The effect of inhaled inactived Mycobacterium phlei as a treatment for asthma.

Allergic asthma is a chronic airway disorder characterized by airway inflammation, mucus hypersecretion, and airway hyperresponsiveness (AHR). A murine model of asthma was used to examine the antiasthmatic effect of inhaled inactived Mycobacterium phlei (M. phlei). AHR, neutrophil levels, eosinophil levels and levels of interleukin (IL)­17 and IL­23 receptor (IL­23R) were monitored. The results demonstrated that inactivated M. phlei alleviates the IL­17+γδT cell­mediated immune response and attenuates airway inflammation and airway hyperresponsiveness in the asthmatic murine lung, partially through inhibiting the expression of IL­23R. In conclusion, inactivated M. phlei may be an effective antiasthmatic treatment, regulating IL­17­producing Î³Î´T (IL­17+γδT) cell­mediated airway inflammation and airway hyperresponsiveness to relieve the symptoms of mice with asthma.

Inhalation of inactivated­Mycobacterium phlei prevents asthma­mediated airway hyperresponsiveness and airway eosinophilia in mice by reducing IL­5 and IL­13 levels.

The present study aimed to investigate whether inhalation of inactivated­Mycobacterium phlei could prevent airway hyperresponsiveness and airway eosinophilia. A total of 24 male Balb/c mice were randomly divided into three groups: Normal control group (group A), asthma model group (group B) and the intervention group (group C), (8 mice/group). Group A mice were sensitized and with challenged saline and group B with ovalbumin (OVA). Group C mice were administered with aerosol Mycobacterium phlei once daily prior to the allergen challenge. Airway responsiveness in each group was assessed. All the animals were sacrificed and lung tissues, blood samples and bronchoalveolar lavage fluid (BALF) were harvested. Cell fractionation and differential cells were counted in serum and BALF. HE staining and alcian blue/periodic acid Schiff staining were used to measure airway eosinophilic inflammation and mucus production. The levels of the cytokines IL­5, IL­13 and IgE were measured in lung and BALF as determined by ELISA and reverse transcription­quantitative polymerase chain reaction assays. The results indicated that inactivated­Mycobacterium phlei suppressed the airway hyperresponsiveness and mitigated airway eosinophilia induced by a methacholine challenge, and significantly reduced the levels of cytokines IL­5 and IL­13 in lung tissue and IgE level in BALF when compared with the OVA­sensitized mice. In conclusion, inhalation of inactivated­Mycobacterium phlei could reduce OVA­induced airway hyperresponsiveness and may be a potential alternative therapy for allergic airway diseases.

[Effect of Mycobacterium phlei F.U.36 on balance of CD4⁺CD25⁺ regulatory T cells and Th17 cells in asthmatic mice].

OBJECTIVE: To evaluate the effect of early intervention with Mycobacterium phlei F.U.36 injection on the balance of CD4⁺CD25⁺ regulatory T cells and Th17 cells in asthmatic mice, and to investigate the immunomodulatory effect of Mycobacterium phlei F.U.36. METHODS: Thirty female BALB/c mice were randomly divided into three groups: normal control (n=10), asthma model (n=10) and Mycobacterium phlei F.U.36 treatment groups (n=10). A mouse model of asthma was prepared by injection and aerosol inhalation of chicken ovalbumin in the asthma model and Mycobacterium phlei F.U.36 treatment groups, while mice in the normal control group were given normal saline instead. The treatment group was intraperitoneally injected with Mycobacterium phlei F.U.36 (0.57 µg, once every other day) three times in the first two weeks after the first sensitization. All mice were sacrificed at 24 hours after the last challenge. Left lung tissues of these mice were obtained and made into sections for observation of inflammatory changes. The percentages of CD4⁺CD25⁺ regulatory T cells and Th17 cells in CD4⁺ T cells among splenic mononuclear cells were determined by flow cytometry. The levels of interleukin (IL)-10 and IL-17 in serum and bronchoalveolar lavage fluid were measured using ELISA. RESULTS: Compared with the normal control group, the asthma model group had significantly decreased percentages of CD4⁺CD25⁺ regulatory T cells and IL-10 levels (P<0.05) and significantly increased percentages of Th17 cells and IL-17 levels (P<0.05). Compared with the asthma model group, the Mycobacterium phlei F.U.36 treatment group had significantly increased percentages of CD4⁺CD25⁺ regulatory T cells and IL-10 levels (P<0.05) and significantly decreased percentage of Th17 cells and IL-17 levels (P<0.05). CONCLUSIONS: Early intervention with Mycobacterium phlei F.U.36 can promote development of CD4⁺CD25⁺ regulatory T cells and production of IL-10 and inhibit generation of Th17 cells and production of IL-17 in asthmatic mice.

Effect of inhaled inactivated Mycobacterium phlei in children with moderate asthma.

Bacillus Calmette-Guérin and other mycobacterial vaccines are important therapeutic methods in a series of chronic inflammatory disorders characterized by Th1/Th2 imbalance in which Th2 type cells and cytokines often increase. However, few studies have investigated whether it can reduce or prevent the symptoms and attacks in children with asthma. This study evaluated the effect of inactivated Mycobacterium phlei inhaled by an atomizing device placed on asthmatic children. In this randomized, single-center, Seretide-controlled study, children aged 4-12 years with newly diagnosed, moderate, persistent asthma were treated with either inhaled inactivated M. phlei or inhaled Seretide patch. The efficacy of inhaled inactivated M. phlei was related with the alleviation of asthma symptoms, improvement of lung function and reduction of bronchial hyper-responsiveness and total serum IgE, which was similar with Seretide. These findings may have important clinical value in confirming inhaled inactivated M. phlei as a new therapeutic method in moderately asthmatic children.

Inhaled inactivated-Mycobacterium phlei modulates γδT cell function and alleviates airway inflammation in a mouse model of asthma.

BACKGROUND: Mycobacterium bovis Bacille Calmette-Guérin (BCG) and other mycobacterial infections suppress airway hyperresponsiveness and eosinophilic inflammation in asthma.γδT cells are important modulators of airway function and allergic inflammation. Vγ1+γδT cells increase eosinophilic airway inflammation and airway hyperresponsiveness, while Vγ4+γδT reduce airway hyperresponsiveness. The objective of this investigation was to determine the role of γδT cells and its subsets in the treatment of inhaled inactivated-Mycobacterium phlei for asthma. METHODS: OVA-sensitised mice were treated with aerosol Mycobacterium phlei after OVA challenge. Pathological HE staining was performed to measure lung inflammation, flow cytometric analysis was used to assess the intracellular cytokines of each γδT cell, and quantitative real-time PCR was performed to assess Vγ1 mRNA and Vγ4 mRNA expression. RESULTS: Airway inflammation was attenuated by treatment with inhaled inactivated-Mycobacterium phlei. IL-10+γδT cells, IFN-γ+γδT cells,Vγ4 mRNA expression were significantly increased. CONCLUSIONS: Our results indicate that inhalation of Mycobacterium phlei can modulate γδT cell function, the proportion of different γδT cell subsets and reduce airway inflammation in asthmatic mice.

Low levels of residual oil fly ash (ROFA) impair innate immune response against environmental mycobacteria infection in vitro.

Epidemiological studies have shown that pollution derived from industrial and vehicular transportation provokes adverse health effects causing broad spectrum of ambient respiratory diseases. Therefore, air pollution should be taken into account when microbial diseases are evaluated. Environmental mycobacteria (EM) are opportunist pathogens in a variety of immunocompromised patients eliciting significant impact on human morbidity and mortality. The aim of this study was to evaluate the in vitro effects of residual oil fly ash (ROFA) on the alveolar macrophages (AMs) response to opportunistic bacteria. AMs from young Wistar rats were obtained by bronchoalveolar lavage and co-cultured with Mycobacterium phlei (MOI 10). We exposed AM cultures to ROFA to characterize the effect of low ROFA concentrations (0, 2.5, and 5µg/ml) and evaluated the response of pre-exposed AM against the bacilli. Low ROFA concentrations induced superoxide anion and nitrites production (p<0.001). Pre-exposure to ROFA (2.5 and 5µg/ml) caused a significant reduction on TNFα (p<0.001) and superoxide anion (p<0.001) production but, did not modify the nitrite production when AM were co-cultured with M. phlei. In addition, ROFA significantly diminished AM killing ability in culture (p<0.001). Hence, our results indicate that pre-exposure to low levels of ROFA modifies the innate pulmonary defence mechanisms against environmental mycobacteria.

Effects of inhaled inactivated Mycobacterium phlei on airway inflammation in mouse asthmatic models.

BACKGROUND: Corticosteroids are the most efficacious anti-inflammatory drugs for asthma therapy; however, steroids are not always completedly effective for asthma. Studies have shown Mycobacterium bovis Bacille Calmette-Guérin (BCG) and other mycobacterial infections suppress airway hyperresponsiveness and inflammation in asthma. We use a murine model of Ovalbumin (OVA)-induced asthma to study whether nebulized inhalation of inactivated Mycobacterium phlei can alleviate asthmatic airway inflammation through influencing cytokine production and determine whether it can prevent and treat asthma. METHODS: Fifth male Balb/c mice were randomly divided into four groups: normal control group (A), asthma model group (B0, B3, B4, B5), the treatment group (C0, C3, C4, C5), and prevention group (D). Mice were sensitizated and challenged with Ovalbumin to make a murine asthma model. Group C were given treatment of aerosol Mycobacterium phlei once daily after OVA challenge. Groups C3, C4, and C5 were treated for 3 days, 4 days, and 5 days, respectively. Group D inhaled the solution of inactivated Mycobacterium phlei daily before each time of OVA challenge. All the animals were killed and lung tissue and bronchoalveolar lavage fluid (BALF) were harvested. Pathological HE staining and AB-PAS staining were done to measure lung inflammation and mucus production. Total cell numbers and differential cell count in BALF were performed. Cytokines IL-4, IL-10, and IFN-γ levels in BALF were quantified by ELISA. RESULTS: In groups C4, C5, and D, IL-4 production in BALF was decreased and IL-10 and IFN-γ were increased (p<0.05).The number of total inflammatory cells and the mean percentage of eosinophils and lymphocytes in the BALF of group D, group C4, and group C5 was lower than in the corresponding group B (p<0.05). Histological examination of the lungs showed airway inflammation of group D and group C5 were attenuated. CONCLUSION: The inhalation of Mycobacterium phlei can reduce airway inflammation in asthmatic mice. This ability was associated with its immunomodulatory effect on regulating IL-4, IL-10, and IFN-γ secretion. Aerosol administration of inactivated Mycobacterium phlei may be accepted as an alternative method with less risk of adverse reactions in treatment of asthma.

Evolution of intravesical immunotherapy for bladder cancer: mycobacterial cell wall preparation as a promising agent.

BACKGROUND: The intra-cavitary administration of antineoplastic agents for the treatment of non-invasive bladder cancer has met with variable results. Mytomicin-C [corrected] is effective in the prevention of tumor recurrence when administered in the immediate post-resection period, but also exhibits activity against papillary tumors. It lacks efficacy in carcinoma in-situ (CIS) of the bladder. Bacillus Calmette-Guérin (BCG) has been shown to be effective against papillary tumors, but particularly in the treatment of CIS. Unfortunately, live BCG has serious safety limitations. OBJECTIVE: To review the current situation with the use of non-viable preparations of mycobacteria (M. phlei) that have been investigated for the treatment of superficial bladder cancer in limited open-label clinical trials. CONCLUSION: MCC (Urocidin) [corrected] has shown activity against a variety of tumor cells, both in vitro and in animal cancer models. Limited clinical trials have also shown it to be active against non-muscle invasive bladder tumors in patients who have previously failed one or more courses of chemotherapy and/or immunotherapy with BCG. An unique dual immunomodulatory and apoptotic mechanism of action has been proposed for MCC.

[Inhibitory effect of combined bacterin on growth of sarcoma 180 in mice].

BACKGROUND & OBJECTIVE: It was reported that the symptoms of tumor patients may be alleviated markedly and even the tumor may be regressed completely after acute infection. Bacterin OK-432 has notable inhibitory effect on the growth of various tumors in animals. At present, OK-432 has been used in clinical immunotherapy for tumors with no other adverse events besides fever and leucocytosis. This study was to investigate the effects of combined bacterin on the serum level of interleukin 12(p70)[IL-12(p70)] and the growth of sarcoma 180 (S180) in mice. METHODS: After transplantation of S180, the mice were randomized into 5 groups, and received injection of Staphylococcus aureus, Salmonella typhimurium, Mycobacteria phlei, and combined bacterin containing the 3 bacteria strains, respectively, or received no treatment (blank control). The weight of S180 xenografts, the thymus, and the spleen in mice was measured. The serum level of IL-12(p70) was detected by ELISA. RESULTS: The mean weight of S180 tumors was 1.39 g in Staphylococcus aureus group, 1.50 g in Salmonella typhimurium group, 1.36 g in Mycobacteria phlei group, 0.62 g in combined bacterin group, and 2.40 g in blank control group; the differences among the 5 groups were significant (F=66.73, P<0.001). The mean weight of S180 tumors was significantly lower in the 4 bacterin groups than in control group, and significantly lower in combined bacterin group than in the 3 single bacterin groups (q test, P<0.001). The weights of the thymus and the spleen among the 5 groups had no significant difference (F=2.36, P>0.05; F=1.89, P>0.05). The inhibition rate of tumor growth was significantly higher in combined bacterin group than in Staphylococcus aureus, Salmonella typhimurium, and Mycobacteria phlei groups (74.17% vs. 42.08%, 37.50%, and 43.33%, P<0.01). The mean serum level of IL-12(p70) was 19.44 pg/ml in combined bacterin group, 12.41 pg/ml in Staphylococcus aureus group, 10.35 pg/ml in Salmonella typhimurium group, 11.68 pg/ml in Mycobacteria phlei group, and 4.45 pg/ml in control group; the difference among the 5 groups was significant (F=15.76, P<0.0001), but the difference among the 3 single bacterin groups was not significant (q test, P>0.05), while the differences between other groups were significant (q test, P<0.01). CONCLUSIONS: The chosen bacterins in this study can induce the mice to produce IL-12(p70) and suppress the growth of S180. The effect of the combined bacterin is much better than the single bacterins.

The activity of milk leukocytes in response to a water-soluble fraction of Mycobacterium phlei in bovine subclinical mastitis.

The effect of a water-soluble fraction (WSF) of a non-pathogenic strain of Mycobacterium phlei was studied in bovine subclinical mastitis (SCM) by measuring the myeloperoxidase and acid phosphatase enzyme levels in the milk leukocytes. Forty-five cows were divided into three equal groups. Group I, consisting of 15 healthy cows, served as the control, whereas groups II and III each contained 15 cows with subclinical mastitis on the basis of a positive reaction in the California mastitis test (CMT). The cows in group II received 100 microg of WSF in 5 ml sterile phosphate-buffered saline, pH 7.4 (PBS) once only, while those in group III received 5 ml sterile PBS daily for 7 days, both treatments being given by the intramammary route. Observations were made up to 30 days after treatment (AT). The CMT of the healthy milk was negative (0), whereas it ranged between 1 and 2 points in SCM. The somatic cell count (SCC) increased significantly (p < 0.05) on day 3, then fell steeply from day 7 up to day 30 AT in the cows in group II. A steady decrease in the total bacterial count (TBC) was observed in the group treated with WSF but the bacterial counts remained high in the groups treated with PBS. The mean acid phosphatase level was enhanced by 119% on day 3 AT in group II but only by 18.7% in the cows in group III. The mean myeloperoxidase level was enhanced by 100% in the cows in group II but only by 18% in those in group III on day 3 AT. This significant reduction in the bacterial load in infected cows caused by intramammary infusion of WSF may be due to activation of the microbicidal activity of the neutrophils, but this requires confirmation.

Evaluation of immune responses of cattle as a means to identify high or low responders and use of a human microarray to differentiate gene expression.

An immune response (IR) index to identify cows with high (H) and low (L) antibody-mediated immune responses (AMIR) had been previously devised. High AMIR associated with decreased mastitis and improved response to vaccination. Measurement of cell-mediated immune response (CMIR) was not included in the index; therefore various antigen/adjuvant combinations were evaluated as inducers of DTH to be added to the IR-index. The Bacillus Calmette Guérin (BCG)-induced/purified protein derivative (PPD)-elicited tuberculin skin test is a reliable measure of DTH; however, its use to identify livestock with high CMIR may be confounded due to previous exposure to Mycobacteria tuberculosis. DTH to BCG/PPD was therefore compared with that induced by Mycobacteria phlei (saprophyte) and its derivative phlein as the test antigen. Antibody to OVA was also evaluated. The results indicated that BCG/PPD and M. phlei/phlein induced similar DTH, but cross reaction to PPD was evident following induction of DTH using M. phlei making it a less than ideal alternative for testing livestock. Nonetheless, cows could be ranked for both AMIR and CMIR. RNA from two cows with the highest and lowest IR ranks was then used to probe a human 1.7 kD microarray to determine the ability of a human array to provide information on bovine genes associated with H and L.

Development of short non-CpG phosphodiester oligonucleotides as immune stimulatory agents.

We have previously reported that DNA isolated from Mycobacterium phlei (M. phlei) stimulates the synthesis of cytokines by monocytes and macrophages independently of the presence of unmethylated CpG motifs. Oligonucleotides as small as five to six bases isolated from M. phlei DNA have been found to induce cytokine synthesis. In the present study, we have investigated the potential for such CpG-lacking DNA to act as an immune stimulant. A series of six base length phosphodiester oligonucleotides derived from the genome of M. phlei were synthesised and tested for their ability to induce the synthesis of cytokines by murine, non-human primate (rhesus macaques and chimpanzee) and human peripheral blood mononuclear cells. The results show that phosphodiester oligonucleotides with a 5'GGGxGG3' sequence where x is A, C, G or T have the ability to induce the synthesis of IL-1beta, IL-6, IL10 or IL-12 by non-human primate and human PBMC, murine cells being unresponsive. The phosphodiester 5'GGGxGG3' oligonucleotides were shown to be stable in human serum, with a half-life of approximately 72 h. The addition of aluminium hydroxide to these 5'GGGxGG3' oligonucleotides potentiated, in a concentration-dependent manner, the synthesis of IL-12 by human peripheral blood mononuclear cells. These phosphodiester six base length non-CpG motif oligonucleotides may have potential as immunopotentiators for vaccines.

IFN-gamma diagnostic tests in the context of bovine mycobacterial infections in Belgium.

In countries where cattle tuberculosis caused by Mycobacterium bovis (Mbov) and paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (Mptb) are present, testing strategies for the Mbov eradication have to discriminate between these two infections. Present indirect tests are based on the analysis of the specific cellular immune response (DTH, IFN-gamma) against crude mycobacterial antigens (avian and bovine PPD). In this study, we compared the evolution of the IFN-gamma responses of animals experimentally infected with Mbov, Mptb, or inoculated with Mycobacterium phlei. Mbov inoculation induced a strong IFN-gamma response that allows rapid classification of the status of the animals following interpretation criteria set up by us. Experimental inoculation with M. phlei induced sensitisation to mycobacterial antigens as detected by the IFN-gamma test but these reactions were of short duration, therefore, repeated testing allows us to define these animals as aspecific reactors. IFN-gamma response induced after oral inoculation of calves with Mptb was of low intensity and ratio of responses measured against avian versus bovine PPD did not allow a clear diagnostic at least for the six first month of infection.

Mycobacterium phlei as an oral immunomodulator with Newcastle disease vaccine.

Experiments were conducted in chickens to understand the effects of oral immunomodulation. Heat inactivated M phlei, a commensal Mycobacterium and a non-specific immunomodulator, was administered orally prior to live Newcastle disease F (ND F) strain vaccination. In experimental birds it lead to an enhanced cell mediated Immune response (CMI) against the vaccine. There was a reduction in the Haemagglutination inhibiting (HI) antibodies. However, it did not affect the protection against a virulent challenge, as the protection percentage was more or less same in vaccinated birds irrespective of the M.phlei administration. M. phlei administration could not enhance the immune response to inactivated ND F vaccine administered orally. The results indicate that M. phlei favours a CMI response to orally administered live ND F vaccine. It may be of potential use in enhancing CMI against vaccines and a cheaper alternative to costlier recombinant cytokines.

The mannose receptor mediates uptake of pathogenic and nonpathogenic mycobacteria and bypasses bactericidal responses in human macrophages.

The mannose receptor (MR) is involved in the phagocytosis of pathogenic microorganisms. Here we investigated its role in the bactericidal functions of human monocyte-derived macrophages (MDMs), using (i) trimannoside-bovine serum albumin (BSA)-coated latex beads and zymosan as particulate ligands of the MR, and (ii) mannan and mannose-BSA as soluble ligands. We show that phagocytosis of mannosylated latex beads did not elicit the production of O2-. Zymosan, which is composed of alpha-mannan and beta-glucan, was internalized by the MR and a beta-glucan receptor, but the production of O2- was triggered only by phagocytosis through the beta-glucan receptor. Activation and translocation of Hck, a Src family tyrosine kinase located on lysosomes, has previously been used as a marker of fusion between lysosomes and phagosomes in human neutrophils. In MDMs, Hck was activated and recruited to phagosomes containing zymosan later than LAMP-1 and CD63. Phagosomes containing mannosylated latex beads fused with LAMP-1 and CD63 vesicles but not with the Hck compartment, and the kinase was not activated. We also demonstrate that the MR was unable to distinguish between nonpathogenic and pathogenic mycobacteria, as they were internalized at similar rates by this receptor, indicating that this route of entry cannot be considered as a differential determinant of the intracellular fate of mycobacteria. In conclusion, MR-dependent phagocytosis is coupled neither to the activation of NADPH oxidase nor to the maturation of phagosomes until fusion with the Hck compartment and therefore constitutes a safe portal of entry for microorganisms.

Fusion of azurophil granules with phagosomes and activation of the tyrosine kinase Hck are specifically inhibited during phagocytosis of mycobacteria by human neutrophils.

Pathogenic mycobacteria parasitize macrophages and reside within phagosomes, which do not fuse with lysosomal granules. Mycobacteria are also internalized by neutrophils, which possess at least two types of granules, specific and azurophil granules, the latter being specialized lysosomes. Here, we investigated the ability of mycobacteria to inhibit the fusion of these granules with their phagosomes in human neutrophils. It was found that when pathogenic (Mycobacterium kansasii and Mycobacterium avium) or nonpathogenic (Mycobacterium smegmatis and Mycobacterium phlei) mycobacteria were internalized by neutrophils, they induced the inhibition of azurophil granule fusion with phagosomes even when they were serum opsonized. In contrast, secretion of specific granule content and production of O2-, both of which contribute to the neutrophil bactericidal response, were triggered. Hck is a Src family tyrosine kinase associated with azurophil granules. During internalization of zymosan, azurophil granules fused with phagosomes and Hck was activated and translocated to the phagosomal membrane, whereas in neutrophils engulfing mycobacteria, Hck did not translocate and remained unactivated. The activation of the tyrosine kinase Fgr was not affected. These results indicate that 1) pathogenic and nonpathogenic mycobacteria trigger similar bactericidal responses in neutrophils, 2) phagocytosis and fusion of azurophil granules can be uncoupled by mycobacteria, and 3) Hck could be one of the key elements of the azurophil secretory pathway that are altered during phagocytosis of mycobacteria.

Effect of immunostimulation on cytotoxic activity of intestinal intraepithelial lymphocytes of chickens in infectious bursal disease and Eimeria tenella infections.

In chickens, intestinal intraepithelial lymphocytes (iIELs) exhibit spontaneous natural killer (NK) cell like activity, by which they are active in the first line of defence on gut epithelial linings. In the present study, the cytotoxic activity of iIELs was found to be drastically suppressed in chickens experimentally infected with infectious bursal disease (IBD) virus at the age of 5 weeks and also in chickens experimentally infected with Eimeria tenella oocysts at the age of 8 weeks (p < 0.01). As nonspecific immunostimulation is gaining importance, immunostimulants such as immunostimulating Mycobacterium phlei (ISMP) and bone marrow culture supernatant (BMCS) were tested for their influence on the functional activity of iIELs of chickens in IBD and E. tenella infections. In chickens primed with ISMP a week prior to respective experimental infections, it was found that the cytotoxic activity of iIELs was restored (p < 0.01) in both IBD and E. tenella infections. At the same time, in chickens primed with BMCS a week prior to respective experimental infections, the cytotoxic activity of iIELs was restored to a certain extent (p < 0.01) in E. tenella but not at all in IBD infection. These results showed that application of immunostimulation helped potentiate and restore the functional activity of iIELs of chickens in IBD and E. tenella infections.

Effect of Mycobacterium phlei on the development of immunity to Babesia bigemina.

The immunomodulatory role of Mycobacterium phlei against intracellular blood protozoan Babesia bigemina was demonstrated following experimental immunisation and challenge in bovine calves. A lysate of erythrocytes infected (6 x 10(9)) with B. bigemina was used as a source of dead antigen either with Freund's complete adjuvant (FCA) or with a trypsinised culture of M. phlei as a non-specific immunomodulation (NSI) agent with appropriate controls. Following virulent challenge with B. bigemina infected erythrocytes (1 x 10(7)), the NSI printed calves showed 100% protection, while the dead antigen alone with FCA afforded 75% protection. The protective status of the immunising regimes was studied by clinicopathological parameters and assessment of humoral and cell-mediated immune responses. The role of babesial dead antigen and the effects of M. phlei on the development of immunity to B. bigemina is discussed.

Evaluation of an enzyme-linked immunosorbent assay licensed by the USDA for use in cattle for diagnosis of ovine paratuberculosis.

A commercially available Mycobacterium phlei-absorbed enzyme-linked immunosorbent assay (ELISA) approved to detect antibodies to Mycobacterium paratuberculosis in cattle was evaluated for its applicability in sheep. The potential for interference with ELISA results from cross-reacting antibodies to Corynebacterium pseudotuberculosis was also investigated. Serum samples were randomly selected from a collection of samples obtained in 1986-1991 from 6 infected and 5 noninfected sheep flocks varying in breed, age, and geographic origin. Tests were performed on sera from 27 paratuberculous sheep, confirmed by histopathology, bacteriologic culture, and/or acid-fast staining of ileal mucosal smears, and on sera from 246 noninfected sheep. The optical density of each sample was expressed as a percentage of the optical density of a known positive sheep serum sample tested on the same plate. These values were log-transformed to achieve normality of distribution, and sensitivity and specificity estimates were calculated based on 2 and 3 standard deviations above the mean of the percent positive value (PPV) of the noninfected sheep. A cutoff value of PPV > or = 55.74 resulted in an estimated sensitivity of 0.48 and a specificity of 0.95. Sera from 10 noninfected sheep with PPV above the cutoff level of 55.74% were absorbed with heat-treated C. pseudotuberculosis organisms in addition to M. phlei antigens. Sera from 14 ELISA-positive paratuberculous sheep and 23 ELISA-negative noninfected sheep were similarly treated, and results were compared. Absorption with C. pseudotuberculosis resulted in a significant decrease in PPV in all 3 groups of sheep sera, but a greater decrease was observed in the noninfected sheep with PPV above the cutoff level when compared with noninfected sheep with PPV below that level. Results of this study suggest that ELISA may be of value in screening sheep flocks for paratuberculosis, but further experimentation is needed to optimize the sensitivity and specificity of the assay. Exposure to C. pseudotuberculosis may confound results obtained by M. phlei-absorbed ELISA for paratuberculosis.