Mycobacterium Avium Subspecies Paratuberculosis DNA Research Articles

Mycobacterium avium subsp. paratuberculosis and Crohn's disease: characterization of the interaction with different aspects of the disease.

Crohn's disease (CD) is a chronic granulomatous inflammatory bowel disease with no fully understood etiology and cure. Mycobacterium avium subspecies paratuberculosis (MAP), the etiologic agent of paratuberculosis, is also isolated from samples from human patients with CD. Paratuberculosis is characterized by persistent diarrhea and progressive weight loss and primarily affects ruminants, which eliminate the agent via feces and milk. The involvement of MAP in the pathogenesis of CD and other intestinal diseases is unclear. Thus, the present study aimed to analyze immunological, socioepidemiological, biochemical, and therapeutic variables that may be related to the occurrence of MAP in blood samples and CD patients. The sampling was random, and the population of origin was the patients from the Bowel Outpatient Clinic of the Alpha Institute of Gastroenterology (IAG), Hospital das Clínicas, Universidade Federal de Minas Gerais (HC-UFMG). Blood samples were collected from 20 patients with CD, eight with ulcerative rectocolitis (UCR), and 10 control patients without inflammatory bowel diseases. Samples were subjected to real-time PCR for detection of MAP DNA, oxidative stress analyses, and socioepidemiological variables. MAP was detected in 10 (26.3%) of the patients, seven (70%) were CD patients, 2 (20%) were URC patients, and one (10%) was a non-IBD patient. MAP was found more frequently among CD patients, but not restricted to CD patients. The presence of MAP in the blood of these patients occurred simultaneously with an inflammatory response with an increase in neutrophils and significant alterations in the production of antioxidant enzymes such as catalase and GST.

Molecular Detection and Typing of Mycobacterium avium Subspecies paratuberculosis from Fecal Samples of Small Ruminants

The chronic nature of Jhone’s disease and prolonged incubation period of the agent often complicate the diagnosis of this disease. The present study was undertaken with an aim to compare fecal microscopy using Ziehl-Nielsen (ZN) staining method and Polymerase Chain Reaction assay for detection of Mycobacterium avium subspecies paratuberculosis (MAP) in small ruminants. A total of 205 fecal samples of sheep and goat were first microscopically examined using ZN staining method and later tested for presence of MAP DNA through IS900 PCR. Win Episcope 2.0 software was used to determine agreement between the test assays. Of the total 205 fecal samples, 78 (38%) were positive by ZN staining method and 48 (23.4%) by IS900 PCR. Moderate level of agreement (0.517) was observed between the test assays. Restriction enzyme analysis of DNA amplicons from IS1311 PCR revealed that all positive samples belonged to Bison type MAP strain. Single predominant strain (Bison type) among all ruminants indicates its ability to infect multiple host species, possibility of interspecies transmission and endemic co-circulation.

Clinical, histopathological, and molecular findings for Mycobacterium avium subspecies paratuberculosis (MAP) in dairy goats under semiarid conditions

The objective of this study was to report the occurrence of Mycobacterium avium subspecies paratuberculosis (MAP) in dairy goats, via description of their clinical presentation, histopathological findings, and molecular identification of the infectious agent. Screening was performed using IS900 real-time PCR (qPCR) in milk samples from 179 properties in the semiarid of Northeast region of Brazil. Pooled milk samples from all lactating goats from processing plants were submitted to molecular diagnosis. One property had a positive result at qPCR. The production unit which had the positive sample for MAP was located, and an on-site visit to this property was performed to collect individual milk samples, seven of which tested MAP positive by IS900 qPCR. With permission from the owner, two goats (Animal 1 was positive and Animal 2 was negative on first qPCR for MAP) were acquired and euthanized. Animals 1 and 2 had milk and portions of the duodenum, ileum, colon, and mesenteric lymph nodes positive at qPCR for MAP. Animal 1 also had MAP DNA detected in part of the jejunum and cecum. In animal 2, the ileocecal valve tested positive. MAP was not detected in the blood or feces of either animal; however; it was confirmed for the association of clinical findings, histopathology, and qPCR. The gene IS900 from the positive samples were sequenced and showed a 99% similarity with MAP. The MAP was identified for the first time in the goat milk and tissues in the semiarid region of Northeast Brazil.

Analysis of Colostrum and Udder Skin Swabs from a Dairy Goat Herd in Germany regarding the Occurrence of Mycobacterium avium Subsp. paratuberculosis.

Simple SummaryThe analysis of transmission routes for paratuberculosis as well as their prevention are critical for effective disease control. The aim of the present study was to assess the importance of colostrum and the udder skin as routes for transmission of Mycobacterium avium subspecies paratuberculosis (MAP) within a dairy goat herd. Swabs of the udder skin and colostrum samples were collected from goats of a paratuberculosis-infected herd during lambing season and analyzed for MAP DNA. Additionally, cultivation of the swabs was performed. MAP could not be detected in the colostrum. A low number of udder skin swabs was positive for MAP DNA but no bacteria could be cultured. Because the detection methods are imperfect, the occurrence of MAP in colostrum or on the udder skin can never be completely excluded. Therefore, present recommendations concerning colostrum and youngstock management should still be re-evaluated, but applied in goat herds within a paratuberculosis control program until the role of colostrum and udder skin for within-herd transmission of MAP is further investigated.Oral intake of Mycobacterium avium subspecies paratuberculosis (MAP) in first days of life is considered to be the main route of infection for paratuberculosis. This can be related to a direct contact to contaminated feces or feeding of MAP containing colostrum. Colostrum is believed to become contaminated either by lactogenic shedding or introduction of MAP from environmental sources. In this pilot study, the presence of MAP in individual and bulk colostrum samples from a paratuberculosis-infected, vaccinated dairy goat herd in Germany and the effect of udder skin disinfection on the MAP load of colostrum were examined. In order to distinguish between lactogenic shedding and fecal contamination, 49 udder skin swabs were cultivated on solid medium whereas 29 swabs were additionally analyzed by qPCR. qPCR was applied on 110 individual colostrum samples collected from 55 goats, one before and one after disinfection with a mycobactericidal disinfectant, and 14 bulk colostrum samples. MAP DNA was detected in 10.3% (3/29) of the swab samples, but no viable MAP was cultivated from any sample. These results indicate a low-level MAP contamination of the udder skin and colostrum of milking goats suggesting a low risk of MAP transmission via these routes.

Antibody detection and molecular analysis for Mycobacterium avium subspecies paratuberculosis (MAP) in goat milk: Systematic review and meta-analysis

Paratuberculosis is an incurable infectious disease that affects several species, including goat (Capra hircus). The etiologic agent is Mycobacterium avium subspecies paratuberculosis (MAP) that has tropism for the intestine, causing anorexia, progressive weight loss and death. In goats, the main transmission route is the ingestion of water and food contaminated by infected feces. Affected animals also eliminate the agent through milk, with a potential biological risk to public health. Thus, the aim of this study was to conduct a research of the literature available in electronic media for a systematic review, followed by a meta-analysis of the results found on prevalence and diagnostic tests adopted in the detection of MAP antibodies and DNA in goat milk. The following search parameters were used: “Mycobacterium avium subsp. paratuberculosis” AND (goat OR small ruminant) AND (milk OR pasteurized milk). Strictly obeying pre-established criteria, 437 articles were selected from the respective electronic databases of scientific content: ScienceDirect (285), PubMed (68), Web of Science (60) and Scopus (24), of which nine papers were elected to the construction of the systematic review and meta-analysis. The prevalence of MAP antibodies in milk detected by milk-ELISA ranged from 1.1 to 67.7% and the prevalence of MAP DNA in goat milk detected by MAP-specific polymerase chain reaction (PCR) ranged from 1.94 to 37.74%. A meta-analysis indicated a combined MAP infection prevalence of 8.24%, but with high heterogeneity among study findings (I2 = 98.7%). The identification of the MAP in goat milk implies the need for surveillance of the agent in order to prevent economic losses and impact on public health.

Genomic Detection of Mycobacterium avium subspecies paratuberculosis in Blood Samples of Patients with Inflammatory Bowel Disease in Southern Iran.

Background: Inflammatory bowel disease (IBD), of which Crohn’s disease (CD) and ulcerative colitis (UC) are the two main clinicopathological subtypes, is a group of digestive system diseases of unknown etiology. Risk factors for IBD are environmental factors, genetics, and immune system agents. Mycobacterium avium subspecies paratuberculosis (MAP) is one of the most important infectious factors and a suspected cause of IBD. The present study aimed to determine the prevalence of MAP in both IBD patients and non-IBD people as well as to investigate the relationship between the presence of this bacterium and IBD.Methods: A cross-sectional study was conducted during May-December 2017 among 146 IBD patients (32 with CD and 114 with UC) at the Motahari Clinic affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. For comparison, the blood samples of 146 non-IBD volunteers (the control group) were tested for the presence of MAP using the polymerase chain reaction method (specific IS900 fragment). The data were analyzed using the SPSS software (version 19.0). The Kolmogorov-Smirnov test was used to evaluate the normal distribution of variables. The χ2 test was used to compare the qualitative variables between the groups.Results: MAP was present in 104 (71.2%) IBD patients out of which 24 (75%) had CD and 80 (70.2%) had UC. In the control group, MAP was present in 63 (43.2%) non-IBD volunteers. There was a significant association between the presence of IBD and MAP (P<0.001).Conclusion: A high prevalence of MAP was observed in the South of Iran. MAP DNA was detected in the blood samples of CD and UC patients as well as non-IBD volunteers. The high prevalence of MAP indicated a possible role of MAP in stimulating IBD.

Mycobacterium avium Subspecies paratuberculosis DNA and Antibodies in Dairy Goat Colostrum and Milk.

Mycobacterium avium subspecies paratuberculosis (MAP) is endemic in the Dutch dairy goat population causing economic loss, and negatively influencing welfare. Moreover, there are concerns about a potential zoonotic risk. Therefore the industry’s objectives are to decrease MAP prevalence, limit economic losses as well as reduce the concentration of MAP in (bulk) milk. To diminish within-farm spread of infection, vaccination, age dependent group housing with separation of newborns from adults, as well as rearing on artificial or treated colostrum and milk replacers are implemented. However, the importance of MAP contaminated colostrum and milk as a route of infection in dairy goat herds is unknown. Therefore the aim of this study was to detect the presence of MAP DNA in colostrum and milk from dairy goats in infected herds. A convenience sample of 120 colostrum samples and 202 milk samples from MAP infected dairy goat herds were tested by IS900 real-time Polymerase Chain Reaction (PCR) for MAP DNA. Furthermore, 22 colostrum samples and 27 post mortem milk samples of goats with clinical signs consistent with paratuberculosis from known infected herds were tested. The majority of samples were from goats vaccinated against MAP. Positive or doubtful PCR results were obtained in none of the 120 and two of the 22 colostrum samples, and in eight of the 202 and four of the 27 milk samples Negative PCR results were obtained in the remaining 140 (99%) colostrum samples and 217 (95%) milk samples.

Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples.

The rapid identification of Mycobacterium avium subspecies paratuberculosis (MAP) infected animals within the herd is essential for preventing the spread of the disease as well as avoiding human exposure. Although culture is seen as the gold standard, there are various molecular assays available i.e., polymerase chain reaction (PCR) or isothermal amplification technique (recombinase polymerase amplification (RPA)) for the detection of MAP. The accuracy of the molecular assays is highly dependent on the DNA extraction method. In order to establish a rapid point of need system for the detection of MAP DNA from stool samples, we developed a rapid DNA extraction protocol (MAP DNA SpeedXtract) specified for use in combination with the RPA. The whole procedure from “sample in” to “result out” was conducted in a mobile suitcase laboratory. The DNA extraction is based on reverse purification by magnetic beads, which reduces the required technical demand. The MAP DNA SpeedXtract was performed within 25 min and only three pipetting steps were needed. The amplification and detection time were 20 min in RPA. The sensitivity and specificity of the developed protocol in comparison with the lab-based silica membrane column extraction and real-time PCR were 90.9% (n = 22) and 100% (n = 23), respectively. In conclusion, we established a rapid and reliable protocol for the extraction and detection of MAP DNA. All reagents are cold chain independent. The entire setup is ideal for point of need identification of MAP infected cases.

Bio-incidence and Bio-type of Mycobacterium avium subspecies paratuberculosis in diarrheic dairy cattle and buffaloes of Punjab area in India

Dairy cattle and buffaloes (n=101) suffering from chronic diarrhea (&gt;3 weeks) with complete history of duration of diarrhea, systemic effects, body condition and faecal abnormalities were included in this study. Faecal samples, whole blood and rectal biopsies from selected animals were collected for the detection of Mycobacterium avium-subspecies paratuberculosis (MAP). Faecal smears were prepared and examined for the load of acid fast bacilli (AFB). Acid fast clumps were recorded in 40.6 per cent (41/101) of diarrheic animals and PCR confirmation was made byIS900 primers. MAP was detected in blood samples of four animals (4/41), while faecal samples were positive only in two of these 4 animals. All the four animals had persistent watery diarrhea and progressive loss in body weights and productivity. Rectal biopsies of two animals were positive by PCR. Remission of clinical signs was observed in these animals within 2-months. Biotyping of MAP-DNA from blood samples by IS1311 PCR-REA revealed restriction pattern of 67, 218 and 325 bp suggesting that MAP DNA belonged to ‘Bison type’, which is a major biotype infecting domestic livestock in India.

Pathophysiology of rheumatoid arthritis is associated with polymorphisms in Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) and susceptibility to Mycobacteria

Abstract Single nucleotide polymorphisms (SNPs) in T-cell regulator genes such as Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) have been associated with many inflammatory autoimmune disorders including rheumatoid arthritis (RA). Along with these SNPs and an environmental trigger of a mycobacterial infection from Mycobacterium avium subspecies paratuberculosis (MAP), we hypothesis that this will lead to chronic inflammation of the synovial joints of RA. We gathered 132 consented subjects blood samples, where genotyping was done for nine SNPs found in PTPN2/22 using TaqMan™ genotyping. Quantification of the effect of the SNPs on gene expression of PTPN2/22 and IFN-γ expression was determined through real time PCR. T-cell proliferation assays were done to examine the response of phytohematoagglutonin (PHA) and mycobacterial antigens in subject samples by the use of BrdU proliferation assays. Detection of MAP bacterial IS900 DNA was done by nested PCR. Out of the nine SNPs examined, PTPN2:rs478582/PTPN22:rs2476601 were found significant in RA compared to the healthy controls (p-values &amp;lt; 0.05). Gene expression of PTPN2/22 was downregulated in RA overall compared to healthy controls. IFN-γ expression was upregulated in samples with significant SNPs. T-cell proliferation increased when T-cells tested had significant SNPs (p-values &amp;lt; 0.05). IS900 MAP DNA was detected more significantly in RA than in healthy controls, where samples with either significant SNPs had higher presence of MAP (p-values &amp;lt; 0.05). This study suggests that SNPs in PTPN2/22 dysregulates T-cell regulation and increases susceptibility to Mycobacteria, leading to increased proliferation of T-cells and higher amounts of IFN-γ expression in RA.

Immunopathological changes and apparent recovery from infection revealed in cattle in an experimental model of Johne’s disease using a lyophilised culture of Mycobacterium avium subspecies paratuberculosis

Johne’s disease (JD) or paratuberculosis is an economically significant, chronic enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Experimental models of JD in cattle are logistically challenging due to the need for long term monitoring, because the clinical disease can take years to manifest. Three trials were undertaken, the largest involving 20 cattle exposed orally to a low dose of C strain MAP and 10 controls studied for 4.75 years. Frequent blood and faecal sampling was used to monitor immunological and infection parameters, and intestinal biopsies were performed at two time points during the subclinical disease phase. Although clinical disease was not seen, there was evidence of infection in 35% of the animals and at necropsy 10% had histopathological lesions consistent with JD, similar to the proportions expected in naturally infected herds. Faecal shedding occurred in two distinct phases: firstly there was intermittent shedding <∼9 months post-exposure that did not correlate with disease outcomes; secondly, in a smaller cohort of animals, this was followed by more consistent shedding of increasing quantities of MAP, associated with intestinal pathology. There was evidence of regression of histopathological lesions in the ileum of one animal, which therefore had apparently recovered from the disease. Both cattle with histopathological lesions of paratuberculosis at necropsy had low MAP-specific interferon-gamma responses at 4 months post-exposure and later had consistently shed viable MAP; they also had the highest loads of MAP DNA in faeces 4.75 year s post-exposure. In a trial using a higher dose of MAP, a higher proportion of cattle developed paratuberculosis. The information derived from these trials provides greater understanding of the changes that occur during the course of paratuberculosis in cattle.

Role of PTPN2/22 polymorphisms in pathophysiology of Crohn's disease.

AIMTo establish the relationship of protein tyrosine phosphatase non-receptor type 2 and 22 (PTPN2/22) polymorphisms and mycobacterial infections in Crohn’s disease (CD).METHODSAll 133 subjects’ blood samples were genotyped for nine single nucleotide polymorphisms (SNPs) in PTPN2/22 using TaqMan™ genotyping, while the effect of the SNPs on PTPN2/22 and IFN-γ gene expression was determined using RT-PCR. Detection of Mycobacterium avium subspecies paratuberculosis (MAP) IS900 gene was done by nPCR after DNA extraction from the isolated leukocytes of each subjects’ blood samples. T-cells isolated from the patient samples were tested for response to phytohematoagglutonin (PHA) mitogen or mycobacterial antigens by BrdU proliferation assays for T-cell activity.RESULTSOut of the nine SNPs examined, subjects with either heterozygous (TC)/minor (CC) alleles in PTPN2:rs478582 occurred in 83% of CD subjects compared to 61% healthy controls (P-values < 0.05; OR = 3.03). Subjects with either heterozygous (GA)/minor (AA) alleles in PTPN22:rs2476601 occurred in 16% of CD compared to 6% healthy controls (OR = 2.7). Gene expression in PTPN2/22 in CD subjects was significantly decreased by 2 folds compared to healthy controls (P-values < 0.05). IFN-γ expression levels were found to be significantly increased by approxiately 2 folds in subjects when either heterozygous or minor alleles in PTPN2:rs478582 and/or PTPN22:rs2476601 were found (P-values < 0.05). MAP DNA was detected in 61% of CD compared to only 8% of healthy controls (P-values < 0.05, OR = 17.52), where subjects with either heterozygous or minor alleles in PTPN2:rs478582 and/or PTPN22:rs2476601 had more MAPbacteremia presence than subjects without SNPs did. The average T-cell proliferation in CD treated with PHA or mycobacteria antigens was, respectively, 1.3 folds and 1.5 folds higher than healthy controls without any significant SNP.CONCLUSIONThe data suggests that SNPs in PTPN2/22 affect the negative regulation of the immune response in CD patients, thus leading to an increase in inflammation/apoptosis and susceptibility of mycobacteria.

Isothermal DNA amplification with functionalized graphene and nanoparticle assisted electroanalysis for rapid detection of Johne’s disease

Johne’s disease (JD), which is caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a bacterial infection of the intestinal tract of ruminants. JD is a cause of significant economic and animal loss throughout the world. Sensitive, selective, and on-site detection of MAP in clinical samples has always been a problem. This study outlines a loop-mediated isothermal DNA amplification (LAMP) and electrochemical analysis-based point-of-care (POC) detection methodology for MAP. LAMP contributed to the selective amplification of MAP DNA, and electrochemical analysis assisted in the rapid and sensitive analysis of LAMP products. A graphene and tetracycline (TET)-functionalized screen printed carbon electrode was used for the selective detection of magnesium pyrophosphate (Mg2P2O7) produced during the LAMP. The Mg2P2O7 obtained from the LAMP was sandwiched on the electrode between TET and zirconium dioxide nanoparticles (ZrO2). The complexation of Mg2P2O7 triggered an electrochemical change that was monitored using electrochemical techniques. The complexation mechanism and functionalized electrodes were characterized using several microscopic, spectroscopic, and electrochemical techniques. The optimized MAP biosensor was employed to detect a range of MAP DNA concentrations. Using electrochemical impedance spectroscopy, a detection limit of 6.37 aM (20 fg/μL) with a detection range of 6.37 aM (20 fg/μL) to 6.37 pM (20 ng/μL) for MAP DNA was obtained. The application of the biosensor was also assessed by MAP detection in clinical fecal samples. The biosensor could easily detect the presence of MAP in bovine fecal samples and showed good co-relation with other conventional techniques. Therefore, the developed biosensor has the potential to be used for POC detection of JD in animals.

Polymorphisms in Protein Tyrosine Phosphatase Non-receptor Type 2 and 22 (PTPN2/22) Are Linked to Hyper-Proliferative T-Cells and Susceptibility to Mycobacteria in Rheumatoid Arthritis.

A shared genetic pre-disposition, chronic inflammation, and treatment with similar biologics between Rheumatoid arthritis (RA) and Crohn's disease (CD) have intrigued us to investigate whether the two disorders share trigger association or possible causation. We hypothesized earlier that Single Nucleotide Polymorphisms (SNPs) in the negative regulators Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) lead to a dysregulated immune response, susceptibility to environmental triggers, and continued apoptosis as seen in chronic inflammation in RA and CD. To test the hypothesis, peripheral leukocytes samples from 132 consented subjects were genotyped for 9 SNPs in PTPN2/22 using TaqMan™ genotyping. The effect of the SNPs on PTPN2/22 and IFN-γ expression was determined using real time PCR. T-cell proliferation and response to phytohematoagglutonin (PHA) mitogen and mycobacterial antigens were determined by BrdU proliferation assay. Blood samples were also analyzed for the Mycobacterium avium subspecies paratuberculosis (MAP) IS900 gene by nPCR. Out of 9 SNPs examined, heterozygous (TC) or minor (CC) alleles of PTPN2:rs478582 occurred in 79% RA compared to 60% healthy controls (p-values ≤ 0.05; OR = 2.28). Similarly, heterozygous (GA) or minor (AA) alleles of PTPN22:rs2476601 occurred in 29% RA compared to 6% healthy controls (p-values ≤ 0.05; OR = 5.90). PTPN2/22 expression in RA was decreased by 1.2-fold compared to healthy controls. PTPN2:rs478582 upregulated IFN-γ in RA by 1.5-fold. Combined PTPN2:rs478582 and PTPN22:rs2476601 increased T-cell proliferation by 2.7-fold when treated with PHA. Surprisingly, MAP DNA was detected in 34% of RA samples compared to 8% healthy controls, (p-values ≤ 0.05, OR = 5.74). RA samples with PTPN2:rs478582 and/or PTPN22:rs2476601 were more positive for MAP than samples without polymorphisms. Combined occurrence of PTPN2:rs478582 and PTPN22:rs2476601 in association with the presence of MAP has significantly increased T-cell response and elevated IFN-γ expression in RA samples. The data suggest that genetic polymorphisms may play vital role in T-cell regulation, susceptibility to mycobacteria and ultimately response to treatment. This is the first study to report the detection of MAP DNA in the blood of RA patients; further studies are needed using larger number of samples.

Culture-Independent Identification of Mycobacterium avium Subspecies paratuberculosis in Ovine Tissues: Comparison with Bacterial Culture and Histopathological Lesions.

Johne’s disease is a chronic debilitating enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Current abattoir surveillance programs detect disease via examination of gross lesions and confirmation by histopathological and/or tissue culture, which is time-consuming and has relatively low sensitivity. This study aimed to investigate whether a high-throughput quantitative PCR (qPCR) test is a viable alternative for tissue testing. Intestine and mesenteric lymph nodes were sourced from sheep experimentally infected with MAP and the DNA extracted using a protocol developed for tissues, comprised enzymatic digestion of the tissue homogenate, chemical and mechanical lysis, and magnetic bead-based DNA purification. The extracted DNA was tested by adapting a previously validated qPCR for fecal samples, and the results were compared with culture and histopathology results of the corresponding tissues. The MAP tissue qPCR confirmed infection in the majority of sheep with gross lesions on postmortem (37/38). Likewise, almost all tissue culture (61/64) or histopathology (52/58) positives were detected with good to moderate agreement (Cohen’s kappa statistic) and no significant difference to the reference tests (McNemar’s Chi-square test). Higher MAP DNA quantities corresponded to animals with more severe histopathology (odds ratio: 1.82; 95% confidence interval: 1.60, 2.07). Culture-independent strain typing on tissue DNA was successfully performed. This MAP tissue qPCR method had a sensitivity equivalent to the reference tests and is thus a viable replacement for gross- and histopathological examination of tissue samples in abattoirs. In addition, the test could be validated for testing tissue samples intended for human consumption.

DETECTION OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS IN ROCK HYRAXES ( PROCAVIA CAPENSIS) IMPORTED FROM SOUTH AFRICA.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic, progressive, and consecutively fatal enteritis, especially in ruminants. MAP distribution among wildlife is not yet clear. In this study, three wild-born rock hyraxes ( Procavia capensis) had been imported from South Africa to a German zoological garden. During the quarantine period, four young animals were born. The wild-born animals showed symptoms of mild diarrhea shortly after their arrival in the zoological garden, but all routine parasitological and bacteriologic tests performed were negative. Therefore, the animals were additionally tested for MAP infection. MAP DNA was detected by seminested PCR (snPCR) in a pooled fecal sample of the seven animals. Subsequent PCR analysis of the individual feces samples confirmed the excretion of MAP in two rock hyraxes (one wild-born and one born in captivity). Sequence analysis of the corresponding 278-bp amplicons revealed 100% homology to the reference MAP-K10 IS900 sequence. No antibody response against MAP was detected in the individual serum samples. MAP-specific postmortem lesions were not observed by gross pathology and histology, neither after death nor after euthanization of the animals. Nevertheless, MAP was detected by snPCR and culture in the gastrointestinal tract, urogenital tract, cardiovascular system, and/or respiratory system of three other animals of the group (one wild-born and two born in captivity). This study is the first report confirming MAP occurrence in rock hyraxes. Therefore, it is recommended that veterinarians and zoo employees consider rock hyraxes as a possible source of MAP infection for domestic livestock in South Africa and the valuable animal stock of zoological facilities.

Detection of Mycobacterium avium subspecies paratuberculosis in powdered infant formula using IS900 quantitative PCR and liquid culture media

Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated in Crohn's disease in humans resulting in public concern over the presence of MAP in powdered infant formula, which could contribute towards early human exposure to MAP or MAP components. Testing of representative powdered infant formula samples using effective tests is required to provide information on contamination of infant formula with MAP, so that consumers can make informed decisions. This study aimed to test representative powdered infant formula samples for the presence of MAP using a quantitative PCR and liquid culture method. For this purpose, an efficient DNA extraction method was developed and an optimum decontamination protocol for culture method was identified. A total of 122 powdered infant formula samples were tested, comprising 72 brands produced by 12 manufacturers from 9 countries. Powdered infant formula samples were reconstituted and centrifuged to separate the casein pellet, cream layer and whey fraction. A sensitive qPCR test was performed on DNA extracted from the casein pellet. In addition, the cream layer and casein pellet were cultured in liquid media, following decontamination with the optimum protocol. Of the 122 samples tested, 6 were positive for MAP DNA but none were positive for growth in culture at 12 and 20 weeks. The limit of detection of the quantitative PCR was less than 5 MAP organisms per 1.5g milk powder. The methods developed in the study could be used for quality assurance testing for infant formula and calf milk replacers. The low contamination level of MAP and absence of viable forms in our study suggests a relatively low risk of exposure of infants to MAP components.

The investigation of the truncated mbtA gene within the mycobactin cluster of Mycobacterium avium subspecies paratuberculosis as a novel diagnostic marker for real-time PCR

The inability of Mycobacterium avium subspecies paratuberculosis (MAP) to produce endogenous mycobactin in-vitro is most likely due to the presence of a truncated mbtA gene within the mycobactin cluster of MAP. The main goal of this study was to investigate this unique mbtA truncation as a potential novel PCR diagnostic marker for MAP.Novel primers were designed that were located within the truncated region and the contiguous MAP2179 gene. Primers were evaluated against non-MAP isolates and no amplicons were generated. The detection limit of this mbtA-MAP2179 target was evaluated using a range of MAP DNA concentrations, MAP inoculated faecal material and 20 MAP isolates. The performance of mbtA-MAP2179 was compared to the established f57 target. The detection limits recorded for MAP K-10 DNA and from MAP K-10 inoculated faecal samples were 0.34pg and 104CFU/g respectively for both f57 and mbtA-MAP2179. A detection limit of 103CFU/g was recorded for both targets, but not achieved consistently. The detection limit of MAP from inoculated faecal material was successful at 103CFU/g for mbtA-MAP2179 when FAM probe real-time PCR was used. A MAP cell concentration of 102CFU/g was detected successfully, but again not consistently achieved. All 20 mycobacterial isolates were successfully identified as MAP by f57 and mbtA-MAP2179. Interestingly, the mbtA-MAP2179 real-time PCR assay resulted in the formation of a unique melting curve profile that contained two melting curve peaks rather than one single peak. This melting curve phenomenon was attributed towards the asymmetrical GC% distribution within the mbtA-MAP2179 amplicon.This study investigated the implementation of the mbtA-MAP2179 target as a novel diagnostic marker and the detection limits obtained with mbtA-MAP2179 were comparable to the established f57 target, making the mbtA-MAP2179 an adequate confirmatory target. Moreover, the mbtA-MAP2179 target could be implemented in multiplex real-time PCR assays and with its unique melting curve profile adds increased specificity to MAP diagnostic tests.

PCR Inhibition of a Quantitative PCR for Detection of Mycobacterium avium Subspecies Paratuberculosis DNA in Feces: Diagnostic Implications and Potential Solutions.

Molecular tests such as polymerase chain reaction (PCR) are increasingly being applied for the diagnosis of Johne’s disease, a chronic intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Feces, as the primary test sample, presents challenges in terms of effective DNA isolation, with potential for PCR inhibition and ultimately for reduced analytical and diagnostic sensitivity. However, limited evidence is available regarding the magnitude and diagnostic implications of PCR inhibition for the detection of MAP in feces. This study aimed to investigate the presence and diagnostic implications of PCR inhibition in a quantitative PCR assay for MAP (High-throughput Johne’s test) to investigate the characteristics of samples prone to inhibition and to identify measures that can be taken to overcome this. In a study of fecal samples derived from a high prevalence, endemically infected cattle herd, 19.94% of fecal DNA extracts showed some evidence of inhibition. Relief of inhibition by a five-fold dilution of the DNA extract led to an average increase in quantification of DNA by 3.3-fold that consequently increased test sensitivity of the qPCR from 55 to 80% compared to fecal culture. DNA extracts with higher DNA and protein content had 19.33 and 10.94 times higher odds of showing inhibition, respectively. The results suggest that the current test protocol is sensitive for herd level diagnosis of Johne’s disease but that test sensitivity and individual level diagnosis could be enhanced by relief of PCR inhibition, achieved by five-fold dilution of the DNA extract. Furthermore, qualitative and quantitative parameters derived from absorbance measures of DNA extracts could be useful for prediction of inhibitory fecal samples.

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.