Evaluation of cell mediated immune diagnostic tests to detect Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne\u27s disease, a chronic progressive fatal disease of ruminants. Similar to tuberculosis, diagnostic tests based on detecting the organism or antibodies are not capable of detecting infected animals in the early stages. Consequently, diagnostic tests based upon the cell mediated immune response, such as the skin test and gamma interferon enzyme-linked immuno absorbent assay (ELISA), which have been successful at detecting tuberculosis need to be investigated further for paratuberculosis. The first paper evaluated the skin test in sheep along with two antibody based diagnostic tests using Bayesian statistical methods that estimate the sensitivity and specificity of diagnostic tests in multiple populations without a gold standard. The second paper used receiver operating characteristic (ROC) analysis to evaluate the accuracy of the gamma interferon (IFN-gamma) ELISA in subclinically infected sheep. These 2 studies similarly concluded the skin test and IFN-gamma ELISA had an estimated sensitivity of around 70% and a specificity of 98%. The next paper evaluated the environmental parameters necessary for handling whole blood for the IFN-gamma ELISA and assessed the validity of positive controls. The conclusion was blood should be maintained at room temperature, stimulated within 8 hours and phytohemaglutinin A (PHA) was the most accurate positive control tested in cattle. Finally the last paper attempts to further characterize the meaning of a positive response to the skin test or the IFN-gamma ELISA by assessing the consequences of environmental exposure to dead MAP. This study found that inhaled or ingested dead MAP does not stimulate a detectable response on the skin test or IFN-gamma ELISA

Johne’s Disease Status of the McNay Sheep Flock

Johne’s disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) results in chronic weight loss and eventual death in sheep as well as in other ruminants. The disease has spread throughout the U.S.sheep flock, and it is estimated that 10% or possibly more of the flocks in the U.S. are infected. Sheep can be infected and spread the organism for years before succumbing to the disease. Current diagnostic tests are unable to detect animals in the early stages of infection, and because there is a lack of “known negative” sheep in production settings, the development and subsequent validation of new diagnostic tests are hindered. The objective of this report is to describe the process the McNay flock went through to ensure researchers that this population was not infected with MAP

Induction of B Cell Responses upon Experimental Infection of Neonatal Calves with Mycobacterium avium subsp. paratuberculosis

The objective of this study was to determine if experimental infection of neonatal calves with Mycobacterium avium subsp. paratuberculosis would invoke changes in the percentages of total B cells in the peripheral blood mononuclear cell population and of subpopulations of B cells as determined by CD5, CD25, and CD45RO markers during a 12-month period. Experimental infection groups included control (noninfected), oral (infected with M. avium subsp. paratuberculosis strain K-10), oral/DXM (pretreatment with dexamethasone before oral inoculation), i.p. (intraperitoneal inoculation), and oral/M (oral inoculation with mucosal scrapings from a cow with clinical disease) groups. Over the course of the study, the percentages of total B cells in nonstimulated and antigen-stimulated cell cultures increased for oral and i.p. group calves, with the highest percentages noted at 3 and 6 months. Oral/M group calves had increased percentages of activated B cells, as determined by CD5dim and CD5bright markers, at 9 and 12 months. Experimental infection by all methods resulted in increased expression of CD25 and CD45RO B cells early in the study, but the most significant results were observed at 12 months for oral/DXM and oral/M group calves. Immunoblot analyses with a whole-cell sonicate of M. avium subsp. paratuberculosis demonstrated the most reactivity with sera from i.p. group calves and the least reactivity with sera from oral group calves. Further evidence of M. avium subsp. paratuberculosis-specific antibody responses in the i.p. group calves was demonstrated using the ethanol vortex enzyme-linked immunosorbent assay (EvELISA) method. In summary, an induction of B cell responses was noted after experimental infection with M. avium subsp. paratuberculosis, with differences in responses noted according to the method of experimental inoculation

Optimization of Methods for the Detection of Mycobacterium avium subsp. paratuberculosis in Milk and Colostrum of Naturally Infected Dairy Cows

Two decontamination chemicals, hexadecylpyridinium choride (HPC) and N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH), were compared for their efficacy of reducing the growth of non-specific microorganisms in milk while minimally affecting the recovery of Mycobacterium avium subsp. paratuberculosis (MAP). In addition three culture mediums, Bactec 12B and Trek-ESP para-JEM, and Herrold’s egg yolk media (HEYM), were compared for the ability to suppress growth of non-specific microorganisms as well as their sensitivity of detection of low levels of MAP in milk. Results indicated that exposing the milk to 1.5% NALC-NaOH for 15 minutes most effectively reduced nontarget microorganisms without reducing MAP viability. In addition, the Bactec 12B medium detected the lowest levels of MAP more rapidly and more consistently than the other two mediums

Shedding of Mycobacterium avium subsp. paratuberculosis into Milk and Colostrum of Naturally Infected Dairy Cows over Complete Lactation Cycles

The primary mode of transmission of Mycobacterium avium subsp. paratuberculosis (MAP) is fecal-oral. However, MAP is also shed into the milk and colostrum of infected cows. The objective of this study was to identify ifan association exists between stage of MAP infection and days in lactation with the amount of MAP present in milk and colostrum of naturally infected cows. Results indicated that MAP is primarily shed in early lactation and in cows with advanced infection. This experiment provides crucial information to dairy producers pertaining to the threat of MAP transmission via milk and colostrum. Producers now know that allowing a calf to suckle even once is exposing it to the highest concentrations of MAP and therefore possibly infecting the newest generation of animal

Identification and Characterization of a Spore-Like Morphotype in Chronically Starved Mycobacterium avium Subsp. Paratuberculosis Cultures

Mycobacteria are able to enter into a state of non-replication or dormancy, which may result in their chronic persistence in soil, aquatic environments, and permissive hosts. Stresses such as nutrient deprivation and hypoxia provide environmental cues to enter a persistent state; however, a clear definition of the mechanism that mycobacteria employ to achieve this remains elusive. While the concept of sporulation in mycobacteria is not novel, it continues to spark controversy and challenges our perceptions of a non-replication. We investigated the potential role of sporulation in one-year old broth cultures of Mycobacterium subsp. paratuberculosis (MAP). We show that dormant cultures of MAP contain a mix of vegetative cells and a previously unknown morphotype resembling a spore. These spore-like structures can be enriched for using sporulating media. Furthermore, purified MAP spore forms survive exposure to heat, lysozyme and proteinase K. Heat- treated spores are positive for MAP 16SrRNA and IS900. MAP spores display enhanced infectivity as well as maintain acid-fast characteristics upon germination in a well-established bovine macrophage model. This is the first study to demonstrate a new MAP morphotype possessing spore-like qualities. Data suggest that sporulation may be a viable mechanism by which MAP accomplishes persistence in the host and/or environment. Thus, our current understanding of mycobacterial persistence, pathogenesis, epidemiology and rational drug and vaccine design may need to be reevaluated

Identification of Mycobacterium spp. of veterinary importance using rpoB gene sequencing

<p>Abstract</p> <p>Background</p> <p>Studies conducted on <it>Mycobacterium </it>spp. isolated from human patients indicate that sequencing of a 711 bp portion of the <it>rpoB </it>gene can be useful in assigning a species identity, particularly for members of the <it>Mycobacterium avium </it>complex (MAC). Given that MAC are important pathogens in livestock, companion animals, and zoo/exotic animals, we were interested in evaluating the use of <it>rpoB </it>sequencing for identification of <it>Mycobacterium </it>isolates of veterinary origin.</p> <p>Results</p> <p>A total of 386 isolates, collected over 2008 - June 2011 from 378 animals (amphibians, reptiles, birds, and mammals) underwent PCR and sequencing of a ~ 711 bp portion of the <it>rpoB </it>gene; 310 isolates (80%) were identified to the species level based on similarity at ≥ 98% with a reference sequence. The remaining 76 isolates (20%) displayed < 98% similarity with reference sequences and were assigned to a clade based on their location in a neighbor-joining tree containing reference sequences. For a subset of 236 isolates that received both 16S rRNA and <it>rpoB </it>sequencing, 167 (70%) displayed a similar species/clade assignation for both sequencing methods. For the remaining 69 isolates, species/clade identities were different with each sequencing method. <it>Mycobacterium avium </it>subsp. <it>hominissuis </it>was the species most frequently isolated from specimens from pigs, cervids, companion animals, cattle, and exotic/zoo animals.</p> <p>Conclusions</p> <p><it>rpoB </it>sequencing proved useful in identifying <it>Mycobacterium </it>isolates of veterinary origin to clade, species, or subspecies levels, particularly for assemblages (such as the MAC) where 16S rRNA sequencing alone is not adequate to demarcate these taxa. <it>rpoB </it>sequencing can represent a cost-effective identification tool suitable for routine use in the veterinary diagnostic laboratory.</p

Draft Genome Sequence of a Mycobacterium avium Complex Isolate from a Broadbill Bird

Mycobacterium avium complex (MAC) organisms cause opportunistic infections in humans, yet their epidemiology remains poorly understood. They are slowly growing environmental and animal-associated mycobacteria that have little notoriety except for the strains that cause disseminated infections in HIV- infected humans (1). Most MAC organisms are classified taxonomically as a single species, M. avium, which is divided into at least four subspecies, M. avium subsp. avium, M. avium subsp. hominissuis, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum (2). The only other species in this group is M. intracellulare. Genotyping of this diverse bacterial group has been achieved using intergenic spacers (3) and rpoB sequence analysis (4, 5)

Identification and Characterization of a Spore-Like Morphotype in Chronically Starved Mycobacterium avium Subsp. Paratuberculosis Cultures

Mycobacteria are able to enter into a state of non-replication or dormancy, which may result in their chronic persistence in soil, aquatic environments, and permissive hosts. Stresses such as nutrient deprivation and hypoxia provide environmental cues to enter a persistent state; however, a clear definition of the mechanism that mycobacteria employ to achieve this remains elusive. While the concept of sporulation in mycobacteria is not novel, it continues to spark controversy and challenges our perceptions of a non-replication. We investigated the potential role of sporulation in one-year old broth cultures of Mycobacterium subsp. paratuberculosis (MAP). We show that dormant cultures of MAP contain a mix of vegetative cells and a previously unknown morphotype resembling a spore. These spore-like structures can be enriched for using sporulating media. Furthermore, purified MAP spore forms survive exposure to heat, lysozyme and proteinase K. Heat- treated spores are positive for MAP 16SrRNA and IS900. MAP spores display enhanced infectivity as well as maintain acid-fast characteristics upon germination in a well-established bovine macrophage model. This is the first study to demonstrate a new MAP morphotype possessing spore-like qualities. Data suggest that sporulation may be a viable mechanism by which MAP accomplishes persistence in the host and/or environment. Thus, our current understanding of mycobacterial persistence, pathogenesis, epidemiology and rational drug and vaccine design may need to be reevaluated

Comparative genomic analysis of Mycobacterium avium subspecies obtained from multiple host species

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium </it>(<it>M. avium</it>) subspecies vary widely in both pathogenicity and host specificity, but the genetic features contributing to this diversity remain unclear.</p> <p>Results</p> <p>A comparative genomic approach was used to identify large sequence polymorphisms among <it>M. avium </it>subspecies obtained from a variety of host animals. DNA microarrays were used as a platform for comparing mycobacterial isolates with the sequenced bovine isolate <it>M. avium </it>subsp. <it>paratuberculosis </it>(MAP) K-10. Open reading frames (ORFs) were classified as present or divergent based on the relative fluorescent intensities of the experimental samples compared to MAP K-10 DNA. Multiple large polymorphic regions were found in the genomes of MAP isolates obtained from sheep. One of these clusters encodes glycopeptidolipid biosynthesis enzymes which have not previously been identified in MAP. <it>M. avium </it>subsp. <it>silvaticum </it>isolates were observed to have a hybridization profile very similar to yet distinguishable from <it>M. avium </it>subsp. <it>avium</it>. Isolates obtained from cattle (n = 5), birds (n = 4), goats (n = 3), bison (n = 3), and humans (n = 9) were indistinguishable from cattle isolate MAP K-10.</p> <p>Conclusion</p> <p>Genome diversity in <it>M. avium </it>subspecies appears to be mediated by large sequence polymorphisms that are commonly associated with mobile genetic elements. Subspecies and host adapted isolates of <it>M. avium </it>were distinguishable by the presence or absence of specific polymorphisms.</p