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Michael T. Collins - One of the best experts on this subject based on the ideXlab platform.
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Diagnosis of bovine Paratuberculosis by a novel enzyme-linked immunosorbent assay based on early secreted antigens of Mycobacterium avium subsp. Paratuberculosis.
Clinical and vaccine immunology : CVI, 2008Co-Authors: Sung Jae Shin, Donghee Cho, Michael T. CollinsAbstract:We previously reported that protein antigens of serodiagnostic potential were more abundant in culture filtrates than cellular extracts from liquid cultures of Mycobacterium avium subsp. Paratuberculosis (D. Cho and M. T. Collins, Clin. Vaccine Immunol. 13:1155-1161, 2006). Based on this observation, a novel enzyme-linked immunosorbent assay (ELISA) using antigens secreted by young (early- to mid-log-phase) cultures of M. avium subsp. Paratuberculosis JTC303 (a low-passage isolate originating from the ileum of a Holstein bull) in mycobactin-supplemented Watson-Reid medium (pH 6.0) was developed and evaluated using a previously described panel of bovine sera (M. T. Collins et al., Clin. Diagn. Lab. Immunol. 12:685-692, 2005) that included 444 Paratuberculosis cases and 412 controls. The new assay, called JTC-ELISA, had a significantly higher diagnostic sensitivity and an equivalent specificity compared to those of five commercial Paratuberculosis ELISA kits. By receiver-operating characteristic analysis, the JTC-ELISA had the highest area under the curve of the six assays evaluated. The JTC-ELISA was particularly sensitive at detecting low-level fecal shedders of Mavium subsp. Paratuberculosis (40%; the sensitivity of the commercial kits was 20%). The JTC-ELISA works effectively on both serum and milk samples for the detection of cattle with subclinical M. avium subsp. Paratuberculosis infections, providing a cost-effective diagnostic tool to support Paratuberculosis control programs in cattle herds.
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effect of mycobacterium Paratuberculosis infection on production reproduction and health traits in us holsteins
Preventive Veterinary Medicine, 2007Co-Authors: M G Gonda, Michael T. Collins, Y M Chang, G E Shook, B W KirkpatrickAbstract:Our objective was to estimate the effect of Mycobacterium Paratuberculosis infection on milk, fat, and protein yield deviations, pregnancy rate, lactation somatic cell score, and projected total months in milk (productive life). A serum ELISA and fecal culture for M. Paratuberculosis were performed on 4375 Holsteins in 232 DHIA herds throughout the US. Primarily first through third lactation cows (99% of total) were assayed for infection. Trait information (except productive life) was obtained for the lactation concurrent with disease tests. Productive life was total months in milk through a cow's life, which was projected if a cow was still milking. For most analyses, case definition for M. Paratuberculosis infection was defined as either an ELISA S/P ratio>or=0.25 or a positive fecal culture for M. Paratuberculosis or both. To determine if diagnostic test affected estimates, case definition was redefined to include only cows with ELISA S/P ratios>or=0.25 or only fecal culture-positive cows. Linear models were used to estimate effect of M. Paratuberculosis infection on traits. M. Paratuberculosis-infected cows (7.89% of cows) produced 303.9 kg less milk/lactation, 11.46 kg less fat/lactation, and 9.49 kg less protein/lactation (P
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goat Paratuberculosis in chile first isolation and confirmation of mycobacterium avium subspecies Paratuberculosis infection in a dairy goat
Journal of Veterinary Diagnostic Investigation, 2006Co-Authors: J Kruze, Enrique Paredes, Miguel Salgado, Armin Mella, Michael T. CollinsAbstract:In October 2004, 41 goats >2 years old from a Saanen dairy goat herd located in Purranque County, 10th Region, Chile, were sampled and tested for Paratuberculosis. While collecting samples it was observed that several goats were thin and emaciated. One goat was sufficiently debilitated to warrant humane euthanasia. This animal was brought to the Veterinary School at the Universidad Austral de Chile for necropsy. The goat selected for necropsy was a 12-year-old doe. The animal showed classical clinical signs of caprine Paratuberculosis: emaciation despite willingness to eat, dry and rough hair coat, and no evidence of diarrhea. Gross pathology and histopathology of the necropsied goat were consistent with paucibacillary Paratuberculosis. Bacteriology, serology, and PCR confirmed the diagnosis. This is the first published report of goat Paratuberculosis in Chile confirming a case of caprine paucibacillary Paratuberculosis.
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evaluation of five antibody detection tests for diagnosis of bovine Paratuberculosis
Clinical and Vaccine Immunology, 2005Co-Authors: Michael T. Collins, Scott J Wells, Kristine R Petrini, James E Collins, Ronald D Schultz, R H WhitlockAbstract:Five diagnostic tests based on enzyme-linked immunosorbent assay (ELISA) technology for bovine Paratuberculosis were evaluated by using individual serum or milk samples from 359 dairy cattle in seven Paratuberculosis-free herds and 2,094 dairy cattle in seven Mycobacterium Paratuberculosis-infected dairy herds. Three independent laboratories using three different culture procedures completed fecal cultures for M. Paratuberculosis on these cattle and found 417 cows to be shedding M. Paratuberculosis in their feces. An animal that was fecal culture positive for M. Paratuberculosis by any of the three laboratories was considered a confirmed case of infection. The specificity of three ELISAs (two on serum and one on milk) was ≥99.8%. The specificity of the remaining two ELISAs, both done on serum, was 94.9 and 84.7%. Four of the five ELISAs evaluated produced similar sensitivity in detecting fecal culture-positive cattle (27.8 to 28.9%). Serum ELISA “D” had the lowest specificity (84.7%) and the highest sensitivity (44.5%), but if the cutoff value defining a positive test was changed from 125 to 250% (of the positive control) the sensitivity and specificity, 31.8 and 97.5%, respectively, were comparable to those of the other four assays. If the case definition for M. Paratuberculosis infection was based on the culture results of a single laboratory instead of the combined results of three laboratories, ELISA sensitivity estimates were 45.7 to 50.0%. With the exception of ELISA D, assay agreement was high (kappa 0.66 to 0.85) for categorical assay interpretations (positive or negative), but linear regression of quantitative results showed low correlation coefficients (r2 = 0.40 to 0.68) due to the fact that ELISA results for some cows were high in one assay but low in another assay. Likelihood ratio analysis showed a direct relationship between the magnitude of ELISA result and the odds of a cow shedding M. Paratuberculosis in its feces. If used judiciously and interpreted quantitatively, these ELISAs are useful tools in support of Paratuberculosis control programs in dairy herds.
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mycobacterium avium subsp Paratuberculosis pathogen pathogenesis and diagnosis
Revue Scientifique Et Technique De L Office International Des Epizooties, 2001Co-Authors: Elizabeth J. B. Manning, Michael T. CollinsAbstract:Johne's disease, or Paratuberculosis, is a chronic intestinal infection caused by Mycobacterium avium subsp. Paratuberculosis. The usually fatal disease is characterised by cachexia, and in some species diarrhoea, after a long pre-clinical phase. Treatment is ineffective and economically impracticable. The infection primarily affects domestic and free-ranging ruminants, but has also been reported in primates, rabbits, stoats and foxes. Since Paratuberculosis is often subclinical, under-reporting is suspected, even though the disease is notifiable in numerous countries. Herd prevalence of bovine Paratuberculosis in Europe ranges from 7% to 55%. In the United States of America, herd prevalence is strongly associated with herd size; 40% of herds of more than 300 head were found to be infected. In Australia, reported dairy herd infection rates range between 9% and 22%. Paratuberculosis in domestic livestock entails significant economic losses due to several factors (e.g. reduced production, premature culling and increased veterinary costs). Free-ranging and captive wildlife are also at risk from Paratuberculosis.
John P Bannantine - One of the best experts on this subject based on the ideXlab platform.
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Immunogenicity of proteome-determined Mycobacterium avium subsp. Paratuberculosis-specific proteins in sheep with Paratuberculosis.
Clinical and vaccine immunology : CVI, 2008Co-Authors: Valerie Hughes, John P Bannantine, Susan Denham, Stuart C. Smith, Alfredo Garcia-sanchez, Jill Sales, Michael L. Paustian, Kevin Mclean, Karen StevensonAbstract:Mycobacterium avium subsp. Paratuberculosis causes Paratuberculosis, a chronic granulomatous enteritis. Detecting animals with Paratuberculosis infections is difficult because the currently available tools have low sensitivity and lack specificity; these tools are prone to generating spurious positive test results caused by exposure to environmental M. avium complex organisms. To generate candidate antigens for incorporation into a specific test for Paratuberculosis, subspecies-specific proteins were determined by proteomic comparison of M. avium subsp. Paratuberculosis and M. avium subsp. avium. Analysis was aimed at revealing proteins only expressed (or predominant) in the protein profile of M. avium subspecies Paratuberculosis. Two-dimensional gel electrophoresis resolved approximately 1,000 protein spots from each subspecies. Proteome analysis identified protein spots whose expression profile appeared markedly increased in M. avium subsp. Paratuberculosis, and 32 were identified by analysis of their tryptic peptide profile by matrix-assisted laser desorption ionization-time of flight analysis. Thirty of these proteins were cloned, and their recombinant proteins were expressed. Ovine Paratuberculosis sera were used to assess their immunoreactivity by enzyme-linked immunosorbent assay (ELISA), Western blotting, and dot blot analysis. Seventeen proteins were detected in at least one of the immunoassays, and eleven proteins were detected by ELISA with an optical density in excess of the cutoff of 0.1 in four of six sera tested. The immunoreactivity of these proteins indicates their potential as unique diagnostic antigens for the development of a specific serological detection of Paratuberculosis.
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Early antibody response against Mycobacterium avium subspecies Paratuberculosis antigens in subclinical cattle
Proteome Science, 2008Co-Authors: John P Bannantine, Judith R Stabel, Mitchell V. Palmer, W. Ray Waters, Darrell O Bayles, Michael L. PaustianAbstract:Background Our laboratories have previously reported on the experimental infection of cattle with Mycobacterium avium subsp Paratuberculosis ( M. Paratuberculosis ) using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 M. Paratuberculosis coding sequences. These combined tools have enabled a unique look at the temporal analysis of M. Paratuberculosis antigens within the native host. The primary objective of this study was to identify M. Paratuberculosis antigens detected by cattle early during infection. A secondary objective was to evaluate the humoral immune response in cattle during the initial year of infection. Results Sera from two experimentally infected cattle, taken pre-inoculation and at day 70, 194 and 321 post infection, identified dynamic antibody reactivity among antigens with some showing an increased response over time and others showing declining levels of reactivity over the same time period. A M. Paratuberculosis specific protein, encoded by MAP0862, was strongly detected initially, but the antibody response became weaker with time. The most reactive protein was a putative surface antigen encoded by MAP1087. A second protein, MAP1204, implicated in virulence, was also strongly detected by day 70 in both cattle. Subsequent experiments showed that these two proteins were detected with sera from 5 of 9 naturally infected cattle in the subclinical stage of Johne's disease. Conclusion Collectively these results demonstrate that M. Paratuberculosis proteins are detected by sera from experimentally infected cattle as early as 70 days after exposure. These data further suggest at least two antigens may be useful in the early diagnosis of M. Paratuberculosis infections. Finally, the construction and use of a protein array in this pilot study has led to a novel approach for discovery of M. Paratuberculosis antigens.
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Experimental infection of white-tailed deer (Odocoileus virginianus) with Mycobacterium avium subsp. Paratuberculosis.
Journal of wildlife diseases, 2007Co-Authors: Mitchell V. Palmer, John P Bannantine, Judith R Stabel, W. Ray Waters, Janice M. MillerAbstract:Mycobacterium avium subsp. Paratuberculosis (Map) is the causative agent of Paratuberculosis or Johne's disease, a chronic enteric disease of domestic ruminants as well as some nondomestic ruminants. Paratuberculosis is characterized by a protracted subclinical phase followed by clinical signs such as diarrhea, weight loss, and hypoproteinemia. Fecal shedding of Map is characteristic of both the subclinical and clinical phases, and it is important in disease transmission. Lesions of Paratuberculosis are characterized by chronic granulomatous enteritis and mesenteric lymphadenitis. Animal models of Paratuberculosis that simulate all aspects of the disease are rare. Oral inoculation of 9-day-old white-tailed deer (Odocoileus virginianus) on 3 June 2002 with 1.87×1010 colony-forming units of Map strain K10 resulted in clinical disease (soft to diarrheic feces) as early as 146 days after inoculation; lesions consistent with Paratuberculosis were observed in animals at the termination of the study. Intermitten...
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multilocus short sequence repeat sequencing approach for differentiating among mycobacterium avium subsp Paratuberculosis strains
Journal of Clinical Microbiology, 2004Co-Authors: Alongkorn Amonsin, Srinand Sreevatsan, John P Bannantine, Qing Zhang, Alifiya S Motiwala, Vivek KapurAbstract:We describe a multilocus short sequence repeat (MLSSR) sequencing approach for the genotyping of Mycobacterium avium subsp. Paratuberculosis (M. Paratuberculosis) strains. Preliminary analysis identified 185 mono-, di-, and trinucleotide repeat sequences dispersed throughout the M. Paratuberculosis genome, of which 78 were perfect repeats. Comparative nucleotide sequencing of the 78 loci of six M. Paratuberculosis isolates from different host species and geographic locations identified a subset of 11 polymorphic short sequence repeats (SSRs), with an average of 3.2 alleles per locus. Comparative sequencing of these 11 loci was used to genotype a collection of 33 M. Paratuberculosis isolates representing different multiplex PCR for IS900 loci (MPIL) or amplified fragment length polymorphism (AFLP) types. The analysis differentiated the 33 M. Paratuberculosis isolates into 20 distinct MLSSR types, consistent with geographic and epidemiologic correlates and with an index of discrimination of 0.96. MLSSR analysis was also clearly able to distinguish between sheep and cattle isolates of M. Paratuberculosis and easily and reproducibly differentiated strains representing the predominant MPIL genotype (genotype A18) and AFLP genotypes (genotypes Z1 and Z2) of M. Paratuberculosis described previously. Taken together, the results of our studies suggest that MLSSR sequencing enables facile and reproducible high-resolution subtyping of M. Paratuberculosis isolates for molecular epidemiologic and population genetic analyses.
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molecular epidemiology of mycobacterium avium subsp Paratuberculosis evidence for limited strain diversity strain sharing and identification of unique targets for diagnosis
Journal of Clinical Microbiology, 2003Co-Authors: John P Bannantine, Judith R Stabel, Alongkorn Amonsin, Alifiya S Motiwala, Megan Strother, Beverly Byrum, Saleh A Naser, William P Shulaw, Vivek KapurAbstract:The objectives of this study were to understand the molecular diversity of animal and human strains of Mycobacterium avium subsp. Paratuberculosis isolated in the United States and to identify M. avium subsp. Paratuberculosis-specific diagnostic molecular markers to aid in disease detection, prevention, and control. Multiplex PCR of IS900 integration loci (MPIL) and amplified fragment length polymorphism (AFLP) analyses were used to fingerprint M. avium subsp. Paratuberculosis isolates recovered from animals (n = 203) and patients with Crohn's disease (n = 7) from diverse geographic localities. Six hundred bacterial cultures, including M. avium subsp. Paratuberculosis (n = 303), non-M. avium subsp. Paratuberculosis mycobacteria (n = 129), and other nonmycobacterial species (n = 168), were analyzed to evaluate the specificity of two IS900 integration loci and a newly described M. avium subsp. Paratuberculosis-specific sequence (locus 251) as potential targets for the diagnosis of M. avium subsp. Paratuberculosis. MPIL fingerprint analysis revealed that 78% of bovine origin M. avium subsp. Paratuberculosis isolates clustered together into a major node, whereas isolates from human and ovine sources showed greater genetic diversity. MPIL analysis also showed that the M. avium subsp. Paratuberculosis isolates from ovine and bovine sources from the same state were more closely associated than were isolates from different geographic regions, suggesting that some of the strains are shared between these ruminant species. AFLP fingerprinting revealed a similar pattern, with most isolates from bovine sources clustering into two major nodes, while those recovered from sheep or humans were clustered on distinct branches. Overall, this study identified a high degree of genetic similarity between M. avium subsp. Paratuberculosis strains recovered from cows regardless of geographic origin. Further, the results of our analyses reveal a relatively higher degree of genetic heterogeneity among M. avium subsp. Paratuberculosis isolates recovered from human and ovine sources.
Nackmoon Sung - One of the best experts on this subject based on the ideXlab platform.
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Thermal Tolerance of Mycobacterium Paratuberculosis
Applied and Environmental Microbiology, 1998Co-Authors: Nackmoon Sung, Michael T. CollinsAbstract:D values (decimal reduction time; the time required to kill 1 log concentration of bacteria) were determined for both human and bovine strains (Dominic, Ben, BO45, and ATCC 19698) ofMycobacterium Paratuberculosis in 50 mM lactate solution (pH 6.8) and in milk at four temperatures (62, 65, 68, and 71°C). Viable M. Paratuberculosis organisms were quantified by a radiometric culture method (BACTEC). Thermal death curves for theM. Paratuberculosis strains tested were generally linear, with R2 of ≥0.90, but a few curves (R2, 0.80 to 0.90) were better described by a quadratic equation. The human strains (Dominic and Ben) had similarD values in milk and in lactate solution. However,D values for the bovine strains (BO45 and ATCC 19698) were significantly different depending on the menstruum. Dvalues for low-passage clinical strains (Dominic, Ben, and BO45) were lower than those of the high-passage laboratory strain (ATCC 19698). The D value based on pooled data for clinical strains ofM. Paratuberculosis in milk at 71°C (D71°C) was 11.67 s. PooledD62°C, D65°C, andD68°C of clinical M. Paratuberculosis strains in milk were 228.8, 47.8, and 21.8 s, respectively. The Z value (the temperature required for the decimal reduction time to traverse 1 log cycle) of clinical strains in milk was 7.11°C. The D values of clumped and singleM. Paratuberculosis cells were not significantly different. The D values of all M. Paratuberculosis strains tested were considerably higher than those published forListeria, Salmonella, and Coxiellaspp. and estimated for Mycobacterium bovis, indicating thatM. Paratuberculosis is more thermally tolerant. This study supports the premise that M. Paratuberculosis may survive high-temperature, short-time pasteurization when the initial organism concentration is greater than 101 cells/ml.
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Thermal Tolerance of Mycobacterium Paratuberculosis
Applied and environmental microbiology, 1998Co-Authors: Nackmoon Sung, Michael T. CollinsAbstract:D values (decimal reduction time; the time required to kill 1 log concentration of bacteria) were determined for both human and bovine strains (Dominic, Ben, BO45, and ATCC 19698) of Mycobacterium Paratuberculosis in 50 mM lactate solution (pH 6.8) and in milk at four temperatures (62, 65, 68, and 71 degrees C). Viable M. Paratuberculosis organisms were quantified by a radiometric culture method (BACTEC). Thermal death curves for the M. Paratuberculosis strains tested were generally linear, with R2 of > or = 0.90, but a few curves (R2, 0.80 to 0.90) were better described by a quadratic equation. The human strains (Dominic and Ben) had similar D values in milk and in lactate solution. However, D values for the bovine strains (BO45 and ATCC 19698) were significantly different depending on the menstruum. D values for low-passage clinical strains (Dominic, Ben, and BO45) were lower than those of the high-passage laboratory strain (ATCC 19698). The D value based on pooled data for clinical strains of M. Paratuberculosis in milk at 71 degrees C (D71 degrees C) was 11.67 s. Pooled D62 degrees C, D65 degrees C, and D68 degrees C of clinical M. Paratuberculosis strains in milk were 228.8, 47.8, and 21.8 s, respectively. The Z value (the temperature required for the decimal reduction time to traverse 1 log cycle) of clinical strains in milk was 7.11 degrees C. The D values of clumped and single M. Paratuberculosis cells were not significantly different. The D values of all M. Paratuberculosis strains tested were considerably higher than those published for Listeria, Salmonella, and Coxiella spp. and estimated for Mycobacterium bovis, indicating that M. Paratuberculosis is more thermally tolerant. This study supports the premise that M. Paratuberculosis may survive high-temperature, short-time pasteurization when the initial organism concentration is greater than 10(1) cells/ml.
M T Rowe - One of the best experts on this subject based on the ideXlab platform.
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efficacy of various pasteurization time temperature conditions in combination with homogenization on inactivation of mycobacterium avium subsp Paratuberculosis in milk
Applied and Environmental Microbiology, 2005Co-Authors: Irene R Gra, M T Rowe, Ala G Williams, Donald D MuiAbstract:The effect of various pasteurization time-temperature conditions with and without homogenization on the viability of Mycobacterium avium subsp. Paratuberculosis was investigated using a pilot-scale commercial high-temperature, short-time (HTST) pasteurizer and raw milk spiked with 101 to 105 M. avium subsp. Paratuberculosis cells/ml. Viable M. avium subsp. Paratuberculosis was cultured from 27 (3.3%) of 816 pasteurized milk samples overall, 5 on Herrold's egg yolk medium and 22 by BACTEC culture. Therefore, in 96.7% of samples, M. avium subsp. Paratuberculosis had been completely inactivated by HTST pasteurization, alone or in combination with homogenization. Heat treatments incorporating homogenization at 2,500 lb/in2, applied upstream (as a separate process) or in hold (at the start of a holding section), resulted in significantly fewer culture-positive samples than pasteurization treatments without homogenization (P < 0.001 for those in hold and P < 0.05 for those upstream). Where colony counts were obtained, the number of surviving M. avium subsp. Paratuberculosis cells was estimated to be 10 to 20 CFU/150 ml, and the reduction in numbers achieved by HTST pasteurization with or without homogenization was estimated to be 4.0 to 5.2 log10. The impact of homogenization on clump size distribution in M. avium subsp. Paratuberculosis broth suspensions was subsequently assessed using a Mastersizer X spectrometer. These experiments demonstrated that large clumps of M. avium subsp. Paratuberculosis cells were reduced to single-cell or “miniclump” status by homogenization at 2,500 lb/in2. Consequently, when HTST pasteurization was being applied to homogenized milk, the M. avium subsp. Paratuberculosis cells would have been present as predominantly declumped cells, which may possibly explain the greater inactivation achieved by the combination of pasteurization and homogenization.
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persistence of mycobacterium Paratuberculosis during manufacture and ripening of cheddar cheese
Applied and Environmental Microbiology, 2004Co-Authors: J A Donaghy, N L Totton, M T RoweAbstract:Model Cheddar cheeses were prepared from pasteurized milk artificially contaminated with high 104 to 105 CFU/ml) and low (101 to 102 CFU/ml) inocula of three different Mycobacterium Paratuberculosis strains. A reference strain, NCTC 8578, and two strains (806PSS and 796PSS) previously isolated from pasteurized milk for retail sale were investigated in this study. The manufactured Cheddar cheeses were similar in pH, salt, moisture, and fat composition to commercial Cheddar. The survival of M. Paratuberculosis cells was monitored over a 27-week ripening period by plating homogenized cheese samples onto HEYM agar medium supplemented with the antibiotics vancomycin, amphotericin B, and nalidixic acid without a decontamination step. A concentration effect was observed in M. Paratuberculosis numbers between the inoculated milk and the 1-day old cheeses for each strain. For all manufactured cheeses, a slow gradual decrease in M. Paratuberculosis CFU in cheese was observed over the ripening period. In all cases where high levels (>3.6 log10) of M. Paratuberculosis were present in 1-day cheeses, the organism was culturable after the 27-week ripening period. The D values calculated for strains 806PSS, 796PSS, and NCTC 8578 were 107, 96, and 90 days, respectively. At low levels of contamination, M. Paratuberculosis was only culturable from 27-week-old cheese spiked with strain 806PSS. M. Paratuberculosis was recovered from the whey fraction in 10 of the 12 manufactured cheeses. Up to 4% of the initial M. Paratuberculosis load was recovered in the culture-positive whey fractions at either the high or low initial inoculum.
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effect of commercial scale high temperature short time pasteurization on the viability of mycobacterium Paratuberculosis in naturally infected cows milk
Applied and Environmental Microbiology, 2002Co-Authors: Irene R Gra, Edward Hitchings, Ala Mccartney, Fiona Ferguso, M T RoweAbstract:Raw cows9 milk naturally infected with Mycobacterium Paratuberculosis was pasteurized with an APV HXP commercial-scale pasteurizer (capacity 2,000 liters/h) on 12 separate occasions. On each processing occasion, milk was subjected to four different pasteurization treatments, viz., 73°C for 15 s or 25 s with and without prior homogenization (2,500 lb/in 2 in two stages), in an APV Manton Gaulin KF6 homogenizer. Raw and pasteurized milk samples were tested for M. Paratuberculosis by immunomagnetic separation (IMS)-PCR (to detect the presence of bacteria) and culture after decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h (to confirm bacterial viability). On 10 of the 12 processing occasions, M. Paratuberculosis was detectable by IMS-PCR, culture, or both in either raw or pasteurized milk. Overall, viable M. Paratuberculosis was cultured from 4 (6.7%) of 60 raw and 10 (6.9%) of 144 pasteurized milk samples. On one processing day, in particular, M. Paratuberculosis appeared to have been present in greater abundance in the source raw milk (evidenced by more culture positives and stronger PCR signals), and on this occasion, surviving M. Paratuberculosis bacteria were isolated from milk processed by all four heat treatments, i.e., 73°C for 15 and 25 s with and without prior homogenization. On one other occasion, surviving M. Paratuberculosis bacteria were isolated from an unhomogenized milk sample that had been heat treated at 73°C for 25 s. Results suggested that homogenization increases the lethality of subsequent heat treatment to some extent with respect to M. Paratuberculosis , but the extended 25-s holding time at 73°C was found to be no more effective at killing M. Paratuberculosis than the standard 15-s holding time. This study provides clear evidence that M. Paratuberculosis bacteria in naturally infected milk are capable of surviving commercial high-temperature, short-time pasteurization if they are present in raw milk in sufficient numbers.
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Isolation of Mycobacterium Paratuberculosis from Milk by Immunomagnetic Separation
Applied and environmental microbiology, 1998Co-Authors: Irene R. Grant, H. J. Ball, M T RoweAbstract:An immunomagnetic separation (IMS) technique was developed to facilitate selective isolation of Mycobacterium Paratuberculosis cells from milk. Rabbit polyclonal antibodies against radiation-killed intact M. Paratuberculosis cells were produced and used to coat sheep anti-rabbit immunoglobulin G (IgG) type M-280 Dynabeads. The rabbit anti-M. Paratuberculosis IgG-coated beads (IMB) reacted strongly with laboratory strains of M. Paratuberculosis as determined by slide agglutination, and microscopic examination confirmed that M. Paratuberculosis cells attached to the IMB. The IMB were found to have a maximum binding capacity of 10(4) to 10(5) CFU of M. Paratuberculosis. Studies showed that IMS selectively recovered M. Paratuberculosis from inoculated milk containing as few as 10 CFU of M. Paratuberculosis per ml when 10 microliter IMB (ca. 10(6) beads) was added to 1 ml of milk and the preparation was incubated for 30 min at room temperature with gentle agitation. Larger volumes of milk (10 and 50 ml) were centrifuged and resuspended in 1 ml of phosphate-buffered saline-0.05% Tween 20 prior to IMS in order to increase the sensitivity of the method. Currently, primary isolation of M. Paratuberculosis from a milk sample relies on chemical decontamination, followed by culturing on Herrold's egg yolk medium, which must be incubated at 37 degreesC for up to 18 weeks. The potential value of our IMS method is as an aid for rapid detection of M. Paratuberculosis in milk when it is used in conjunction with end point detection methods, such as IS900 PCR or an enzyme-linked immunosorbent assay.
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inactivation of mycobacterium Paratuberculosis in cows milk at pasteurization temperatures
Applied and Environmental Microbiology, 1996Co-Authors: Irene R Gra, H J All, S D Neill, M T RoweAbstract:The thermal inactivation of 11 strains of Mycobacterium Paratuberculosis at pasteurization temperatures was investigated. Cows' milk inoculated with M. Paratuberculosis at two levels (10(7) and 10(4) CFU/ml) was pasteurized in the laboratory by (i) a standard holder method (63.5 degrees C for 30 min) and (ii) a high-temperature, short-time (HTST) method (71.7 degrees C for 15 s). Additional heating times of 5, 10, 15, 20, and 40 min at 63.5 degrees C were included to enable the construction of a thermal death curve for the organism. Viability after pasteurization was assessed by culture on Herrold's egg yolk medium containing mycobactin J (HEYM) and in BACTEC Middlebrook 12B radiometric medium supplemented with mycobactin J and sterile egg yolk emulsion. Confirmation of acid-fast survivors of pasteurization as viable M. Paratuberculosis cells was achieved by subculture on HEYM to indicate viability coupled with PCR using M. Paratuberculosis-specific 1S900 primers. When milk was initially inoculated with 10(6) to 10(7) CFU of M. Paratuberculosis per ml, M. Paratuberculosis cells were isolated from 27 of 28 (96%) and 29 of 34 (85%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. Correspondingly, when 10(3) to 10(4) CFU of M. Paratuberculosis per ml of milk were present before heat treatment, M. Paratuberculosis cells were isolated from 14 of 28 (50%) and 19 of 33 (58%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. The thermal death curve for M. Paratuberculosis was concave in shape, exhibiting a rapid initial death rate followed by significant "tailing." Results indicate that when large numbers of M. Paratuberculosis cells are present in milk, the organism may not be completely inactivated by heat treatments simulating holder and HTST pasteurization under laboratory conditions.
Vivek Kapur - One of the best experts on this subject based on the ideXlab platform.
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multilocus short sequence repeat sequencing approach for differentiating among mycobacterium avium subsp Paratuberculosis strains
Journal of Clinical Microbiology, 2004Co-Authors: Alongkorn Amonsin, Srinand Sreevatsan, John P Bannantine, Qing Zhang, Alifiya S Motiwala, Vivek KapurAbstract:We describe a multilocus short sequence repeat (MLSSR) sequencing approach for the genotyping of Mycobacterium avium subsp. Paratuberculosis (M. Paratuberculosis) strains. Preliminary analysis identified 185 mono-, di-, and trinucleotide repeat sequences dispersed throughout the M. Paratuberculosis genome, of which 78 were perfect repeats. Comparative nucleotide sequencing of the 78 loci of six M. Paratuberculosis isolates from different host species and geographic locations identified a subset of 11 polymorphic short sequence repeats (SSRs), with an average of 3.2 alleles per locus. Comparative sequencing of these 11 loci was used to genotype a collection of 33 M. Paratuberculosis isolates representing different multiplex PCR for IS900 loci (MPIL) or amplified fragment length polymorphism (AFLP) types. The analysis differentiated the 33 M. Paratuberculosis isolates into 20 distinct MLSSR types, consistent with geographic and epidemiologic correlates and with an index of discrimination of 0.96. MLSSR analysis was also clearly able to distinguish between sheep and cattle isolates of M. Paratuberculosis and easily and reproducibly differentiated strains representing the predominant MPIL genotype (genotype A18) and AFLP genotypes (genotypes Z1 and Z2) of M. Paratuberculosis described previously. Taken together, the results of our studies suggest that MLSSR sequencing enables facile and reproducible high-resolution subtyping of M. Paratuberculosis isolates for molecular epidemiologic and population genetic analyses.
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molecular epidemiology of mycobacterium avium subsp Paratuberculosis evidence for limited strain diversity strain sharing and identification of unique targets for diagnosis
Journal of Clinical Microbiology, 2003Co-Authors: John P Bannantine, Judith R Stabel, Alongkorn Amonsin, Alifiya S Motiwala, Megan Strother, Beverly Byrum, Saleh A Naser, William P Shulaw, Vivek KapurAbstract:The objectives of this study were to understand the molecular diversity of animal and human strains of Mycobacterium avium subsp. Paratuberculosis isolated in the United States and to identify M. avium subsp. Paratuberculosis-specific diagnostic molecular markers to aid in disease detection, prevention, and control. Multiplex PCR of IS900 integration loci (MPIL) and amplified fragment length polymorphism (AFLP) analyses were used to fingerprint M. avium subsp. Paratuberculosis isolates recovered from animals (n = 203) and patients with Crohn's disease (n = 7) from diverse geographic localities. Six hundred bacterial cultures, including M. avium subsp. Paratuberculosis (n = 303), non-M. avium subsp. Paratuberculosis mycobacteria (n = 129), and other nonmycobacterial species (n = 168), were analyzed to evaluate the specificity of two IS900 integration loci and a newly described M. avium subsp. Paratuberculosis-specific sequence (locus 251) as potential targets for the diagnosis of M. avium subsp. Paratuberculosis. MPIL fingerprint analysis revealed that 78% of bovine origin M. avium subsp. Paratuberculosis isolates clustered together into a major node, whereas isolates from human and ovine sources showed greater genetic diversity. MPIL analysis also showed that the M. avium subsp. Paratuberculosis isolates from ovine and bovine sources from the same state were more closely associated than were isolates from different geographic regions, suggesting that some of the strains are shared between these ruminant species. AFLP fingerprinting revealed a similar pattern, with most isolates from bovine sources clustering into two major nodes, while those recovered from sheep or humans were clustered on distinct branches. Overall, this study identified a high degree of genetic similarity between M. avium subsp. Paratuberculosis strains recovered from cows regardless of geographic origin. Further, the results of our analyses reveal a relatively higher degree of genetic heterogeneity among M. avium subsp. Paratuberculosis isolates recovered from human and ovine sources.
