MAP-specific DNA Research Articles

Field study on bovine paratuberculosis using real-time PCR and liquid culture for testing environmental and individual fecal samples implemented in dairy cow management

Bovine paratuberculosis (Johne's disease) is a bacterial, chronic, and wasting intestinal disease caused by Mycobacterium avium ssp. paratuberculosis (MAP). Johne's disease causes severe losses in dairy farm productivity and is also suspected to be a potential trigger for Crohn's disease in humans. The fecal-oral infection of MAP to neonates is recognized as an important within-herd transmission route. Our objective was to recommend diagnostic methods for herds with suspected paratuberculosis requiring fast results, as well as for herds with breeding programs or others that aim at being nonsuspected of paratuberculosis infection. We determined a period of 8 wk from sampling to diagnostic findings suitable for testing of cows during the dry period. We therefore tested environmental and individual fecal samples with one rapid and one highly sensitive diagnostic method. Environmental samples (boot swabs) were taken as a first step in 3 herds and tested using a DNA extraction protocol for feces and subsequent real-time PCR (referred to as fecal PCR). Additionally, cultivation in liquid medium for 6 wk was performed and verified with real-time PCR (referred to as liquid culture). Automation of DNA extraction based on magnetic beads and the PCR setup was performed with pipetting robots. As a result, we successfully detected MAP in boot swabs of all herds by both methods. In a second step, 245 individual fecal samples from the 3 herds were examined using also fecal PCR and liquid culture. The results obtained by fecal PCR were compared with detection of MAP using cultivation in liquid medium for 6 wk. Testing individual cows, we identified MAP-specific DNA in 53 fecal samples using the liquid culture. Using fecal PCR, we revealed 43 positive samples of which 39 also tested positive in the liquid culture, revealing MAP-positive cows in all 3 herds. The fecal PCR procedure allows rapid detection of MAP-specific DNA with 74% of the sensitivity of liquid culture. For the purpose of testing with maximal sensitivity, cultivation in liquid medium is recommended. Cultivation of MAP in liquid medium M7H9C means a significant time gain in comparison to cultivation on solid media, which requires twice as much time. Thus, this testing fits within the 6- to 8-wk dry period of gravid cows and provides test results before calving, a prerequisite to prevent fecal-oral transmission to newborn calves.

Enhanced sensitivity and fast turnaround time in laboratory diagnosis for bovine paratuberculosis in faecal samples

Bovine paratuberculosis (Johne's disease in cattle) is caused by the pathogen Mycobacterium avium subsp. paratuberculosis (MAP) and is a widespread chronic bacterial infectious disease in cattle. Due to the peculiarities of the pathogen, detection of MAP in faeces remains difficult.DNA extraction and real-time PCR for detection of MAP in bovine faeces (direct PCR) have been refined and feasible procedures for rapid, sensitive and automatable detection of the pathogen agent have been developed. Accordingly, in a first step we tested 20 faecal samples using two MAP complete kits (DNA extraction kits based on magnetic beads combined with real-time PCR assays) and six other DNA extraction kits for faeces. MAP-specific DNA was detected by real-time PCR assays. Cultivation of MAP on the solid medium HEYM and in the liquid medium M7H9C served as reference standards.The two complete kits detected significantly more MAP-DNA positive samples than the other procedures applied (p < 0.04). Ct values of 37 and 38 served as cut-off for the respective real-time PCR assays calculated on the basis of standard curves and droplet digital PCR (ddPCR).In a second step, the two MAP complete kits were employed for a comprehensive study including 107 positive and 50 negative faecal samples which had been previously tested on HEYM cultivation. The MAP complete kits yielded sensitivity values of 86% and 89% and specificity values of 100% compared to cultivation of MAP in the liquid medium M7H9C. In detail, cultivation of MAP in M7H9C detected the pathogen in 97% and 100% of the samples tested after an incubation period of six and twelve weeks, respectively. However, the cultivation of MAP on HEYM succeeded in only 74% after twelve weeks of incubation. In all these solid culture positive samples, MAP was also detected using the two complete kits.Additionally, the impact of repeated freezing and thawing of samples on re-cultivation of MAP was tested using 20 faecal samples and resulted in a reduction to 75% and 25% of bacterial growth when using liquid medium M7H9C and solid medium HEYM, respectively.The results of this study show that complete kits with refined automatable protocols for DNA extraction in combination with real-time PCR assays for detection of MAP can compete with sensitive cultivation of the pathogen in liquid medium.

Faecal shedding of Mycobacterium avium subspecies paratuberculosis reduces before parturition in sheep?

Paratuberculosis is a disease affecting mainly ruminants, and it is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Control of the disease relies primarily on test-and-removal that is not easy to apply effectively for reasons associated with its pathogenesis. Therefore the aim of this preliminary study was to investigate whether positivity of sheep to MAP-specific antibody and DNA, detected by ELISA and faecal real time polymerase chain reaction (PCR) respectively, varies between specific stages of breeding i.e. before and after parturition, and before the mating season.Rectal faeces (n=120) and whole blood (n=120) samples were collected from a flock of sheep with no record of clinical paratuberculosis. The sheep under study are maintained throughout the year, in isolated confinement with no access to pasture or other animals, and a continuously monitored diet.The proportion of positive animals to real time PCR before parturition (13.9%) was found to be significantly lower (χ2=10.67, P=0.0015) than the proportion of positive animals after parturition (59.5%), and before the mating season (47.6%). The proportion of positive animals after parturition and before the mating season did not differ significantly. PCR analysis of one sample collected from each animal after parturition, allowed detection of more than 71% of the reactors.In conclusion, faecal shedding of MAP detected by real time PCR seems to be decreased before parturition, which probably makes the specific period less suitable for detection of shedders in sheep with no clinical signs of paratuberculosis.

Prevalence and Association of Mycobacterium avium subspecies paratuberculosis with Disease Course in Patients with Ulcero-Constrictive Ileocolonic Disease.

BackgroundAssociation of Mycobacterium avium subspecies paratuberculosis (MAP) and Crohn’s disease (CD) has been controversial due to contradictory reports. Therefore, we determined the prevalence of MAP in patients with CD and intestinal tuberculosis (ITB) and its association with clinical course.MethodologyBlood and intestinal biopsies were taken from 69 CD, 32 ITB patients and 41 patients with haemorrhoidal bleed who served as controls. qPCR targeting of MAP-specific IS900 gene was used to detect the presence of MAP DNA. qPCR results were further validated by sequencing. Immunohistochemistry (IHC) was used to detect the presence of MAP antigen in biopsy specimens. CD and ITB patients were followed-up for disease course and response to therapy.Principal FindingsThe frequency of MAP-specific DNA in biopsies by qPCR was significantly higher in CD patients (23.2%, p = 0.03) as compared to controls (7.3%). No significant difference in intestinal MAP presence was observed between ITB patients (12.5%, p = 0.6) and controls (7.3%). MAP presence in blood of CD patients was 10.1% as compared to 4.9% in controls while no patients with ITB were found to be positive (p = 0.1). Using IHC for detection of MAP antigen, the prevalence of MAP in CD was 2.9%, 12.5% in ITB patients and 2.4% in controls. However, long-term follow-up of the patients revealed no significant associations between clinical characteristics and treatment outcomes with MAP positivity.ConclusionWe report significantly high prevalence of MAP in intestinal biopsies of CD patients. However, the presence of MAP does not affect the disease course and treatment outcomes in either CD or ITB patients.

Short communication: Viable Mycobacterium avium subspecies paratuberculosis in retail artisanal Coalho cheese from Northeastern Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis and it potentially plays a role in Crohn’s disease. In humans, the main route of transmission of MAP might be the intake of contaminated milk and dairy products. Considering that MAP has already been detected in many types of cheese in different counties, and that Coalho cheese is an important dairy product in northeastern Brazil, the aim of this study was to report the first detection of MAP in retail Coalho cheese in Brazil by PCR and culture. Of 30 retail Coalho cheese samples, 3 (10%) amplified fragments of a similar size to that expected (626 bp) were obtained and viable MAP was recovered by culture from 1 (3.3%) sample. The DNA from the positive culture sample was sequenced and showed 99% identity with the insertion sequence IS900 deposited in GenBank. It was possible to identify the presence of MAP-specific DNA in the analyzed samples for the first time in Brazil, and to recover viable cells from retail Coalho cheese.

Detection of Mycobacterium avium Subspecies Paratuberculosis from Intestinal and Nodal Tissue of Dogs and Cats

Objective. To determine prevalence of MAP in intestinal and nodal tissue from dogs and cats at necropsy at Kansas State University and to determine if an association existed between presence of MAP and gastrointestinal inflammation, clinical signs, or rural exposure. Procedures. Tissue samples were collected from the duodenum, ileum, and mesenteric and colic nodes of adult dogs (73) and cats (37) undergoing necropsy for various reasons. DNA was extracted and analyzed for insertion sequence 900 using nested PCR. Positive samples were confirmed with DNA sequencing. An online mapping system was used to determine if patients lived in an urban or rural environment based on the home address. Medical records were reviewed for clinical signs and histological findings at necropsy. Results. MAP was identified from 3/73 (4.1%) dogs and 3/37 (8.1%) cats. There was no documented association between presence of MAP and identification of histologic-confirmed gastrointestinal inflammation, gastrointestinal clinical signs, or exposure to a rural environment. Conclusion and Clinical Relevance. MAP-specific DNA can be identified within the intestinal and nodal tissue of dogs and cats that do not have pathological lesions or clinical signs consistent with gastrointestinal disease. The significance of this organism's presence without associated gastrointestinal pathology is unknown.

Association of Mycobacterium avium subspecies paratuberculosis With Crohn Disease in Pediatric Patients

The aim of the present study was to determine the prevalence of Mycobacterium avium subsp paratuberculosis (MAP) DNA in intestinal biopsies from pediatric patients with granulomatous Crohn disease (CD) or ulcerative colitis (UC), and matched control subjects without inflammatory bowel disease (IBD). DNA was extracted from formalin-fixed paraffin-embedded colonic and ileal biopsies from patients with CD (n = 22) or UC (n = 20), and from controls without IBD (n = 21). IS900 nested polymerase chain reaction was performed in triplicate to determine the presence of MAP-specific DNA. In mucosal biopsies from terminal ileum, IS900 amplicons were detected in 1 of 19 (5.2%) control subjects, 1 of 20 (5%) patients with UC, and 7 of 20 (35%) patients with CD (P < 0.05 vs controls, odds ratio 9.6). In colonic biopsies, IS900 amplicons were detected in 0 of 19 control subjects, 1 of 19 (5.2%) patients with UC, and 5 of 19 (26.3%) patients with CD (P < 0.05 vs controls, odds ratio 14.8). In patients with CD, there was no correlation between disease activity and the presence of IS900. Our technique enabled sensitive and specific detection of MAP DNA in archival endoscopic biopsy specimens. Although MAP-specific DNA can be detected in about 5% of intestinal biopsies from children with UC or controls without IBD, its presence was significantly associated with pediatric granulomatous CD, being particularly prevalent in ileal tissue. This easily defined clinical subset of patients may be useful for additional studies to determine the role of MAP in CD.

Absence of Mycobacterium avium ss paratuberculosis-specific IS900 sequence in intestinal biopsy tissues of Indian patients with Crohn’s disease

The role of Mycobacterium avium ss paratuberculosis (MAP) in the etiopathology of Crohn's disease (CD) remains controversial, because of conflicting reports demonstrating the presence of MAP-specific insertion sequence from intestinal biopsy tissues of patients clinically diagnosed for the disease. The present study was carried out to investigate the presence of MAP DNA in the intestinal tissues of CD patients to ascertain the relevance of MAP in Indian patients with CD. Patients diagnosed as CD at our institute were recruited. Healthy individuals without inflammatory bowel disease served as controls. Mucosal biopsy specimens were collected from ileum and colon in duplicates and subjected to histopathological examination and polymerase chain reaction (PCR) amplification. Total DNA (81 CD patients, 85 healthy individuals) and total RNA (12 CD patients, 12 healthy individuals) isolated from tissue specimens was used for amplification of MAP-specific IS900 by nested PCR. MAP-specific IS900 DNA and RNA could not be detected by nested PCR in the intestinal tissues of any patient with CD. Our results do not support the etiological role of MAP in the pathogenesis of CD in Indian patients.

Detection of Mycobacterium avium Subspecies Paratuberculosis‐Specific DNA by PCR in Intestinal Biopsies of Dogs

Mycobacterium avium subspecies paratuberculosis (MAP) is the cause of paratuberculosis. MAP infections have not been reliably detected in dogs, but a reemerging debate about the link between MAP and Crohn's disease has renewed interest about the occurrence of MAP in pets. This study was undertaken to examine canine intestinal biopsies for the presence of MAP-specific DNA. Forty-two dogs with chronic vomiting, diarrhea, or both; and 14 dogs with no gastrointestinal disease. All dogs with signs of gastrointestinal disease had a standard work-up for chronic gastrointestinal disease. Endoscopically obtained intestinal biopsies were submitted for histopathologic and molecular investigations. Biopsies were screened for MAP-specific DNA by 3 polymerase chain reaction (PCR) methods (nested, seminested, and triplex real-time PCR). Samples from control dogs were obtained during necropsy. Histopathology of the biopsies was indicative of inflammatory bowel disease (IBD) in 17 and neoplasia in 6 dogs. Six dogs showing nonspecific changes responded to diet and were classified as having food-responsive enteropathy. In 13 dogs a final diagnosis was not established. MAP-specific DNA was detected and confirmed by sequencing in 8 dogs (19%). These dogs were diagnosed with food-responsive enteropathy (n=3), IBD (n=2), and open diagnosis (n=3). MAP-specific DNA was not detected in dogs with no gastrointestinal disease. MAP-specific DNA was detected in approximately one fifth of dogs with chronic gastrointestinal disease and might play a role as a pathogenic agent. Apart from animal welfare, the zoonotic aspect warrants further studies addressing the viability of MAP organism in canine intestinal biopsies by culture.

Incidence of Mycobacterium avium Subspecies paratuberculosis in a Population-Based Cohort of Patients With Crohn's Disease and Control Subjects

To define the incidence of Mycobacterium avium subspecies paratuberculosis (MAP) in patients with Crohn's disease (CD) and in control subjects. Blood samples from 361 CD patients from a previously described population-based inflammatory bowel disease (IBD) cohort and 200 blood donor controls, of known NOD2 genotype, were screened by PCR for MAP-specific IS900 DNA. These results were correlated with NOD2 genotype. The PCR assay was capable of detecting 20 fg of purified MAP DNA, equivalent to roughly 100 MAP cells/mL of blood. MAP-specific IS900 DNA was detected in 33.8% of CD cases and 21.5% of controls (OR 1.86, 95% CI 1.247-2.785, P= 0.002). All study participants were genotyped for the NOD2 mutations 2104C>T (R702W), 2722G>C (G908R), and 3020insC (1007fs). Carriage of one or two NOD2 mutations was not associated with a significantly higher risk of CD (OR 0.75, 95% CI 0.465-1.207, P= 0.234). No significant association was seen in the CD cohort for carriage of one or two NOD2 mutations and MAP status (OR 0.883, 95% CI 0.494-1.579, P= 0.675). Screening peripheral blood using IS900 PCR indicated that MAP DNA could be detected in a significant proportion of CD cases from a large population-based cohort, and also, in control subjects. The over-representation of MAP DNA in CD suggests either a role or a probable role for MAP in the etiology of CD.

Mycobacterium avium subsp. paratuberculosis in Scavenging Mammals in Wisconsin

The presence of Mycobacterium avium subsp. paratuberculosis (MAP) in non-ruminant wildlife has raised questions regarding the role of these species in Johne's disease transmission. In this study we tested 472 tissues from 212 animals of six different species of scavenging mammals. All animals were taken from within a 210-square-mile area in Dane and Iowa counties of south central Wisconsin from September to May in 2003-04 and tested for the presence of MAP. We detected MAP-specific DNA in 81 of 212 (38%) scavenging mammals, in 98 of the 472 (21%) tissues; viable MAP was cultured from one coyote's ileum and lymph node tissue. Despite the low numbers of viable MAP isolated in this study, our data adds to the increasing evidence demonstrating the potential for transmission and infection of MAP in nonruminant species and provides possible evidence of interspecies transmission. The apparently high exposure of nonruminant wildlife provides potential evidence of a spill-over of MAP to wildlife species and raises the question of spillback to domestic and wild ruminants. These results demonstrate the importance of understanding the role of wildlife species in developing management strategies for Johne's disease in domestic livestock.

Detection of Mycobacterium avium subspecies paratuberculosis genetic components in retail cheese curds purchased in Wisconsin and Minnesota by PCR

Research has been focused on the detection of Mycobacterium avium subspecies paratuberculosis (MAP) in pasteurized milk; however, pasteurized milk is a key ingredient in a variety of food products. Therefore, MAP contamination in milk-derived products must be investigated. We undertook a six-month study to investigate the presence of viable MAP and MAP genetic components in cheese curds purchased from retail outlets in the northern and southern regions of Wisconsin and Minnesota. A total of 98 retail cheese curd samples were tested for MAP by PCR prescreen, culture on Herrold's egg yolk agar slants with mycobactin J and amphoteracin B, naladixic acid, and vancomycin, and slant rinse PCR using IS900 and hspX primer sets. Although no viable MAP were able to be cultured, 5% of the samples were PCR positive with both the IS900 and hspX primer sets (MAP-specific DNA) when prescreened and 1% of the samples were PCR positive with both the IS900 and hspX primer sets when culture slants were rinsed and tested.

Absence of Mycobacterium avium subsp. paratuberculosis in the microdissected granulomas of Crohn's disease

The etiology of Crohn's disease remains unknown with inflammatory, infectious, and/or genetic causes suspected. Granulomatous inflammation is a characteristic feature of the disorder, resembling the tissue response to mycobacterium. Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent in Johne's disease, a chronic ulcerative intestinal condition in cattle, and has been implicated as a likely candidate. We carefully microdissected the granulomas from the paraffin-embedded resection specimens of 18 patients with well-established Crohn's disease. The DNA obtained was PCR amplified for the IS900 and IS1311 repeat elements of MAP, PCR product size maintained at 101 and 124 base pairs, respectively. Archival tissue from bovine Johne's disease was used as a positive control. MAP-specific DNA, confirmed by sequencing and comparison with prototype strain sequence, was appropriately amplified from the positive control. None of the Crohn's disease cases yielded a positive amplification product, failing to support a role for the organism in the pathogenesis of this illness.