies exhibited good species-specificity when reacted against crude soluble cystozoite antigens in enzyme immunoassays. However, only two monoclonal antibodies (IgM and IgG2a) exhibited reactivity in Western blots against specific protein bands. Both reacted against S. gigantea antigens of 100,000, 43,000 and 39,000 molecular weight. Neither monoclonal antibody reacted against the heterologous species S. tenella. Ultrastructural studies performed with colloidal-gold conjugated antisera revealed that both monoclonal antibodies reacted against antigens located around micronemes and amylopectin granules in S. gigantea cystozoites. Another monoclonal antibody (IgGI) reacted only against microneme determinants in S. tenella cystozoites. In contrast, polyclonal sheep and rabbit immune sera cross-reacted against a wide range of cystozoite antigens.     

Evaluation of monoclonal antibodies to K88, K99, F41 and 987P fimbrial adhesins for the detection of porcine enterotoxigenic Escherichia coli in paraffin-wax tissue sections

A panel of monoclonal antibodies against fimbrial adhesins of porcine enterotoxigenic Escherichia coli were evaluated for the detection of enteric colibacillosis in paraffin-wax embedded sections of piglet small intestine. Using the immunoperoxidase technique, monoclonal antibodies were used to detect epitopes on the K99 adhesin and on the a and c regions of the K88 adhesin. However, monoclonal antibodies to the F41 and 987P adhesins failed to react in sections with organisms colonising the intestine of gnotobiotic piglets monoinfected with strains bearing those adhesins, whereas corresponding polyclonal antisera gave positive results. In contrast to apparent expression of all K99 organisms, only a proportion of organisms were identified by monoclonal or polyclonal antibodies as expressing K88. In some instances, failure of immunostaining was attributed to prolonged storage of tissue in formalin.     

Chicken IgY: Utilizing the evolutionary difference

Chicken antibodies offer advantages over traditional ones (e.g. rabbit antibodies) due to the evolutionary difference between these immunoglobulins. Chicken give better antibody response against conserved mammalian antigens, they do not react with rheumatoid factors, bacterial or mammalian Fc receptors and can reduce background reactions due to cross-reactivity of anti-IgG antibodies.     

The indirect hemagglutination test for the detection of antibodies in cattle naturally infected mycoplasmas.

Stable mycoplasma antigens for the indirect hemagglutination test (IHA) were prepared employing glutaraldehyde treated sheep erythrocytes sensitized with Mycoplasma agalactiae subsp. bovis and Mycoplasma bovigenitalium antigens. Employing these antigens mycoplasma antibodies were detected in sera from cattle which had mastitic symptoms due to natural infection with either M. agalactiae subsp. bovis or M. bovigenitalium. A total of 200 cows from four herds were examined at varying intervals for the presence of M. agalactiae subsp. bovis and for the detection of antibody using growth inhibition and IHA tests. Mycoplasmas were isolated from 37 animals. Growth inhibiting antibody was detected from 56 of the 200 animals. In the IHA tests, antibody titer greater than or equal to 1:80 were detected in 148 animals, 76 of these having antibody titers greater than or equal to 1:160, while sera of 116 normal control animals had no growth inhibiting antibody and none had IHA antibody titers greater than 1:40. M. bovigenitalium was isolated from the milk of three of 26 animals in a fifth herd during an outbreak of mastitis. Growth inhibiting antibodies were demonstrated in the sera of ten of the 26 animals. However, the IHA test detected antibody titers of greater than or equal to 1:160 in 13 animals and of 1:80 in one of the 26 animals. To determine the specificity of the IHA tests, M. agalactiae subsp. bovis and M. bovigenitalium antigens were reacted with rabbit hyperimmune typing sera produced against 12 species of bovine mycoplasmatales. Homologous antisera showed IHA antibody titers of 1:1280 and 1:2560 against M. agalactiae subsp. bovis and M. bovigenitalium respectively, whereas heterologous antisera showed IHA antibody titers of less than or equal to 1:20. Also eight type-specific bovine antisera were reacted with M agalactiae subsp. bovis and M. bovigenitalium antigens in homologous and heterologous tests. Homoogous reactions showed IHA antibody titers greater than or equal to 1:320, whereas heterologous reactions showed IHA titers of less than or equal to 1:20. This IHA test promises to be useful for the detection of bovine mycoplasma antibodies in sera from cattle infected with M. agalactiae subsp. bovis or M. bovigenitalium. Thes test is sensitive, reproducible and specific and the technique is relatively simple and rapid. The antigens were stable for at least seven months.     

The detection of transmissible gastroenteritis viral antibodies by immunodiffusion.

Precipitating antibodies against transmissible gastroenteritis viral antigens were detected by the immunodiffusion test in two transmissible gastroenteritis viral hyperimmune antisera and in antiserum prepared against haemagglutinating encephalomyelitis virus but not in sera from several species of normal animals, in antisera prepared against a variety of othet viruses and bacteria or sera from swine with bacterial enteritis. When the immunodiffusion test was compared with the virus neutralization test for the detection of transmissible gastroeneritis viral antibodies in 20 swine sera certain samples which contained high titres of virus neutralizing antibodies failed to produce precipitation while other sera were positive in the immunodiffusion test although their virus neutralizing antibody titres were relatively low. Precipitating antibodies were also detected by immunodiffusion in several samples of milk whey from a sow which had been vaccinated with inactivated transmissible gastroenteritis virus.     

Species specificity in thermostable and ethanol insoluble tissue antigens. I. Immunization of rabbits and goats with bovine antigen.

Kidney and spleen antigens from cow, sheep, goat, European moose, reindeer, deer and roe deer were prepared by boiling and ethanol precipitation and tested against rabbit and caprine anti-bovine kidney sera. Two different levels of antigen concentration were used for immunizing animals in the various groups. In the group using high antigen concentrations, the precipitation reaction obtained initially disappeared after further inoculations, probably due to immunological tolerance. Sera tested from animals inoculated with low level antigen concentration showed a variation in the immunological response. The caprine antibovine sera showed distinct species specific reaction, while rabbit antisera showed either species specific or organ specific reaction, with a limited degree of cross reaction. In the production of species specific antisera against ruminant tissue antigens, goat seems to be preferable to rabbit.