Immunoblot analysis of humoral immune responses to Mycobacterium bovis in experimentally infected cattle: early recognition of a 26-kilodalton antigen

Development of a serodiagnostic test for bovine tuberculosis necessitates an understanding of the humoral immune responses of animals following infection with Mycobacterium bovis. The antibody responses in groups of calves challenged intranasally with different doses of M. bovis (approximately 10(2), 10(4), and 10(6) CFU) or placed in contact with the infected animals were analyzed by immunoelectrophoretic blotting in which a whole-cell sonicate of M. bovis was utilized as an antigen. Antibody responses were evident early in infections in which calves were exposed to high doses of M. bovis, while in groups exposed to lower doses, the time until antibody was detected increased as the challenge dose decreased. In cattle exposed to M. bovis, immunoblot analysis showed antibody responses to three main antigens of 26, 22, and 16 kDa. It was further demonstrated that antibody responses to the 26-kDa antigen appeared earliest in the course of infection. Preliminary investigations in this study have identified a 26-kDa antigen for potential use in improved serodiagnosis by enzyme-linked immunosorbent assays.     

Performance of the single intradermal comparative tuberculin test in identifying cattle with tuberculous lesions in Irish herds

The sensitivity of the single intradermal comparative tuberculin test, as applied in the Republic of Ireland, was estimated in 353 cattle with tuberculous lesions. These cattle had been removed from 47 herds which were depopulated owing to chronic or extensive infection with Mycobacterium bovis. The test had a sensitivity of 90.9 per cent, because 321 (90.9 per cent) of the 353 cattle with tuberculous lesions gave a positive or inconclusive result, and 32 gave a negative result. These 32 negative cattle came from 17 (36 per cent) of the 47 depopulated herds.     

Experimentally induced infections bovine keratoconjunctivitis: effectiveness of a pilus vaccine against exposure to homologous strains of Moraxella bovis

A study was conducted to determine whether vaccination with sonicated pili of Moraxella bovis would protect cattle from subsequent infection and disease when experimentally challenged by exposure to homologous cultures of M bovis. Some calves were intramuscularly inoculated 2 times with pili of M bovis suspended in water, and others were subcutaneously inoculated 2 times with pili of M bovis suspended in oil; 21 days were allowed between inculations. Controls were nonvaccinated calves. Fourteen days after the last inculation, all calves were exposed to virulent homologous cultures of M bovis. The results indicated that vaccination with sonicated pili of M bovis may induce protective immunity against homologous strain challenge exposure. Vaccines in oil that were injected subcutaneously protected to a greater extent than did vaccines in water that were injected intramuscularly. The development of inflammatory nodules at the site of inoculation was associated with resistance to infection and disease. Only 1 of the vaccinated calves that resisted disease lacked precipitating antibodies against sonicated pili at the time of the challenge exposure. This calf had antibodies 2 weeks later.     

Mycobacterium paratuberculosis: a potential food-borne pathogen?

Mycobacterium paratuberculosis commonly infects dairy cattle, leading to Johne's disease, which is also known as paratuberculosis. The infection is chronic progressive, and incurable. As the infection progresses, excretion of M. paratuberculosis in feces and milk occurs, and the bacterium spreads through the blood to multiple internal organs. Consequently, raw products originating from cattle may harbor M. paratuberculosis. Thermal treatments, such as pasteurization, are commonly relied on to kill food-borne bacterial pathogens that can infect humans. The small number of studies conducted to determine the thermal resistance of M. paratuberculosis suggest that it is less susceptible to destruction by heat killing than are milkborne zoonotic bacterial pathogens such as Listeria spp. or Mycobacterium bovis. Published reports concerning the thermal resistance of M. paratuberculosis in milk are reviewed herein, and key issues concerning the efficacy of pasteurization for elimination of M. paratuberculosis from milk are summarized.     

Monitoring Babesia bovis infections in cattle by using PCR-based tests

The sensitivity and specificity of PCR tests based on the small-subunit rRNA gene sequence of Babesia bovis were compared in a blind study of experimentally infected cattle with the corresponding parameters of the complement fixation (CF) test currently used in the United States to screen for bovine babesiosis. Cattle were experimentally infected with a single inoculum of a cloned laboratory strain of B. bovis. Blood samples were collected and tested over a period covering from the day of infection to 10 months postinfection. The level of parasitemia (percent infected erythrocytes) present in each sample was estimated from test results and was plotted as a function of time postinfection. These data are the first describing the course of infection by methods capable of detecting parasitemias in the range of 10(-7)%, which frequently occur in the carrier state. Parasitemias in the samples tested strongly influenced the sensitivity and negative predictive value of the PCR-based tests which varied with time postinfection. The average sensitivities of the three PCR-based tests for B. bovis ranged from 58 to 70% for a single determination, while the sensitivity of the CF test was only 6%. Both PCR-based and CF tests for B. bovis had high specificity values ranging from 96 to 100%.     

Informed consent revisited

The resurgence in mycobacterial infection worldwide has led to renewed attention to the pathogenesis of Mycobacterium species. The purpose of this study was to characterize the infection of alveolar macrophages (AMs) by nonopsonized Mycobacterium bovis, and to elucidate the mechanism by which a differential infection of subpopulations of AM may occur. A difference in susceptibility to Mycobacterium bovis infection of subpopulations of AMs was observed, such that the least dense cells were the least susceptible (21.4 +/- 10.7%) and the most dense cells were the most readily infected (61.8 +/- 5.6%). The percentage of AMs staining for CD14 receptors showed a similar differential distribution, with fewer of the least dense cells expressing CD14 and a greater percentage of the most dense cells staining for CD14 receptor expression. To investigate the role of CD14 receptors in the infection of AMs, anti-CD14 antibody was added to the cell cultures. Infection of AM by Mycobacterium bovis was blocked by up to 60.2% by anti-CD14 antibody but not by isotype control antibody. The results of this study suggest that Mycobacterium bovis selectively infects AM subpopulations, specifically those with the greatest expression of CD14, a putative receptor mechanism for Mycobacterium bovis infection of porcine AM.     

Babesial antibody dynamics after cattle immunisation with live vaccines, measured with an indirect immunofluorescence test

The efficacy of vaccination of Argentinean cattle against babesiosis and anaplasmosis using live immunogens was tested to detect specific antibodies in samples obtained about 60 days after vaccination. Under these conditions a higher than expected proportion of cattle failed to show antibodies against Babesia bigemina. Therefore, a study was designed to evaluate if this failure was due to insensitivity of the routine test to detect antibodies to B. bigemina or to lack of infectivity of the live vaccine. Four groups (G) of cattle were each inoculated subcutaneously with 10 million Babesia bovis (vaccinal strain R1A), 10 million B. bigemina (vaccinal strain S1A) and 10 million Anaplasma centrale (strain M1). G1 and G2 consisted of ten Angus bulls 20-24 months old and ten Angus bulls 15-18 months old, respectively; G3 and G4 consisted of ten and 16 Holstein 1-month-old male calves, respectively. Blood samples were obtained on days 0, 10, 20, 30, 40, 50 and 60 after vaccination and the sera were analysed with an indirect immunofluorescent (IFA) test to detect antibodies to B. bovis (baseline dilution for a positive result 1:60) and B. bigemina (baseline dilution 1:120). Positive IFA titres were considered as evidence of the infectivity of the Babesia vaccinal strains contained in the vaccine. All Angus bulls were found positive to antibodies against both Babesia species, by day 20 (B. bovis) and day 30 (B. bigemina), whereas 10-25% of Holstein calves were negative throughout. The partial lack of vaccine infectivity in the calves was considered to be a consequence of innate resistance of young calves to Babesia. Antibody titres to B. bovis and B. bigemina declined by day 60 after vaccination. However, all cattle that were positive to B. bovis antibodies on day 50 were still positive to the IFA test 10 days later while 10%, 30% and 12% of cattle of G1, G2 and G3 that were positive to B. bigemina antibodies on day 50 after vaccination were found negative to the IFA test on day 60. In future, samples taken on days 40-50 will be used for detection of B. bigemina antibodies in vaccinated cattle, on day 60 for A. centrale and on either occasion for B. bovis. The reaction to the inoculation of B. bigemina S1A strain appears to lag behind the reaction to B. bovis R1A strain. It is not certain if this is a normal reaction to this B. bigemina strain or the result of interaction with the B. bovis strain.     

DNA and a CpG oligonucleotide derived from Babesia bovis are mitogenic for bovine B cells

DNAs from bacteria and variety of nonvertebrate organisms, including nematodes, mollusks, yeasts, and insects, cause polyclonal activation of murine B lymphocytes. Similar studies have not been reported for bovine B cells, and to date no studies have reported mitogenic properties of protozoal DNA for any species. However, we and others have observed that protozoal parasite antigens can induce the proliferation of lymphocytes from nonexposed donors. Extending these studies, we now show that the mitogenic property of protozoal antigen preparations is in part attributable to parasite DNA and that Babesia bovis DNA is directly mitogenic for bovine B cells. DNase treatment of B. bovis extracts abrogated B. bovis-induced proliferation of peripheral blood mononuclear cells from nonexposed cattle. Like DNAs from other organisms that were mitogenic for murine B cells, B. bovis DNA is largely nonmethylated and induced a dose-dependent proliferation of bovine B cells, which was reduced upon methylation. Furthermore, B. bovis and E. coli DNAs enhanced immunoglobulin secretion by cultured B cells, inducing moderate increases in immunoglobulin G1 and stronger increases in immunoglobulin G2. Because certain nonmethylated CpG motifs present in bacterial DNA are known to stimulate proliferation of murine and human B cells, an 11-kb fragment of B. bovis DNA was analyzed for CG dinucleotide content and for the presence of known immunostimulatory sequences (ISS) centered on a CG motif. The frequency of CG dinucleotides was approximately one-half of the expected frequency, and several CpG hexameric sequences with known activity for murine B cells were identified. An oligodeoxynucleotide containing one of these ISS (AACGTT), which is present within the rhoptry-associated protein-1 (rap-1) open reading frame, was shown to stimulate B-cell proliferation. These ISS may be involved in host immune modulation during protozoal infection and may be useful as vaccine adjuvants.     

Comparison of PCR, isoenzyme analysis, and antigen detection for diagnosis of Entamoeba histolytica infection

The Babesia bovis merozoite surface antigen-1 (MSA-1) is an immunodominant, neutralization-sensitive merozoite surface antigen encoded by a polymorphic gene family. MSA-1 antigenic polymorphism results in a complete lack of immunologic cross-reactivity among strains. It is unknown how rapidly this antigenic shift occurs, or whether it evolves in the mammalian host. To determine whether the dominant epitopes encoded by a single msa-1 gene copy vary during the course of a single infection, the antibody response to these epitopes was measured after infection of cattle with the Mo7 biologically cloned strain of B. bovis using an Mo7 gene copy-specific enzyme-linked immunosorbent assay. Antibodies against MSA-1 encoded by this gene copy were detected by postinoculation (PI) day 15 in each of 5 experimentally infected animals. Importantly, detectable antibody persisted in all carrier animals without a significant decrease in optical density through 12 mo PI, at which time the experiment was terminated. The results indicate that immunodominant epitopes expressed by a single gene copy of msa-1 do not undergo marked antigenic shift typical of the gene family during the course of a single infection in the mammalian host. The results are compatible with the limited MSA-1 polymorphism reported in some geographically defined endemic populations.     

Effects of vaccination with a Moraxella bovis bacterin on the subsequent development of signs of corneal disease and infection with M bovis in calves under natural environmental conditions

A vaccination study was conducted for infectious bovine keratoconjunctivitis (IBK) in 440 purebred Hereford cattle (cows and their newborn calves) of the USDA Meat Animal Research Center cattle herd at Clay Center, Ne. The cattle were allotted to 4 groups: 60 calves were vaccinated with an autogenous Moraxella bovis bacterin (group 1); 60 calves that were matched with group 1 calves were designated nonvaccinated matched controls (group 2); 99 calves were peer group nonvaccinated controls (group 3); and 219 cows, the dams of the calves, were nonvaccinated consorts (group 4). The infection rates in cattle groups 1, 2, 3, and 4 during the summer were 96.6, 98.3, 100, and 79.1%, respectively, and the disease rates were 90, 93, 85, and 20%. The infection and the disease rates were significantly (P less than 0.01) different between claves and cows. The disease rate was also significantly different between older and younger cows. A larger percentage of the affected calves and cows had mild or moderate (61%) signs of IBK rather than severe (39%) signs. The rate of body weight gain was reduced in calves with severe signs of IBK. The results seemed to indicate that little would be gained by vaccinating cattle against IBK under the conditions of study.     

Piroplasms of domestic animals in the Macedonia region of Greece. 1. Serological cross-reactions

During a serological survey on haemoparasites in Macedonia, serum samples were collected from cattle, sheep and goats. All sera were tested by the indirect immunofluorescence test (IFAT); the cattle sera against Theileria orientalis, T. annulata, Babesia bigemina, B. bovis, B. divergens and B. major antigens; the sheep and goat sera against T. ovis, B. ovis, B. motasi and B. crassa antigens. Parallel tests of negative and positive control sera against all the antigens showed the existence of cross-reactions of different degrees between species of the same genus. In cattle, the most important cross-reactions were obtained against B. bigemina antigen, especially with the anti-B. bovis serum, in small ruminants against B. motasi with the anti-B. crassa serum. In the field sera, there was a high correlation between the antibody titres of B. bigemina and B. bovis, and also between the titres of these two Babesia spp. and B. divergens. A high correlation was also found between B. motasi and B. crassa, and lower ones between these two and B. ovis. The correlations of the sera titres were due to mixed infections or to cross-reactions. Therefore, the use of the IFAT is not always satisfactory for diagnosing infections in regions where animals are infected with different piroplasms.     

Isolation of pathogenic Mycobacterium strains in cattle slaughtered in the abattoir of Bobo-Dioulasso, Burkina Faso

The major cause of meat rejection at the slaughterhouse of Bobo-Dioulasso is bovine tuberculosis. The objective of this work was to confirm the postmortem diagnosis pronounced at the slaughterhouse using laboratory methods (direct examination following Ziehl-Hielsen staining and isolation of Mycobacterium strains following culture in a Loewenstein-Jensen medium. During the study, 39 pathogenic Mycobacterium strains (38 Mycobacterium bovis and 1 Mycobacterium tuberculosis) out of 100 sampled out suspicious strains, were studied. No Mycobacteria typical of M. farcinogenes was found on direct examination. We were then able to differentiate between bovine tuberculosis and bovine farcy. The results confirm the well-founded decision in the slaughterhouse to discard the meat on the grounds of tuberculosis. The majority of cattle is located in villages around Bobo-Dioulasso and its is therefore highly likely that the disease is enzootic in the area with a fairly high level of infection in animals.     

An immunohistochemical method of detecting Mycoplasma species antigens by use of monoclonal antibodies on paraffin sections of pneumonic bovine and caprine lungs

Lung samples from pneumonic lesions in cattle and goats, naturally or experimentally infected with strains of the Mycoplasma mycoides cluster, were fixed in formalin and embedded in paraffin. An immunohistochemical technique using monoclonal or polyclonal antibodies was performed on tissue sections in order to detect Mycoplasma antigens. Four monoclonal antibodies (MAbs), one (2A3) raised against M. mycoides ssp. mycoides small colony (SC) and large colony (LC), two (1D3 and 5E5) against M. mycoides ssp. capri, and one (5A10) against M. bovis, were used. A range of polyclonal antibodies, raised to the individual subspecies of the M. mycoides cluster, and one to Pasteurella haemolytica, was also used. The MAb 2A3 showed positive immunostaining in lung sections from cattle and goats naturally and experimentally infected with M. mycoides ssp. mycoides SC and LC, but not with pneumonic lesions of cattle and goats due to other members of the M. mycoides cluster, M. bovis or Pasteurella spp. The MAb 1D3 showed immunostaining in lung sections from goats naturally and experimentally infected with M. mycoides ssp. capri, but again not with pneumonic lesions caused by other members of the M. mycoides cluster, M. bovis or Pasteurella spp. The MAb 5E5 immunoreacted in sections from pneumonic lesions from all animals infected with one of the three M. mycoides cluster subspecies used in the study, but not with M. bovis or Pasteurella infected tissue. Immunoreaction was mainly found in the cell debris around necrotic areas, as well as in macrophages, neutrophils and epithelial cells. The localization of antigens of the M. mycoides cluster using polyclonal antisera followed basically the same pattern as that obtained with the monoclonals. However, a wide cross reactivity was found between different antisera and relatively high background immunostaining was also seen, especially in necrotic areas. The results suggest that immunohistochemical methods using monoclonal antibodies are useful tools for the diagnosis and study of the pathogenesis of pneumonia caused by the Mycoplasmas of the M. mycoides cluster.     

Endotoxic activity of cell-free rumen fluid from cattle fed hay or grain

The cell-free rumen fluid from cattle fed hay or grain exhibited the following biological characteristics which strongly suggest the presence of endotoxin or a toxic principle similar to endotoxin of gram-negative bacteria: proved lethal to mice when injected with actinomycin D; proved extremely lethal to chick embryo; induced biphasic pyogenic response in rabbits; enhanced susceptibility to bacterial infection in mice; evoked positive epinephrine skin reaction in rabbits and phenol-water or aqueous ether proved lethal to mice and chick embryos. A quantitative difference in concentrations of endotoxin was observed on LD50 in mice and chick embryos and response to the epinephrine skin test in rabbits. Cell-free rumen fluid of grain-fed cattle contained at least twice as much endotoxin as that of hay-fed cattle. Endotoxin in cell-free rumen fluid and in higher concentration in cattle fed grain than in those fed hay support the hypothesis that rumen bacterial endotoxins may participate in the pathogenesis of diseases associated with high grain feeding such as lactic acidosis and the sudden-death syndrome.     

Towards a standardized approach to DNA fingerprinting of Mycobacterium bovis. International Union Against Tuberculosis and Lung Disease, Tuberculosis in Animals Subsection

The Tuberculosis in Animals Subsection of the International Union Against Tuberculosis and Lung Disease (IUATLD) recently identified a need to standardize the deoxyribonucleic acid (DNA) strain typing of Mycobacterium bovis. The standard method for strain typing of M. tuberculosis isolates cannot be directly extrapolated to M. bovis due to the low copy number of IS6110 identified in the majority of M. bovis strains, particularly from cattle. To improve the resolution of M. bovis strains, alternative methods and additional DNA probes have been investigated. In combination with studies of published literature, laboratories performing M. bovis DNA fingerprinting were surveyed. Results of these surveys allowed us to reach consensus and to make recommendations for DNA typing of M. bovis isolates, which hopefully will lead towards a standardized approach to the DNA fingerprinting of this organism. This approach, in conjunction with conventional epidemiological traceback approaches, should facilitate more accurate and effective investigations into the epidemiology, maintenance and transmission of M. bovis within and between man and domesticated, feral and wild animals, both at a local and a global level.     

Nervous signs in bovine coccidiosis

In 2 outbreaks of coccidiosis due to E bovis and/or E zurnii infection in Canadian cattle, nervous signs included opisthotonos, medial strabismus, hypersensitivity, tetanic spasms, and convulsions. All of the affected animals died in convulsions after an illness of one to several days during which time they showed periodic nervous signs. Necropsy revealed a very severe enteritis with the most severe lesions in the spiral colon. Much of the intestinal mucosa in this area had been destroyed by the parasite. None of several suggested causes of such nervous signs was indicated by laboratory findings, but the possibility of toxins produced by the coccidia could not be ruled out.     

HLA-B27 and the seronegative spondyloarthropathies

Cross-reactivity between Babesia bovis and B. bigemina becomes a problem in discrimination of the two infections in endemic areas where the two species usually occur in association. With the aim of identifying candidate proteins for use as specific diagnostic tools, culture-derived components of three geographically different stocks of B. bovis (Lismore, Kwanyanga and Mexico) and one of B. bigemina (Mexico) were analyzed by immunoprecipitation using acrylamide gel electrophoresis. The approach taken was based on the analysis of 35S-methionine-labelled parasite antigens released into culture supernatant. A variety of serum samples were tested, including a panel of calf sera experimentally produced against the different stocks of Babesia, serum samples from cattle naturally infected in the field in Brazil, and a panel of anti-B. bovis monoclonal antibodies, previously characterized by the indirect fluorescent antibody test, ELISA and Western immuno-blotting. Approximately 28 and 23 bands (with molecular weights ranging from 200 to 14 kDa) were detected in total protein profiles of B. bovis and B. bigemina culture supernatants, respectively, whereas no bands were seen in the uninfected red blood cell culture supernatant (negative control). The immunoprecipitation analysis showed antigenic diversity amongst the stocks of B. bovis and resulted in identification of at least five B. bovis specific antigens common to the three stocks (molecular weights of 80, 72, 58, 38 and 24 kDa) and four B. bigemina specific antigens (molecular weights of 240, 112, 50 and 29 kDa).     

Role of infected, non-diseased badgers in the pathogenesis of tuberculosis in the badger

The lungs and kidneys of 15 badgers which had no visible lesions of tuberculosis but from which Mycobacterium bovis was isolated from pooled collections of lymph nodes were serially sectioned. Lesions of tuberculosis were detected by histopathology in the the lungs of 13 and in the kidneys of one of them. The lesions were mostly typical early stage granuloma-lesions which were considered to be the primary foci of infection. These lesions suggest an early containment phase of arrested development previously not observed and provide further evidence on which to propose a hypothesis for the pathogenesis of tuberculosis in the badger.     

Stimulation of nitric oxide production in macrophages by Babesia bovis

Gamma interferon (IFN-gamma)-activated macrophages are believed to play a key role in resistance to Babesia bovis through parasite suppression by macrophage secretory products. However, relatively little is known about interactions between this intraerythrocytic parasite and the macrophages of its bovine host. In this study, we examined the in vitro effect of intact and fractionated B. bovis merozoites on bovine macrophage nitric oxide (NO) production. In the presence of IFN-gamma, B. bovis merozoites stimulated NO production, as indicated by the presence of increased L-arginine-dependent nitrite (NO2-) levels in culture supernatants of macrophages isolated from several cattle. The merozoite crude membrane (CM) fraction stimulated greater production of NO, in a dose-dependent manner, than did the merozoite homogenate or the soluble, cytosolic high-speed supernatant fraction. Stimulation of NO production by CM was enhanced by as little as 1 U of IFN-gamma per ml of culture medium. Upregulation of inducible NO synthase mRNA in bovine macrophages by either B. bovis-parasitized erythrocytes and IFN-gamma or CM was also observed. B. bovis-specific T-helper lymphocyte culture supernatants, all of which contained IFN-gamma, were also found to induce L-arginine-dependent NO2- production. Supernatants that induced the highest levels of NO also contained biologically active TNF. These results show that B. bovis merozoites and antigen-stimulated B. bovis-immune T cells can induce the production of NO, a molecule implicated in both protection and pathologic changes associated with hemoprotozoan parasite infections.     

The safety and efficacy of Australian tick-borne disease vaccine strains in cattle in Paraguay

Glycerol preserved, frozen tick-borne disease vaccine strains developed in Australia were imported into Paraguay to test their safety in pregnant Holando heifers and their efficacy against challenge from inoculated local field strains of Babesia bigemina, B. bovis and Anaplasma marginale in Hereford X Criolla heifers. The two Babesia strains proved to be safe and the B. bovis K strain was very effective in providing immunity to a local field strain of B. bovis. The B. bigemina efficacy trial was inconclusive, possibly due to the avirulent nature of the local field strain used in challenge. The A. centrale strain did not prove to be as safe as would be desirable in safety trials, neither did it provide as good protection as the Babesia strains in the efficacy trial. It was concluded that the Babesia strains provided good protection against field challenge in Paraguay and were safe to use in highly susceptible cattle, however an alternative to A. centrale should be sought to provide protection against local strains of A. marginale.