Spontaneous murine thymocyte comitogenic activity consistent with interleukin-1 in cattle naturally infected with Mycobacterium paratuberculosis

The production of comitogenic activity consistent with interleukin-1 (IL-1) activity by blood monocytes from cattle with naturally acquired paratuberculosis was examined by murine thymocyte proliferation. In addition, IL-1-like activity in response to homologous and heterologous antigens was determined. Activity was determined in nine cattle naturally infected with Mycobacterium paratuberculosis and six non-infected cattle. Comitogenic properties were measured in response to M. paratuberculosis antigen (johnin), bacterial lipopolysaccharide (LPS) as a positive control, and culture media as a negative control. Monocytes from infected cattle spontaneously released high levels of IL-1-like activity in the absence of stimuli and significantly (P less than 0.05) increased activity in response to LPS. With johnin, M. bovis PPD and KLH stimulation, comitogenic activity was similar to spontaneous levels. Non-infected cattle had significantly (P less than 0.05) increased comitogenic activity when blood monocytes were stimulated with KLH, M. bovis PPD, johnin, and lipopolysaccharide when compared with non-stimulated cells in that group. Johnin produced the greatest response in non-infected animals. The data suggest that blood monocytes in infected cattle are non-specifically activated with respect to IL-1 production. Alternatively, a defective regulatory mechanism for IL-1 may be operative in infected cattle. In addition, the previous observation that mycobacterial antigens are potent inducers of IL-1 was also verified.     

Effects of dialyzable lymph node extracts on lymphoblast proliferative capacity of blood mononuclear cells in cattle with chronic paratuberculosis.

Dialyzable lymph node extracts (DLE) containing transfer factor prepared from calves sensitized to Mycobacterium paratuberculosis and keyhole-limpet hemocyanin (KLH) were administered to 4 adult cows with chronic paratuberculosis. Cutaneous delayed hypersensitivity, lymphocyte blastogenesis, monocyte migration-inhibition, and lymphoblast proliferative capacity as a reflection of interleukin-2 (IL-2) activity were measured in response to M bovis purified protein derivative, johnin, and KLH before and after treatment with DLE. Change in cutaneous delayed hypersensitivity was not evident after DLE treatment. Alterations in histologic features of pre- and posttreatment sections of ileum and mesenteric lymph nodes were not detected. Lymph node extract treatment significantly (P less than 0.05) increased IL-2 activity and migration-inhibition in response to johnin and KLH in vitro. Treatment had no effect on lymphocyte blastogenesis. The data indicate that cattle with chronic paratuberculosis may benefit from DLE treatment, by virtue of increased IL-2 activity, and that effects of DLE are at least partially mediated by an increase in IL-2 activity.     

MHC class II-restricted, CD4(+) T-cell proliferative responses of peripheral blood mononuclear cells from Mycobacterium bovis-infected white-tailed deer

White-tailed deer are significant wildlife reservoirs of Mycobacterium bovis for cattle, predators, and, potentially, humans. Infection of cattle with M. bovis stimulates an antigen-specific T-cell response, with both CD4(+) and CD8(+) cells implicated in protective immunity. Few studies, however, have examined lymphocyte subset responses to experimental M. bovis infection of white-tailed deer. In this study, a flow cytometric proliferation assay was used to determine the relative contribution of individual peripheral blood mononuclear cell subsets of M. bovis-infected white-tailed deer in the recall response to M. bovis antigen. Naive deer were challenged with M. bovis by cohabitation with infected deer. These M. bovis-challenged deer developed significant in vivo (delayed-type hypersensitivity) and in vitro (proliferative) responses to M. bovis purified protein derivative (PPD). At necropsy, typical tuberculous lesions containing M. bovis were detected within lungs and lung-associated lymph nodes of infected deer. The predominant subset of lymphocytes that proliferated in response to in vitro stimulation with PPD was the CD4(+) subset. Minimal proliferative responses were detected from CD8(+), gamma delta TCR(+), and B-cells. Addition of monoclonal antibodies specific for MHC II antigens, but not MHC I or CD1 antigens, abrogated the proliferative response. Together, these findings indicate that while CD4(+) cells from infected deer proliferate in the recall response to M. bovis antigens, this response is not sufficient to clear M. bovis and immunologic intervention may require stimulation of alternate subsets of lymphocytes.     

Lymphocyte subset proliferative responses of Mycobacterium bovis-infected cattle to purified protein derivative

Despite highly successful eradication efforts in several countries, Mycobacterium bovis infection of cattle remains a significant health concern worldwide. Immune mechanisms of resistance to and/or clearance of M. bovis infection of cattle, however, are unclear. Recent studies have provided evidence supporting a role for CD4(+), CD8(+), and gammadelta TCR(+) T cells in the response of cattle to M. bovis. In the present study, we utilized a flow cytometric-based proliferation assay to determine the relative contribution of individual lymphocyte subsets in the response to M. bovis infection and/or sensitization with mycobacterial purified protein derivative (PPD). Peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle proliferated in response to in vitro stimulation with M. bovis PPD. CD4(+) T cells and gammadelta TCR(+) cells were the predominate subsets of lymphocytes responding to PPD. gammadelta TCR(+) cells also proliferated in non-stimulated cultures; however, the gammadelta TCR(+) cell proliferative response of infected cattle was significantly (p<0.05) greater in PPD-stimulated cultures as compared to non-stimulated cultures. Intradermal injection of PPD for comparative cervical testing (CCT) induced a boost in the in vitro proliferative response of CD4(+) but not gammadelta TCR(+) cells of infected cattle. Administration of PPD for CCT also boosted interferon-gamma (IFN-gamma) production by PBMC of infected cattle following in vitro stimulation with M. bovis PPD. Injection of PPD for CCT did not, however, elicit a proliferative or IFN-gamma response in cells isolated from non-infected cattle. These data indicate that CD4(+) and gammadelta TCR(+) cells of M. bovis-infected cattle proliferate in a recall response to M. bovis PPD and that the CD4(+) cell response is boosted by intradermal injection with PPD for CCT.     

Cytokine gene expression in ileal tissues of cattle infected with Mycobacterium paratuberculosis

Cytokine gene expression in ileal tissues of cattle infected with Mycobacterium paratuberculosis was evaluated. The effects of infection with M. paratuberculosis on cytokine production may influence immune regulation at the site of colonization, resulting in the chronic inflammatory state associated with the latter stages of this disease. Ileal samples were obtained at necropsy from noninfected control cows (n=8) and clinically infected cows (n=7) and processed for immunohistochemistry and in situ hybridization. Cows infected with M. paratuberculosis were in the latter stages of disease with clinical signs such as weight loss, watery diarrhea, and inappetence. Among cytokines we studied, interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, and interferon-gamma (IFN-gamma) were expressed significantly more in infected animals than in noninfected control animals. The expression of tumor necrosis factor-alpha (TNF-alpha), however, was not different between the two groups of cattle. In addition, immunohistochemical staining demonstrated that the number of resident macrophages in the ileum of infected animals was three times greater than that of noninfected cows. In contrast to this, ileal tissues from noninfected control animals contained 1.5 times more neutrophils than the ileal tissues from cows infected with M. paratuberculosis. These data demonstrate that localized ileal cytokine production is different between cows chronically infected with M. paratuberculosis and noninfected control cows.     

Differential expression of CD5 on B lymphocytes in cattle infected with Mycobacterium avium subsp. paratuberculosis

CD5 is a cell surface molecule involved in antigen recognition and is present on all T lymphocytes and a subset of B lymphocytes. The purpose of this study was to examine CD5+ expression on peripheral blood B cells from healthy, noninfected cattle and cattle with subclinical and clinical paratuberculosis. Peripheral blood mononuclear cells (PBMC) were freshly isolated or cultured for 7 days in the presence or absence of live Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis), and then analyzed by flow cytometry for CD5 expression within the B cell subpopulation. Analysis demonstrated a significant increase (P<0.01) in B cells in clinical animals as compared to healthy control cows and subclinically infected cows. In addition, three subpopulations within the CD5+ B cell population were identified: CD5dim, CD5bright, and a minor population that was characterized as CD5extra bright. A decrease in the CD5dim B cell population along with a concomitant increase in CD5bright B cells was observed in infected cows, an effect that was highly significant (P<0.01) for subclinically infected cows in cultured PBMC. In vitro infection with live M. avium subsp. paratuberculosis did not affect CD5+ expression patterns on B cells, regardless of animal infection status. Addition of exogenous IL-10 to PBMC cultures resulted in decreased numbers of CD5(bright) B cells for healthy control cows, whereas, a synergistic effect of IL-10 and infection with live M. avium subsp. paratuberculosis resulted in increased CD5bright B cells for subclinically infected cows. These results suggest that differential expression of CD5bright and CD5dim subpopulations on B cells in animals with paratuberculosis may reflect a shift in host immunity during the disease process.     

Analysis by ELISA and Western blotting of antibody reactivities in cattle infected with Mycobacterium paratuberculosis after absorption of serum with M phlei

Preabsorption of cattle serum with Mycobacterium phlei was of value in eliminating falsely positive reactions in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against M paratuberculosis. Specific antibody titres from 16 animals naturally infected with M paratuberculosis were unaffected by absorption. Analysis by Western blotting indicated that a different set of antigens of M paratuberculosis were recognised by serum from falsely positive reactors compared with that from animals with established infection. After experimental infection the time required for seroconversion in the ELISA in nine calves lay between 10 and 28 months, although one animal had not seroconverted after 30 months when the experiment ended. All animals shed M paratuberculosis in their faeces before seroconversion.     

Specific antibody responses to Mycobacterium bovis in infected cattle analysed with six mycobacterial antigens in enzyme-linked immunosorbent assays

A total of 23 (15.3 per cent) of 150 cattle infected with Mycobacterium bovis and which had never been tuberculin tested showed specific antibody responses to M bovis. Their sera may be important keys to the identification of unique M bovis antigens for use in specific serodiagnostic tests. Assessment of specific and non-specific responses was done by screening sera in six indirect anti-IgG enzyme-linked immunosorbent assays using whole cell sonicates of M bovis and five members of the Mycobacterium avium-intracellulare-scrofulaceum complex as respective antigens. Sera from 16 infected cattle that had been tuberculin tested positive and nine uninfected cattle (never tuberculin tested) were also assayed for specific and non-specific responses. Three other findings emerged. First, 43 of the 150 infected animals (28.7 per cent) showed no antibody responses to any of the mycobacterial antigens used. Secondly, the cattle showing the highest antibody levels were associated with the greatest cross reactivity. Lastly, the results indicated that tuberculin injections may increase antibody responses to shared, rather than specific, M bovis antigens in infected cattle.     

Identification of bovine T cell stimulatory antigens using a cosmid library of Mycobacterium bovis in Mycobacterium smegmatis

Culture filtrates derived from a Mycobacterium bovis cosmid library in Mycobacterium smegmatis were screened for T cell antigens. Recognition and reactivity were measured by the levels of lymphocyte proliferation and the levels of gamma interferon (IFN-gamma) produced when the culture filtrates were incubated with peripheral blood mononuclear cells (PBMC) taken from cattle immunised with M. bovis BCG. The screening system was optimised to distinguish between M. bovis secreted antigens and normal M. smegmatis secreted proteins. From ten culture filtrates screened, two were identified that induced lymphocyte proliferation and IFN-gamma production. Analysis of the DNA inserts from the recombinant cosmids suggest that they may code for different proteins. The results demonstrate that screening recombinant M. smegmatis culture filtrates can be used to identify M. bovis T cell antigens that are recognised by immunised cattle. These antigens may be important for the development of vaccines with protective ability against bovine tuberculosis.     

Polymerase chain reaction analysis of TNF-alpha and IL-6 mRNA levels in whole blood from cattle naturally or experimentally infected with Mycobacterium paratuberculosis.

Johne's disease is characterized by a chronic enteritis that results in granulomatous inflammation, cachexia, and eventual death of cattle infected with Mycobacterium paratuberculosis. The cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) have been associated with granuloma formation and wasting in other disease syndromes. The potential role of these cytokines in the development and progression of Johne's disease has not been investigated. Using the polymerase chain reaction (PCR) and specific bovine oligonucleotide cytokine primers and probes, we examined the expression of messenger RNA for these cytokines in whole blood from M. paratuberculosis infected and uninfected cattle. Cytokine mRNA levels were examined before and after in vitro incubation with E.coli lipopolysaccharide (LPS) and lipoarabinomannan (LAM) purified from M. paratuberculosis. Uninfected calves, experimentally infected calves, and naturally infected cattle all displayed similar cytokine mRNA expression patterns. However, individual animals demonstrated variability in the levels of IL-6 and TNF-alpha mRNA expression as determined by a semiquantitative PCR method using 32P-labelled oligonucleotide probes.     

Cytokine secretion by peripheral blood mononuclear cells from cows infected with Mycobacterium paratuberculosis

OBJECTIVE: To compare cytokine secretion patterns of peripheral blood mononuclear cells (PBMC) from healthy cows and cows subclinically and clinically infected with Mycobacterium paratuberculosis. ANIMALS: 5 noninfected cows, 6 cows with subclinical paratuberculosis, and 4 cows with clinical paratuberculosis. PROCEDURE: PBMC were isolated, and concentrations or activities of secreted interleukin (IL)-1, IL-2, IL-6, tumor necrosis factor (TNF), and interferon-gamma (IFN-gamma) were measured after in vitro stimulation of cells with concanavalin A (ConA), lipopolysaccharide (LPS), or a whole-cell sonicate of M paratuberculosis (MpS). Proliferative responses of PBMC were also determined after stimulation with ConA, phytohemagglutinin, pokeweed mitogen (PWM), or MpS. RESULTS: After stimulation with ConA, cells from subclinically infected cows secreted significantly more, and cells from clinically infected cows secreted significantly less, IFN-gamma, compared with cells from control cows. Cells from cows with subclinical paratuberculosis produced significantly more TNF and IFN-gamma in response to MpS than cells from the other 2 groups. Stimulation of PBMC from subclinically infected cows with ConA or MpS resulted in significantly higher proliferative responses, compared with cells from control and clinically infected cows. In contrast, clinically infected cows had significantly higher proliferative responses to PWM than cells from the other 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE: A decrease in T-cell responses to mitogens or MpS was observed in cows clinically infected with M paratuberculosis, compared with subclinically infected cows, suggesting that activated T cells may delay the progression of paratuberculosis.     

Phytomitogen- and antigen-induced blast transformation of feline lymphocytes.

A semiautomated microassay of blast transformation of feline lymphocytes was developed and used in a study of the response of normal cats to phytomitogen- and antigen-induced stimulation. Peripheral blood lymphocytes were isolated from defibrinated blood by ficolldiatrizoate gradient centrifugation and incubated at a concentration of 1 X 10(5) cells/culture. Blast transformation was measured by the incorporation of tritiated thymidine, to which the cells were exposed during the last 18 hours of incubation. Phytomitogens induced maximal transformation after 3 days' incubation. Greatest stimulation produced by 10 mug of concanavalin A (38- to 183-fold) followed by 4 mug of pokeweek mitogen (nine- to 51-fold), and finally phytohemagglutinin-P (three- to sixfold). Three of 4 cats sensitized to keyhole limpet hemocyanin responded when exposed to 50 mug of keyhole limpet hemocyanin in vitro, maximal transformation occurring after 4 days' incubation. Lymphocyte blast tranformation provides a convenient technique to assess lymphocyte function serially during experimentally induced immunologic alterations in the cat.     

Immunological responses of Eurasian badgers (Meles meles) vaccinated with Mycobacterium bovis BCG (bacillus calmette guerin)

Wildlife species, such as the badger (Meles meles), may act as maintenance hosts for Mycobacterium bovis and contribute to the spread and persistence of tuberculosis in associated cattle populations. Targeted vaccination of badgers against tuberculosis is an option that, if successfully employed, could directly facilitate the advancement of bovine tuberculosis eradication in affected areas. In this study, the immunological responses of a group of badgers vaccinated subcutaneously with low doses of Mycobacterium bovis bacillus calmette guerin (BCG) were measured in vitro and compared with non-vaccinated control animals over a period of 42 weeks. Peripheral blood mononuclear cells (PBMC) from badgers which had received repeated booster injections of BCG proliferated in response to culture with PPD-bovine (purified protein derivative of tuberculin). The proliferation was significantly greater than that seen in the non-vaccinated control group. In contrast, the proliferative response of PBMC from vaccinated badgers to PPD-avian declined relative to the control group. These results demonstrate that repeated vaccination of badgers with M. bovis BCG induced a population of T-lymphocytes responsive to specific antigens in PPD-bovine. Throughout the course of the study, the sera from all animals were tested (BrockTest) by an enzyme-linked immunosorbent assay (ELISA) system for the presence of antibodies to MPB83, a serodominant antigen whose expression is high in M. bovis, but very low in BCG (Pasteur). No animals at any stage showed seroconversion to the antigen, consistent with the tuberculosis-free status of the badgers under study.     

The specificity of antibody response in experimental and natural bovine paratuberculosis studied by crossed immunoelectrophoresis with intermediate gel

The sonicate antigen (MPS) of a local strain (IVRI) of Mycobacterium paratuberculosis and a commercial lysate of Strain 18 were analysed using hyperimmune rabbit and calf antisera to MPS in crossed immunoelectrophoresis with intermediate gel (CIE-ig) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The rabbit antiserum was more potent than the calf antiserum and it precipitated 35 and 15 antigens, respectively, among MPS and lysate antigens. SDS-PAGE resolved 50 and 32 peptides among these antigens respectively, of which, 35 and 15 were precipitated by rabbit antiserum. A CIE-ig reference system, with 30 MPS antigens, was standardized and used to analyse antibody specificities among sera derived from animals experimentally and naturally infected with bovine paratuberculosis. Fourteen antigens of MPS were found to be reactive with these sera and among these, Antigens 2 and 5 were found to be serodominant; sonicate antigens of M. bovis BCG and M. avium did not contain these antigens. Both were high molecular weight (greater than 60 kDa) antigens which may be of serodiagnostic value.     

Mucosal immune response in cattle with subclinical Johne's disease

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease, a chronic granulomatous enteritis of wild and domestic ruminants. During a long subclinical period, the organism persists in the intestine despite systemic cellular and humoral immune responses. To explore the mucosal immune response in Johne's disease, we isolated mononuclear leukocytes from the ileum of cows naturally infected with M. avium subsp. paratuberculosis and from cows that were not infected. We evaluated the immunophenotype of these cells and the proliferative responses after the addition of M. avium subsp. paratuberculosis sonicate or B-cell or T-cell mitogens. Although the percentage of T cells was increased in infected cows, these cells consisted mostly of memory (CD2+CD62L-) and regulatory (CD4+CD25+) T cells. Further evidence of immune hyporesponsiveness included a decrease in the percentage of T cells with an activated phenotype and a decrease in cells expressing major histocompatibility factor class II (MHC class II). Unlike the spleen, ileal lymphocytes from infected cows failed to proliferate in response to M. avium subsp. paratuberculosis sonicate. Additionally, ileal lymphocytes from infected cows proliferated poorly in response to concanavalin A and pokeweed mitogen, suggesting generalized T cell and B cell hyporesponsiveness. These results indicate that a state of tolerance may exist in the intestine of cows subclinically infected with M. avium subsp. paratuberculosis organisms in subclinically infected cows. This effect may be induced, at least in part, by proliferation of regulatory T cells that nonspecifically suppress mucosal immune responsiveness.     

Assessment of diagnostic tools for eradication of bovine tuberculosis in cattle co-infected with Mycobacterium bovis and M. avium subsp. paratuberculosis

The intradermal tuberculin (IDTB) test and the interferon-gamma (IFN-gamma) assay are used worldwide for detection of bovine tuberculosis in cattle, but little is known about the effect of co-infecting agents on the performance of these diagnostic tests. This report describes a field trial conducted in a cattle herd with dual infection (bovine tuberculosis and paratuberculosis) during 3.5 years. It has been based on a strategic approach encompassing serial parallel testing (comparative IDTB test, the IFN-gamma assay and serology of paratuberculosis) that was repeated 8 times over the period, and segregation of animals into two herds. The IDTB test detected 65.2% and the IFN-gamma test detected 69.6% of the Mycobacterium bovis culture-positive cattle. However, the IDTB test performed better during the first part of the trial, while the IFN-gamma test was the only method that detected infected animals during the following three samplings. The number of false positive reactors with the IDTB and/or the IFN-gamma tests was remarkably high compared to other reports, and could be caused by cross-reactivity with M. avium subsp. paratuberculosis. Also, the M. bovis isolates from cattle and wildlife from the same property were characterised using molecular techniques to disclose an epidemiological link. The IDTB test may not be appropriate to eradicate bovine tuberculosis in herds with dual mycobacterial infections. This report highlights the need to use several diagnostic techniques for the accurate detection of M. bovis infected animals in these herds.     

Heat-shock protein-specific T-cell responses in various stages of bovine paratuberculosis.

Bovine paratuberculosis is characterized by a chronic inflammation of the small intestine, caused by infection with Mycobacterium avium ssp. paratuberculosis. Research regarding diagnostic as well as immunopathogenic aspects of paratuberculosis are hampered by the lack of specific antigens. The aim of the present study was to evaluate the potential of mycobacterial heat-shock proteins, as specific antigens, to measure cell-mediated immune responses during various stages of the disease. In a cross-sectional study, peripheral blood mononuclear cells of 179 cows in different stages of M. avium ssp. paratuberculosis infection, vaccinated against paratuberculosis or noninfected, were used to evaluate lymphoproliferative responses to mycobacterial heat-shock protein of 70 kD (HSP70) and 65 kD (HSP65). In addition, lymphoproliferative responses were measured using purified protein derivate (PPD) preparations from M. avium ssp. paratuberculosis, M. avium and M. bovis as antigens. Responses to HSP70 were higher in the vaccinated animals and in asymptomatic animals that shed the organism in their faeces. Compared with these animals, responses were lower in cows with clinical signs of paratuberculosis. Mycobacterial HSP65 induced less prominent responses compared with HSP70, but showed a similar pattern with regard to the stages of disease. Vaccinated and shedding animals also showed the highest responses to PPD derived from M. avium ssp. paratuberculosis (PPD-P). Observations with short-term cell lines raised to PPD-P and to HSP70 indicated that the similarity between those two antigens was not due to the presence of HSP70 in PPD-P. In conclusion, our study indicated that, as for PPD antigens the mycobacterial heat-shock protein-specific cell-mediated immune responses decrease when comparing the asymptomatic stage to the clinical stage in bovine paratuberculosis. Furthermore, this study shows that HSP70, being a well-defined antigen in comparison with PPD antigens, can be used to monitor cell-mediated immune responses in studies regarding the immunopathogenesis of bovine paratuberculosis.     

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.     

Time delay, temperature effects and assessment of positive controls on whole blood for the gamma interferon ELISA to detect paratuberculosis

Our objective was to evaluate the effects of time and temperature on whole blood used in the gamma interferon enzyme-linked immunosorbent assay (IFN-gamma ELISA) for paratuberculosis along with evaluating four potential positive controls, and four different mycobacterial antigens for the ELISA. Nine adult Holstein cattle naturally infected with Mycobacterium avium ssp. paratuberculosis were used in a randomized complete block design. Forty-nine blood tubes were collected from each animal and held at 48.9, 37.8, 26.7, 21.1, 15.6 and 4.4 degrees C for 0, 4, 8, 12, 18, 24, 32, 48 and 72 h. Each blood tube was tested with four mycobacterial antigens (two johnin PPDs, an avain PPD and a whole cell sonicate) and four potential positive controls [concanavalin A (conA), phytohaemagglutinin A (PHA), pokeweed mitogen (PWM) and Staphylococcus enterotoxin A (SEA)]. After incubation for 24 h, the plasma was assayed with a commercial IFN-gamma ELISA. Blood stored at 21.1 and 15.6 degrees C maintained the highest ELISA optical densities (OD) over time with severe reduction in OD values at or above 37.8 degrees C. None of the potential positive controls exactly mimicked the antigen response. SEA and PWM were able to elicit a response after the whole blood quit responding to the antigen and conA underestimated the responsiveness. Phytohemagglutinin A was similar to the antigens on an average, but there was significant disagreement among samples. The PPDs were more potent at stimulating IFN-gamma production than the whole cell sonicate. In conclusion, whole blood should be stored/transported at ambient room temperature and stimulated within 12 h of collection.