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

Genetic profiling of Mycobacterium bovis strains from slaughtered cattle in Eritrea

<div><p><i>Mycobacterium bovis</i> (<i>M</i>.<i>bovis</i>) is the main causative agent for bovine tuberculosis (BTB) and can also be the cause of zoonotic tuberculosis in humans. In view of its zoonotic nature, slaughterhouse surveillance, potentially resulting in total or partial condemnation of the carcasses and organs, is conducted routinely. Spoligotyping, VNTR profiling, and whole genome sequencing (WGS) of <i>M</i>. <i>bovis</i> isolated from tissues with tuberculosis-like lesions collected from 14 cattle at Eritrea’s largest slaughterhouse in the capital Asmara, were conducted.The 14 <i>M</i>. <i>bovis</i> isolates were classified into three different spoligotype patterns (SB0120, SB0134 and SB0948) and six VNTR profiles. WGS results matched those of the conventional genotyping methods and further discriminated the six VNTR profiles into 14 strains. Furthermore, phylogenetic analysis of the <i>M</i>. <i>bovis</i> isolates suggests two independent introductions of BTB into Eritrea possibly evolving from a common ancestral strain in Europe.This molecular study revealed the most important strains of <i>M</i>. <i>bovis</i> in Eritrea and their (dis)similarities with the strains generally present in East Africa and Europe, as well as potential routes of introduction of <i>M</i>. <i>bovis</i>. Though the sample size is small, the current study provides important information as well as platform for future in-depth molecular studies on isolates from both the dairy and the traditional livestock sectors in Eritrea and the region. This study provides information onthe origin of some of the <i>M</i>. <i>bovis</i> strains in Eritrea, its genetic diversity, evolution and patterns of spread between dairy herds. Such information is essential in the development and implementation of future BTB control strategy for Eritrea.</p></div

Genomics reveals historic and contemporary transmission dynamics of a bacterial disease among wildlife and livestock

Whole-genome sequencing has provided fundamental insights into infectious disease epidemiology, but has rarely been used for examining transmission dynamics of a bacterial pathogen in wildlife. In the Greater Yellowstone Ecosystem (GYE), outbreaks of brucellosis have increased in cattle along with rising seroprevalence in elk. Here we use a genomic approach to examine Brucella abortus evolution, cross-species transmission and spatial spread in the GYE. We find that brucellosis was introduced into wildlife in this region at least five times. The diffusion rate varies among Brucella lineages (∼3 to 8 km per year) and over time. We also estimate 12 host transitions from bison to elk, and 5 from elk to bison. Our results support the notion that free-ranging elk are currently a self-sustaining brucellosis reservoir and the source of livestock infections, and that control measures in bison are unlikely to affect the dynamics of unrelated strains circulating in nearby elk populations

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

Chemical Decontamination with <i>N</i> -Acetyl- <scp>l</scp> -Cysteine–Sodium Hydroxide Improves Recovery of Viable Mycobacterium avium subsp. paratuberculosis Organisms from Cultured Milk

ABSTRACT Mycobacterium avium subsp. paratuberculosis is shed into the milk and feces of cows with advanced Johne's disease, allowing the transmission of M. avium subsp. paratuberculosis between animals. The objective of this study was to formulate an optimized protocol for the isolation of M. avium subsp. paratuberculosis in milk. The parameters investigated included chemical decontamination with N -acetyl- l -cysteine–sodium hydroxide (NALC-NaOH), alone and in combination with antibiotics (vancomycin, amphotericin B, and nalidixic acid), and the efficacy of solid (Herrold's egg yolk medium [HEY]) and liquid (Bactec 12B and para -JEM) culture media. For each experiment, raw milk samples from a known noninfected cow were inoculated with 10 2 to 10 8 CFU/ml of live M. avium subsp. paratuberculosis organisms. The results indicate that an increased length of exposure to NALC-NaOH from 5 to 30 min and an increased concentration of NaOH from 0.5 to 2.0% did not affect the viability of M. avium subsp. paratuberculosis . Additional treatment of milk samples with the antibiotics following NALC-NaOH treatment decreased the recovery of viable M. avium subsp. paratuberculosis cells more than treatment with NALC-NaOH alone. The Bactec 12B medium was the superior medium of the three evaluated for the isolation of M. avium subsp. paratuberculosis from milk, as it achieved the lowest threshold of detection. The optimal conditions for NALC-NaOH decontamination were determined to be exposure to 1.50% NaOH for 15 min followed by culture in Bactec 12B medium. This study demonstrates that chemical decontamination with NALC-NaOH resulted in a greater recovery of viable M. avium subsp. paratuberculosis cells from milk than from samples treated with hexadecylpyridinium chloride (HPC). Therefore, it is important to optimize milk decontamination protocols to ensure that low concentrations of M. avium subsp. paratuberculosis can be detected. </jats:p

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 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