Thermal inactivation of Mycobacterium paratuberculosis in milk

Thermal inactivation of Mycobacterium paratuberculosis, a suspected human pathogen, was determined in ultrahigh-temperature whole milk. Three strains of M. paratuberculosis were examined for survival at temperatures from 55 to 75 degrees C using a submerged glass capillary tube method. Clumped and declumped suspensions of the cultures were used to determine the rate of heat inactivation and survival at pasteurization temperatures. Methods for declumping M. paratuberculosis included the use of glass beads, vortexing, and passing the cells through a 26-gauge needle. The latter procedure was found to be superior over other methods and did not affect the viability of cells. Capillary tubes filled with milk containing 4 x 10(6) to 3 x 10(7) CFU/ml were heated at temperatures ranging from 55 to 75 degrees C. At 55 degrees C, minimal thermal inactivation was observed for clumped and declumped cells. At 58 degrees C, thermal inactivation ranging from 0.3 to 0.7 log reduction was observed for both clumped and declumped suspensions. D values at 60 degrees C ranged from 8.6 to 11 min and 8.2 to 14.1 min for clumped and declumped cells, respectively. At 63 degrees C, the D values ranged from 2.7 to 2.9 and 1.6 to 2.5 min for clumped and declumped cells, respectively. Survival of M. paratuberculosis at initial levels ranging from 44 to 10(5) CFU/ml at pasteurization treatment (63 degrees C for 30 min and 72 degrees C for 15 s) was also determined. No survivors were observed after incubating plates for up to 4 months on Middlebrook 7H11 agar and up to 2 months on Herrold's egg yolk medium. The sensitivity of the plating method was 1 CFU/250 microliters. These results demonstrate that low levels of M. paratuberculosis, as might be found in raw milk, will not survive pasteurization treatments.     

Diagnosis of paratuberculosis in dairy cattle, using enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in milk

An ELISA containing lipoarabinomannan (LAM) antigen was used to detect antibodies in milk and serum for diagnosis of Mycobacterium paratuberculosis infection in dairy cattle. In experiment 1, milk and serum samples were obtained from 25 cows, and subjected to LAM ELISA testing immediately, and after 1 year of storage at -70 C. Milk samples, with and without a commonly used chemical preservative, were tested. There was no significant difference in LAM ELISA results between fresh and frozen samples or between preserved and unpreserved milk samples. In experiment 2, milk samples were collected daily from 30 cows over a 14-day period. The day-to-day coefficient of variation was 0.19 for milk LAM ELISA and was 0.15 for serum LAM ELISA, with no statistically significant time effect detected. In experiment 3, single milk, serum, and fecal samples were obtained from 764 cows. The fecal samples were cultured for M paratuberculosis to identify infected cows, and the serum and milk samples were subjected to LAM ELISA testing. Results were compared, using the area under the receiver operating characteristic curves. The milk LAM ELISA had specificity (+/- 95% confidence limits) of 87 +/- 8.1% when the cutoff was set at 50% sensitivity, and specificity of 83 +/- 9.1% when sensitivity was set at 60%. The area under the receiver operating characteristic curve was 0.85 +/- 0.03 for the milk ELISA and 0.75 +/- 0.02 for the serum ELISA. In this population of cattle, the milk LAM ELISA had comparable accuracy to serum LAM ELISA, although the milk LAM ELISA was slightly less reproducible (higher coefficient of variation).     

Identification and characterization of a novel extracellular ferric reductase from Mycobacterium paratuberculosis

Diaspirin cross-linked hemoglobin (DCLHb) is an intramolecularly cross-linked hemoglobin-based oxygen carrier being developed as a therapy for acute blood loss. We report here the absence of immunogenicity of DCLHb in patients enrolled in phase II and III clinical trials of DCLHb. Two very sensitive immunoassays, an enzyme-linked immunosorbent assay (ELISA) and a Western blot assay, were developed and validated for this assessment. The DCLHb-antibodies used in these assays were raised in monkeys, had similar affinities for DCLHb and native human hemoglobin (SFHb), and showed cross-reactivity for subunits of DCLHb and SFHb on the Western blot, suggesting that these antibodies were elicited as a xenogenic response to the protein. In the ELISA, the optical density of a patient sample exposed to DCLHb-coated wells was compared with that of the patient sample exposed to carbonate buffer-coated wells; an optical density ratio of 1.4 was established for discriminating between a positive (reactive) or negative DCLHb antibody response. To date, all of the more than 300 patient specimens (preinfusion and postinfusion) from clinical trials have exhibited a ratio of less than 1.4, confirming the lack of preexisting antibodies to DCLHb and clearly showing the absence of DCLHb antibodies after exposure to this new biologic entity. There has been no requirement for use of the confirmatory Western blot assay. Taken together, the results from this study indicate DCLHb is not immunogenic in humans at doses evaluated clinically.     

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.     

Dietary calcium modulates Mycobacterium paratuberculosis infection in beige mice

A 6-month study was conducted to evaluate the effects of feeding different levels of dietary calcium (Ca) on the persistence of Mycobacterium paratuberculosis infection using a mouse model. Beige mice, averaging 8 weeks of age, were randomly assigned to one of the following dietary treatments: 1) 0.02% Ca, 2) 0.15% Ca, 3) 0.45% Ca, and 4) 1.0% Ca. Mice were infected intraperitoneally with 10(8) CFU viable M. paratuberculosis for 1, 3, and 6 month periods. Plasma Ca levels was unaffected by dietary Ca (x = 7.3 mg/dl). Plasma levels of 1,25(OH)2D3 was elevated significantly in 0.02% and 0.15% Ca groups compared to other treatments at the end of each period, with the highest levels observed for 0.02% Ca mice and intermediate values for 0.15% Ca mice. One month after infection, numbers of viable M. paratuberculosis cultured from the spleen were significantly reduced for 0.15% Ca mice, whereas the number of bacteria isolated from the liver and mesenteric lymph node (MLN) were higher for the 0.02% Ca group. There were no differences in bacterial numbers in the ileum although they tended to be higher for the 0.02% Ca group. Three months after infection, bacterial numbers in the spleen, ileum, and MLN did not differ across treatments, however, significantly lower numbers were found in the liver of 1.0% Ca mice. Reduced bacterial counts were also observed in the liver of 0.15%. 0.45%, and 1.0% Ca mice after a 6-month infection period compared to the 0.02% Ca group, with the lowest numbers isolated from the 1.0% Ca mice. Numbers of viable bacteria cultured from the ileum and MLN after 6 months of infection were also significantly reduced in 1.0% Ca mice. These results suggest that Ca metabolism is an important modulator of M. paratuberculosis infection.     

Isolation and biological properties of osteopontin from bovine milk

The right gastroepiploic artery has been increasingly used as a coronary bypass graft. Short- and mid-term patency rates support the supposition that the right gastroepiploic artery is a satisfactory bypass conduit. However, conclusive angiographic data on long-term patency rates are still lacking. An echo-colour Doppler method was used to detect patency of the right gastroepiploic artery grafts through an upper abdominal approach. A group of 24 patients with a right gastroepiploic artery graft to the right or posterior descending coronary artery, all of whom also had a postoperative angiographic study which showed 100% patency of the graft were used as a reference group. A second group of 89 patients was also investigated only with echo-colour Doppler during the postoperative period (mean 8.0 (range 1-48) months). A patent right gastroepiploic artery graft showed a biphasic velocity pattern. Systolic peak velocity ranged from 8 to 26 cm and diastolic peak velocity from 4 to 13 cm. The right gastroepiploic artery diameter ranged from 1.7 to 2.4 mm and flow from 10.2 to 58.8 ml. Among the second group were three patients who had, at their echo-colour Doppler examination, a possible occlusion of the right gastroepiploic artery graft; an angiographic study was conducted and the graft closure confirmed in all cases. Serial echo-colour Doppler evaluation of the right gastroepiploic artery blood flow pattern and diameter is a non-invasive and safe method to check the patency and flow capacity of the artery graft in follow-up studies.     

Detection of Mycobacterium bovis in milk by polymerase chain reaction

A simple method was developed for DNA extraction of Mycobacterium bovis in milk and further detection of the bacterium by polymerase chain reaction (PCR). Milk previously seeded with M. bovis was used as the starting material. The procedure involved overnight digestion of a milk sample with proteinase K at 56 degrees C and phenol extraction, followed by ethanol precipitation and PCR. The amplification pattern obtained was analyzed with primers BW8-BW9 which amplify a 248 bp in strains of M. bovis. By using the BW8-BW9 primers, 10(3) CFU were detected on silver-stained PAGE gels. The procedure was validated by PCR analysis of milk in tuberculin-positive animals. It is anticipated that this method can be used for routine diagnosis of M. bovis in milk samples.     

Differentiation of Australian isolates of Mycobacterium paratuberculosis using pulsed-field gel electrophoresis

OBJECTIVES: To examine strain variation amongst Australian isolates of Mycobacterium paratuberculosis. DESIGN: Pulsed field gel electrophoresis was optimised for differentiation of M paratuberculosis strains, and this typing technique was then applied to a collection of Australian isolates. PROCEDURE: DNAs from 35 Australian isolates of M paratuberculosis and a UK reference strain were digested with one or other of three restriction endonucleases. The banding patterns obtained after pulsed field gel electrophoresis of the DNA fragments were compared. RESULTS: The Australian isolates were divided into two groups on the basis of their DNA banding pattern. Both were different from the UK reference strain. Seven isolates from cattle in Victoria and the Northern Territory had the same pattern as five isolates from alpacas in Victoria and Western Australia. Another 20 isolates from cattle in Victoria, Western Australia and the Northern Territory had the same pattern as isolates from two sheep and a goat in New South Wales. CONCLUSION: Pulsed field gel electrophoresis was a useful tool for strain typing of M paratuberculosis, and could be used to study the transmission of strains in Australia.     

The isolation and detection of Escherichia coli O157 by use of immunomagnetic separation and immunoassay procedures

Pelvic inflammatory disease (PID) has been associated with the use of intrauterine devices (IUDs) ever since they were introduced. In several mostly retrospective studies the incidence of PID was suggested to be as high as three- to ninefold in IUD users compared to non-users. Later epidemiological prospective studies showed a considerably lower risk comparing different types of contraception. Compared to non-contraceptive users the relative risk ranged from 1.8 in patients with copper-containing IUDs to 3.3 in patients using the older insert IUDs like the Dalkon Shield. More recent studies indicate that PID among IUD users is strongly related to the insertion process and to background risk of sexually transmissible disease. The incidence rate of PID decreases from 9.66 per 1000 woman years during the first 20 days following insertion to 1.38 per 1000 woman years beyond the first 20 days. This incidence is similar to the incidence in non-IUD users. PID is more prevalent in younger single women with different sexual partners. Nulliparous women with stable monogamous relationships are not at higher risk of PID than parous women. In conclusion, IUD users, selected for low risk of sexually transmissible disease, do not have excess PID. Proper counselling and selection is of the utmost importance. As there is a higher risk shortly after insertion, limiting IUD replacements will help diminish PID incidence.     

Isolation and characterization of a 70 kDa protein from Mycobacterium avium

Mycobacterium avium complex (MAC) is an intracellular pathogen which causes disseminated bacterial infection in immunocompromised individuals. This organism predominantly infects macrophages. Attachment of MAC to macrophages is the first step prior to invasion. We have previously shown that a 70 kDa protein of M. avium (Ma) is one of nine monocyte-binding proteins. In the present study, we have purified this protein from sonic extracts of Ma and studied some of its properties. The N-terminal sequence of this protein was identified and found to exhibit a strong homology to the 70 kDa heat shock protein (hsp) of M. leprae (Ml) and M. tuberculosis (Mtb). This protein was found to be present on the surface of the organism and was able to inhibit the attachment of intact Ma to human monocyte derived macrophages (MDM) up to 49% in an in vitro attachment assay using intact fluorescein isothiocyanate (FITC)-labelled Ma. Bovine serum albumin (BSA) and recombinant 70 kDa hsp from Mtb, which were used as controls, inhibited this attachment by 9.8 and 18%, respectively. These results suggest that the 70 kDa protein may have a role in the attachment of intact Ma to MDM. When tested in lymphocyte activation assays, this protein did not appear to significantly stimulate proliferation. However, it was found to stimulate the production of tumor necrosis factor (TNF)-alpha by MDM. This protein may be one of several Ma antigens that trigger host immune response by binding to MDM and stimulating the production of inflammatory cytokines such as TNF-alpha by these cells.     

Isolation and characterisation of glutamate dehydrogenase from Mycobacterium smegmatis CDC 46

Glutamate dehydrogenase (L-glutamate:NADP+ oxidoreductase (deaminating), EC 1.4.1.4) has been purified from Mycobacterium smegmatis CDC 46 using (NH4)2SO4 precipitation, negative adsorption on DEAE-cellulose, 2',5'-ADP-Sepharose affinity chromatography and Sephadex G-200. The enzyme was purified 1041.6-fold and the preparation was found to be homogeneous on column chromatography, polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. Alanine and threonine were identified as the N- and C-terminal amino acids of glutamate dehydrogenase from M. smegmastis. The enzyme kinetics and regulation of glutamate dehydrogenase activity by different nutritional factors has been studied. Initial velocity plots showed that the reaction mechanism of glutamate dehydrogenase from M. smegmatis followed an ordered sequential ter-bi mechanism.     

Analysis of paraffin sections of Crohn''s disease for Mycobacterium paratuberculosis using polymerase chain reaction

Guided tissue regeneration has been shown to permit osteoconduction in otherwise nonhealing cranial defects. The relative importance of preventing the prolapse of soft tissue versus the infiltration of individual connective tissue cells has not been determined. A fibrillar form of polylactic acid (PLA) was tested in 13-mm-diameter defects in the parietal bones of 12 sheep. The polymer was hypothesized to prevent the prolapse of dura and periosteum but allow entrance of individual cells. Control defects in the same sheep were either filled with autogenous bone shavings or left unfilled. The animals were killed at times ranging from 6 to 25 weeks and the defects examined grossly, radiologically, and histologically. The autogenous bone-filled defects were spanned by trabeculated bone by 6 weeks. The unfilled defects demonstrated prolapse of soft tissues into the defect; however, progressive centripetal bone growth was evident. The fibrillar PLA-filled defects were occupied by a full-thickness mixture of fibrous tissue interspersed with PLA. After 19 weeks, small "fingers" of bone were seen to minimally infiltrate the fibrous tissue. Although separation of the dura and periosteum was maintained by the fibrillar PLA, invasion of fibrous tissue restricted osteoconduction.     

Isolation and characterization of nocardia-like variants of Mycobacterium smegmatis

Orange-red-pigmented (OR) colonies were isolated from cream-yellow-pigmented Mycobacterium smegmatis after exposure to either mycobacteriophage MC4 or ultraviolet light; these variant strains were designated OR4 and ORuv, respectively. Early subculture of OR-colonies did not show any segregation of parental-type cells. However, colonies resembling the parental strains, possibly representing a back mutant (REV-OR4), were occasionally found during subculture of established OR-colonies or upon treatment with N-nitrose-N'-nitro-N-methylguanidine. The OR-variants were characterized by their lytic response to nocardiophage, but not to mycobacteriophages, presence of alpha-branched, beta-hydroxylated fatty acids of the Nocardia-type, and a guanine plus cytosine value of deoxyribonucleic acid (DNA) between 62 and 64 mol%. They were more resistant to the lethal action of both ultraviolet light and mitomycin C treatment than the parental and back mutant strains. Although the OR-variants in this study possess characteristics common to the genus Nocardia or some of the 'rhodochrous' mycobacteria, evidence is presented that they form a new class of mycobacterial variants.     

Comparison of immunohistochemistry, acid-fast staining, and cultivation for detection of Mycobacterium paratuberculosis in goats

Forty-seven paired specimens of ileum and mesenterial lymph nodes from goats originating from 2 herds with paratuberculosis were investigated. Culture of the specimens for Mycobacterium paratuberculosis was compared with Ziehl-Neelsen staining and with immunohistochemical tests on paraffin-embedded tissue sections, using a M. paratuberculosis immune serum and an avidin-biotin-alkaline phosphatase method. Immunohistochemical techniques detected most positive samples and produced more clearly visible reactions than did acid-fast staining. Eighteen of 47 samples were positive by immunohistochemical techniques may represent a valuable adjunct to standard techniques for diagnosis of paratuberculosis in goats.     

Survival of Mycobacterium paratuberculosis and preservation of immunoglobulin G in bovine colostrum under experimental conditions simulating pasteurization

OBJECTIVE: To determine whether Mycobacterium paratuberculosis could survive in colostrum after pasteurization. Additionally, this study investigated the effect pasteurization had on IgG concentration in colostrum. ANIMALS: Colostrum samples were collected from cattle (beef and dairy) owned by the state of Ohio. PROCEDURE: Colostrum was divided into aliquots and inoculated with variable concentrations of M paratuberculosis (ATCC No. 19698: 10(4), 10(3), and 10(2) colony-forming units/ml). Half the samples at each concentration were subjected to pasteurization temperatures (63 C) for 30 minutes and the remainder were kept at approximately 20 to 23 C. All samples were incubated (Herrold's egg yolk medium with and without mycobactin J) and observed for growth during the next 16 weeks. Additionally, the IgG concentration of colostrum was determined by radioimmunoassay before and after pasteurization. Samples that coagulated at pasteurization temperatures were mechanically resuspended before measurement of IgG concentration. RESULTS: Growth of M paratuberculosis was retarded but not eliminated by pasteurization. Growth was observed in all unpasteurized samples incubated on Herrold's egg yolk medium with mycobactin J but in only 2 of 18 pasteurized samples similarly cultured. Growth from pasteurized samples appeared 5 to 9 weeks after growth was observed from nonpasteurized samples. Mean colostral IgG concentration was 44.4 g/L in nonpasteurized samples and 37.2 g/L in pasteurized samples, a decrease of 12.3%. High-quality colostrum (> 48 g of IgG/L) had a significantly greater loss of IgG concentration than did colostrum of lesser quality (P = 0.002). CONCLUSIONS: Pasteurization lessened, but did not eliminate, growth of M paratuberculosis from experimentally inoculated colostrum samples. Pasteurization resulted in a significant decrease in colostral IgG concentration but not to an unmanageable level that would preclude the colostrum's use for passive transfer of immunity. CLINICAL RELEVANCE: Colostrum is macrophage rich and may serve as a source of M paratuberculosis infection to calves. Pasteurization of colostrum may lessen the risk of infection, but will not totally eliminate M paratuberculosis.     

Isolation and identification of Mycobacterium cultures antagonistic to phytopathogenic microflora

The immunosuppressive activity of extracellular and water-phenol lipopolysaccharides (LPS) of S.minnesota in S- and R-forms, as well as their gel-filtration, polysaccharide and lipid fractions, was studied in mouse experiments on the model of delayed type hypersensitivity (DTH). The study revealed that the extracellular LPS of S-form S.minnesota was capable of suppressing DTH with lipid A playing the decisive role in this immunosuppressive activity. The extracellular LPS of Reform S.minnesota did not possess the capacity for immunosuppression, but acquired it after redox treatment.