RESUMO
Mycobacterium avium paratuberculosis (MAP) is causative agent of Johne's disease (JD) in domestic animals and has broad host range. JD infected animals shed viable MAP in their milk, feces, blood, and tissues which get transmitted to human beings directly or indirectly by consumption of animal products, through contact, animal handling and through contaminated environment, aerosols. In this current study, we developed hydrolysis probe based TaqMan® real-time PCR assay where samples were investigated by targeting IS900 mRNA and ModD gene to differentiate live MAP shedders from inactive/dead MAP bacilli shedding animals. The IS900 mRNA and ModD gene primers were designed using discontiguous unique conserved sequences of IS900 more towards the 3' end and fibronectin attachment protein (FAP) genes, respectively. Two different reporter dyes Cy5 and TexasRed, with compatible quenchers BHQ-1 and BHQ-2, respectively, were used for probe designing of IS900 and ModD genes. Triplex PCR assay was developed by using serially diluted positive MAP culture in log10 dilution and probe and template titration. TaqMan® probe real-time PCR targeting IS900 mRNA and ModD gene detects the MAP infection at early stage with high sensitivity and specificity. The specificity of developed TaqMan probe real-time PCR was found to be high while validated by using Escherichia coli and Staphylococcus aureus in addition to the MAP culture as there is no non-specific signal from other microbes. The sensitivity of developed TaqMan® probe real-time PCR was computed based on copy numbers ranged from 4.14 × 1011 to 4.14 × 104 for IS900 (FAM), 1.27 × 1011 to 1.27 × 104 for IS900 mRNA (Cy5), and 3.68 × 1010 to 3.68 × 104 for ModD (TexasRed), and lowest limit to detect MAP was 4.14 × 104, 1.27 × 104, and 3.68 × 104 copies for respective genes. This assay would be of great aid to contain the MAP infection in the large herd, where silent shedders spread active infection can be differentiated from passive shedding by non-infected animals. This test would also be equivalent to culture test in terms of specificity and hence can be able to be undertaken in molecular epidemiological studies to represent the actual disease prevalence in the future. KEY POINTS: ⢠Multiplex mRNA-based qPCR was developed to identify the actively infective MAP bacilli from passive ones. ⢠ModD and IS900 used as targets to assess active MAP bacilli in fecal samples of suspected animals. ⢠The LOD was computed using copy numbers with 4.14 × 104 and 3.68 × 104 copies for IS900 and ModD, respectively.
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Doenças dos Bovinos , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , DNA Bacteriano/genética , Fezes/microbiologia , Cabras/genética , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/diagnóstico , Paratuberculose/epidemiologia , Paratuberculose/microbiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: A 26-year-old male had a history of frequent bowel movements, mushy stool with mucus and loss of 25 kg body weight in 6 months was diagnosed as a case of inflammatory bowel disease (IBD). The patient did not respond to routine and standard treatment for IBD. His condition was steadily deteriorating, and he was in a very precarious state when he reported to us. METHODS: Upon laboratory investigation by using IS900 specific PCR [which is specific for Mycobacterium avium subspecies paratuberculosis (MAP)], the blood and stool samples were found negative. However, the presence of low titer MAP-antibodies by indigenous ELISA were found followed by detection of the typical acid-fast MAP bacilli (with 3 + or 4 + grade) microscopically. The MAP stool culture was positive after 6 months incubation. The biotyping by IS1311 specific polymerase chain reaction restriction enzyme (PCR-RE) confirmed infection with 'Indian Bison Type Genotype', a dominant biotype infecting the domestic livestock population of India. Standard anti-MAP therapy was initiated under supervision of the treating physician. The drug of choice in prescribed treatment regimen included Isoniazid (5 mg/kg), Rifampicin (10 mg/kg), Ethambutol (15-25 mg/kg) once a day for 24 weeks and Clarithromycin (250 mg)/Levofloxacin (250 mg) twice a day for 6 weeks. RESULTS: Following treatment, the patient started improving progressively with reduction in bowel movement frequency and gained body weight with an enhanced appetite propensity. Upon follow-up of the patient after 1 year of treatment, stool-microscopy and stool-culture were found negative for MAP. Till the recent past, the patient was further monitored for disease relapse, if any. CONCLUSIONS: This patient has experienced a complete resolution of IBD using a combination of anti-MAP antibiotics. The initial detection of heavy shedding of acid-fast MAP bacilli and typical colony morphology with its characterization obtained from culturing of stool sample indicated the infection of MAP. Interestingly, the present case is one more example of the linkage of demonstrable MAP infection treated with anti-MAP therapy in the presence and then absence of disease in the human host.
Assuntos
Doenças Inflamatórias Intestinais/microbiologia , Mycobacterium avium subsp. paratuberculosis/fisiologia , Paratuberculose/microbiologia , Adulto , Peso Corporal , Fezes/microbiologia , Humanos , Masculino , Mycobacterium avium subsp. paratuberculosis/classificaçãoRESUMO
For decades, Mycobacterium avium subspecies paratuberculosis (MAP) has been linked to the pathogenesis of Crohn's disease. Despite many investigations and research efforts, there remains no clear unifying explanation of its pathogenicity to humans. Proponents argue Crohn's disease shares many identical features with a granulomatous infection in ruminants termed Johne's disease and similarities with ileo-cecal tuberculosis. Both are caused by species within the Mycobacterium genus. Sceptics assert that since MAP is found in individuals diagnosed with Crohn's disease as well as in healthy population controls, any association with CD is coincidental. This view is supported by the uncertain response of patients to antimicrobial therapy. This report aims to address the controversial aspects of this proposition with information and knowledge gathered from several disciplines, including microbiology and veterinary medicine. The authors hope that this discussion will stimulate further research aimed at confirming or refuting the contribution of MAP to the pathogenesis of Crohn's disease and ultimately lead to advanced targeted clinical therapies.
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Doença de Crohn/microbiologia , Doença de Crohn/fisiopatologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/microbiologia , Paratuberculose/fisiopatologia , Animais , Ensaios Clínicos como Assunto/métodos , Doença de Crohn/genética , Humanos , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/genética , Reação em Cadeia da Polimerase/métodosRESUMO
BACKGROUND: Both bovine tuberculosis (TB) and paratuberculosis (PTB) are serious and widespread bacterial infections affecting many domestic and wild animal species. However, current vaccines do not confer complete protection and cause interference with other diagnostics tests, including bovine TB. Therefore, the development of "Differentiating Infected from Vaccinated Animals" (DIVA) tests are a pressing need. In this study, we have tested the feasibility of mycobacterial extracellular vesicles (EVs) as potential source of biomarkers to discriminate between Mycobacterium bovis infected, Mycobacterium avium subsp. paratuberculosis (MAP) infected and MAP-vaccinated cows. We have, initially, characterized vesicle production in the two most medically relevant species of mycobacteria for livestock, MAP and M. bovis, for being responsible for tuberculosis (TB) and paratuberculosis (PTB). RESULTS: Our results indicate that these two species produce EVs with different kinetics, morphology and size distribution. Analysis of the immunogenicity of both type of EVs showed some cross reactivity with sera from PTB+ and TB+ cows, suggesting a limited diagnostic capacity for both EVs. Conversely, we noticed that Mycobacterium tuberculosis (Mtb) EVs showed some differential reactivity between sera from MAP-vaccinated or PTB+ cows from TB+ ones. Mass spectrometry analysis (MS) identified a 19-kDa EV-associated lipoprotein as the main source of the differential reactivity. CONCLUSIONS: LpqH could be a good plasma biomarker with capacity to distinguish PTB+ or MAP-vaccinated cows from cows infected with TB.
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Doenças dos Bovinos/microbiologia , Vesículas Extracelulares/química , Lipoproteínas/análise , Mycobacterium tuberculosis/química , Paratuberculose/diagnóstico , Tuberculose Bovina/diagnóstico , Animais , Vacinas Bacterianas , Biomarcadores/sangue , Bovinos , Reações Cruzadas , Mycobacterium avium subsp. paratuberculosis/química , Mycobacterium bovis/química , Vacinação/veterináriaRESUMO
This technical research communication describes the first study to use quantitative polymerase chain reaction (qPCR) to investigate the presence of Mycobacterium avium complex (MAC) in Australian pasteurised milk. MAC is the most common NTM responsible for human illnesses and includes M. avium subspecies paratuberculosis (MAP). MAC is a causative agent of lymphadenitis in children, with contaminated food and water considered as a likely source. As such the presence of MAC in milk would have public health significance. MAP has been linked to Crohn's disease and is also the causative agent of Johne's disease in cattle. Previous studies have detected MAP in pasteurised milk from Brazil, India, Czech Republic, USA, Argentina, UK, Iran, Ireland and the United Kingdom. This study investigated a total of 180 commercially available Australian pasteurised milk samples which were tested for MAC DNA in triplicate using PCR. All samples were negative for MAC DNA. An additional 14 milk samples were tested, incubated for 3 weeks at 37 °C to potentially increase the concentration of any viable MAC that may be present and then retested. All samples were again negative for MAC DNA. This could be due to concentrations below the limit of detection, limited sample size or could be reflective of the Australian biosecurity control protocols and surveillance of Johne's disease in ruminant animals.
Assuntos
Leite/microbiologia , Complexo Mycobacterium avium/genética , Complexo Mycobacterium avium/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Animais , Austrália , Bovinos , Doença de Crohn/microbiologia , DNA Bacteriano/análise , Humanos , Limite de Detecção , Paratuberculose/microbiologia , PasteurizaçãoRESUMO
The control of Johne's disease requires the identification of Mycobacterium avium ssp. paratuberculosis (MAP)-positive herds. Boot swabs and liquid manure samples have been suggested as an easy-to-use alternative to sampling individual animals in order to diagnose subclinical Johne's disease at the herd level, but there is a need to evaluate performance of this approach in the field. Using a logistic regression model, this study aimed to calculate the threshold level of the apparent within-herd prevalence as determined by individual faecal culture, thus allowing the detection of whether a herd is MAP positive. A total of 77 boot swabs and 75 liquid manure samples were taken from 19 certified negative and 58 positive dairy herds. Faecal culture, three different polymerase chain reaction (PCR) methods and the combination of faecal culture with PCR were applied in order to detect MAP. For 50% probability of detection, a within-herd prevalence threshold of 1·5% was calculated for testing both matrices simultaneously by faecal culture and PCR, with the threshold increased to 4·0% for 90% probability of detection. The results encourage the use of boot swabs or liquid manure samples, or a combination both, for identifying MAP-positive herds and, to a certain extent, for monitoring certified Johne's disease-negative cattle herds.
Assuntos
Doenças dos Bovinos/epidemiologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/epidemiologia , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Feminino , Alemanha/epidemiologia , Modelos Logísticos , Esterco/microbiologia , Paratuberculose/microbiologia , Prevalência , Reação em Cadeia da Polimerase em Tempo Real/veterináriaRESUMO
Recognition of bacterial peptidoglycan-derived muramyl-dipeptide (MDP) by nucleotide oligomerization domain 2 (NOD2) induces crucial innate immune responses. Most bacteria carry the N-acetylated form of MDP (A-MDP) in their cell membranes, whereas N-glycolyl MDP (G-MDP) is typical for mycobacteria. Experimental murine studies have reported G-MDP to have a greater NOD2-stimulating capacity than A-MDP. As NOD2 polymorphisms are associated with Crohn's disease (CD), a link has been suggested between mycobacterial infections and CD. Thus, the aim was to investigate if NOD2 responses are dependent upon type of MDP and further to determine the role of NOD2 gene variants for the bacterial recognition in CD. The response pattern to A-MDP, G-MDP, Mycobacterium segmatis (expressing mainly G-MDP) and M. segmatisΔnamH (expressing A-MDP), Listeria monocytogenes (LM) (an A-MDP-containing bacteria) and M. avium paratuberculosis (MAP) (a G-MDP-containing bacteria associated with CD) was investigated in human peripheral blood mononuclear cells (PBMCs). A-MDP and M. segmatisΔnamH induced significantly higher tumour necrosis factor (TNF)-α protein levels in healthy wild-type NOD2â PBMCs compared with G-MDP and M. segmatis. NOD2 mutations resulted in a low tumour necrosis factor (TNF)-α protein secretion following stimulation with LM. Contrary to this, TNF-α levels were unchanged upon MAP stimulation regardless of NOD2 genotype and MAP solely activated NOD2- and Toll-like receptor (TLRs)-pathway with an enhanced production of interleukin (IL)-1ß and IL-10. In conclusion, the results indicate that CD-associated NOD2 deficiencies might affect the response towards a broader array of commensal and pathogenic bacteria expressing A-MDP, whereas they attenuate the role of mycobacteria in the pathogenesis of CD.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/imunologia , Doença de Crohn/imunologia , Leucócitos Mononucleares/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Infecções por Mycobacterium/imunologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Mycobacterium smegmatis/imunologia , Proteína Adaptadora de Sinalização NOD2/genética , Acetilação , Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Acetilmuramil-Alanil-Isoglutamina/química , Células Cultivadas , Doença de Crohn/etiologia , Doença de Crohn/microbiologia , Citocinas/metabolismo , Análise Mutacional de DNA , Predisposição Genética para Doença , Glicóis/química , Humanos , Imunidade Inata/genética , Espaço Intracelular/microbiologia , Leucócitos Mononucleares/microbiologia , Listeriose/complicações , Listeriose/microbiologia , Ativação Linfocitária/genética , Mutação/genética , Infecções por Mycobacterium/complicações , Infecções por Mycobacterium/microbiologia , Mycobacterium smegmatis/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Polimorfismo de Nucleotídeo Único , Transdução de Sinais , Especificidade da Espécie , Receptores Toll-Like/metabolismoRESUMO
Crohn's disease (CD) is a multifactorial chronic disorder that involves a combination of factors, including genetics, immune response, and gut microbiota. Therapy includes salicylates, immunosuppressive agents, corticosteroids, and biologic drugs. International guidelines do not recommend the use of antibiotics for CD patients, except in the case of septic complications. Increasing evidence of the involvement of gut bacteria in this chronic disease supports the rationale for using antibiotics as the primary treatment for active CD. In recent decades, several pathogens have been reported to be involved in the development of CD, but only Escherichia coli (E. coli) and Mycobacterium avium paratubercolosis (MAP) have aroused interest due to their strong association with CD pathogenesis. Several meta-analyses have been published concerning antibiotic treatment for CD patients, but randomized trials testing antibiotic treatment against E. coli and MAP have not shown prolonged benefits and have generated conflicting results; several questions are still unresolved regarding trial design, antibiotic dosing, the formulation used, the treatment course, and the outcome measures. In this paper, we provide an overview and update of the trials testing antibiotic treatment for active CD patients, taking into account the role of pathogens, the mechanisms by which different antibiotics act on harmful pathogens, and antibiotic resistance. Finally, we also present new lines of study for the future regarding the use of antibiotics to treat patients with active CD.
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Paratuberculosis (PTB) has been reported in the Sudan in cattle and goats for more than 50 years but has never been reported in sheep. However, suspicion of the disease in a breeding flock of sheep in Khartoum North locality was made due to a history of unknown cause of loss of weight. Blood and faecal samples were collected from all animals (N = 59): harvested sera were tested for anti-Mycobacterium avium subsp. paratuberculosis (MAP) antibodies by Enzyme Linked Immunosorbent Assay (ELISA); faeces were screened for acid-fast bacilli by Ziehl-Neelsen staining, tested for MAP DNA by recombinase polymerase amplification (RPA) and some faecal samples were cultured for MAP isolation. Typical MAP acid-fast bacilli were seen in 10.2% (6/59) of the faecal smears, 37.5% of the tested faecal samples (12/32) were positive for MAP DNA and only 3 (5.1%) animals were seropositive for MAP. MAP positive cultures were obtained from 2 out the 6 samples showing typical MAP acid-fast bacilli; the isolates were confirmed by real-time PCR and sequencing. As sheep are animals of utmost economic importance as the main export animals for the country, this first report of ovine PTB warrants special considerations and more investigations for planning control programmes of the disease.
RESUMO
Uncoated tosyl-activated magnetic beads were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Centrifugation and uncoated magnetic beads recovered more than 99% and 93%, respectively, of 1.68 × 107 CFU/mL, 1.68 × 106 CFU/mL and 1.68 × 105 CFU/mL M. smegmatis cells resuspended in phosphate buffer saline. The use of magnetic beads was more efficient to concentrate cells from 1.68 × 104 CFU/mL of M. smegmatis than centrifugation. Likewise, the F57-qPCR detection of MAP cells was different whether they were recovered by beads or centrifugation; cycle threshold (Ct) was lower (p < 0.05) for the detection of MAP cells recovered by beads than centrifugation, indicative of greater recovery. Magnetic separation of MAP cells from milk, urine, and feces specimens was demonstrated by detection of F57 and IS900 sequences. Beads captured no less than 109 CFU/mL from feces and no less than 104 CFU/mL from milk and urine suspensions. In another detection strategy, M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29. Plaque forming units were observed after 24 h of incubation from urine samples containing 2 × 105 and 2 × 103 CFU/mL M. smegmatis. The results of this study provide a promising tool for diagnosis of tuberculosis and Johne's disease.
Assuntos
Doenças dos Bovinos , Micobacteriófagos , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Fezes/microbiologia , Fenômenos Magnéticos , Leite/microbiologia , Micobacteriófagos/genética , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium smegmatis/genética , Paratuberculose/diagnóstico , Paratuberculose/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e EspecificidadeRESUMO
Wild carnivores are at the top of the trophic chain. They are predators and carrion consumers, and thus, prone to come in contact with disease agents contaminating the environment or infecting live or dead animals. We hypothesized that wild canids could be used as sentinels for the detection of regions with higher Mycobacterium avium paratuberculosis (MAP) prevalence in wild and domestic animals. To test this hypothesis, we set up an ELISA to test 262 wolf (Canis lupus) and fox (Vulpes vulpes) sera for MAP-specific antibodies and processed a subset of samples for culture (n = 61), MAP-specific PCR (15) and histopathology (14). In wolves, the optical density (OD) values in the ELISA were continuously distributed. Ten fox sera (4%) had OD readings of over twice the mean, suggesting contact with mycobacteria. However, all samples tested by PCR were negative for both IS900 and ISMAP02 sequences, and samples cultured for MAP yielded no growth. No visible paratuberculosis or tuberculosis-compatible lesions were recorded. On histopathological examination, no lesions compatible with mycobacterial diseases were observed. These results suggest that wild canids show little or no evidence of paratuberculosis and are unlikely to be useful sentinels for the detection of MAP in Southwestern Europe.
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BACKGROUND: The role of Mycobacterium avium paratuberculosis [MAP] in inflammatory bowel disease [IBD], especially Crohn's disease [CD] is controversial due conflicting results and lack of reproducibility and standardised tests. The current study focuses on the role of MAP in disease progression and genetic susceptibility, as MAP is likely one of many factors involved in the complex pathogenesis of IBD, potentially affecting a subgroup depending on genetic susceptibility. METHODS: Serum from 812 patients was evaluated with seven immunoglobulin [Ig] isotype-specific serology tests assessing humoral response to three different MAP antigens. For each of these in total 21 tests, the intra-assay and inter-assay coefficients were used to evaluate test accuracy. Reliable assays were subsequently analysed in relation to disease characteristics and need for biologic therapy/surgery. Genome-wide genotyping was available for all participants. Genetic determinants of humoral response to MAP antigens were evaluated using genome-wide association analysis and polygenic risk scores [PRS]. RESULTS: High IgA or IgM response to MAP2609 was associated with increased use of biologic therapy in CD and ulcerative colitis [UC] [odds ratios 2.69; 95% confidence interval 1.44-5.01; and 2.60, 1.46-4.64, respectively]. No associations were seen for risk of surgery [p-valuesâ >â 0.29]. We could not identify genetic determinants nor polygenic risk scores for MAP response with genome-wide significance. CONCLUSIONS: Extensive assays for serological response to MAP were evaluated using stringent criteria for reliability. Increased IgA and IgM response to MAP antigens was seen in patients exposed to biologic therapy, but no genetic determinants underlying this humoral response were found.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Terapia Biológica , Doenças Inflamatórias Intestinais/tratamento farmacológico , Mycobacterium avium subsp. paratuberculosis/imunologia , Estudos de Coortes , Estudos Transversais , Feminino , Estudo de Associação Genômica Ampla , Humanos , Imunoglobulina A/sangue , Imunoglobulina M/sangue , Doenças Inflamatórias Intestinais/imunologia , Masculino , Pessoa de Meia-Idade , Mycobacterium avium subsp. paratuberculosis/genética , Reprodutibilidade dos TestesRESUMO
Paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic wasting disease mainly of domestic and wild ruminants. It occurs worldwide, causing significant economic losses through decreased productivity, low fertility, increased cull rates and mortality. It is listed by the OIE (World Organization for Animal Health) as a disease of concern to trade in animals. Prevalence of this disease can be studied by detecting anti-MAP antibodies by Enzyme linked immunosorbent Assay (ELISA). The aim of this study was to investigate the current prevalence of MAP infection in cattle in Khartoum State. The overall apparent prevalence of MAP infection was found to be 6.3% and 18.9% at animal and herd levels, respectively. All seropositive animals were cross-bred females of good body condition; most of them (>90%) were >3 years old and >50% were from medium-sized herds in Omdurman. No significant association (p > 0.05) was found between seropositivity and animal herd size. The prevalence of MAP infection in Khartoum State is still low to medium compared to other parts of the world, but it is comparable to those reported from other African countries. Further studies with the view of designing nationwide surveys in domestic ruminants and camels in other states of the country are needed for establishing control programmes.
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Crohn's disease is increasing in incidence and prevalence in younger people and is of a particularly aggressive nature. One emerging treatment targets Mycobacterium avium paratuberculosis (MAP), an organism implicated in the causation of Crohn's disease. This study reviewed a cohort of paediatric patients with active Crohn's disease treated with Anti-Mycobacterial Antibiotic Therapy (AMAT). Sixteen paediatric patients, the majority of whom had failed conventional immunosuppressive therapy, were treated with AMAT. Endoscopic remission was scored using the Simple Endoscopic Score for Crohn's Disease and clinical remission was assessed using the Weighted Paediatric Crohn's Disease Activity Index (wPCDAI). Inflammatory blood markers were also routinely recorded. Patients were followed up clinically and endoscopically during treatment after an average of two months (range 1-6) and 17 months (range 2-49), respectively. A significant reduction in both scores assessing clinical improvement (p < 0.001) and mucosal healing (p < 0.0078) was observed at these timepoints; 47% of patients had achieved clinical remission and 63% endoscopic remission. Haemoglobin and serum inflammatory markers normalised for more than 50% of the cohort by six months of treatment. No adverse effects were reported throughout treatment. This is the first report of Anti-Mycobacterial Antibiotic Therapy offering a safe and efficacious therapy for paediatric patients with Crohn's disease. Further larger randomised studies are required in order to validate these findings.
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We present and discuss a 30-month investigation of a patient that presented with abdominal pain, postprandial diarrhea, bloating, and night sweats and was treated for Crohn's disease without significant improvement. The patient underwent an ileocecetomy with removal of an atonic segment with resolution of functional gastrointestinal symptoms, but profound night sweats continued postoperatively. The patient was presumptively treated for a mixed mycobacterial infection, blood cultures later grew Mycobacterium avium paratuberculosis (MAP), and she improved over time. We discuss MAP and its possible relationship to Crohn's disease.
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Mycobacterium avium paratuberculosis (Map) is a pathogen which causes a chronic progressive granulomatous enteritis known as paratuberculosis or Johne's disease and it primarily affects wild and domestic ruminants. The aim of this research was to examine a flock which consisted of 294 goats and was located in Garfagnana district (Tuscany, Italy) performing ELISA tests, culture and IS900 PCR assay; direct diagnostic methods were carried out not only on bulk tank milk and cheese samples but also on individual milk and tissue specimens collected from nine subjects positive to ELISA tests. Out of 294 animals, 20 goats (6.8%) were positive to ELISA surveys. Bulk tank milk samples were negative to culture and to PCR assay carried out on the DNA extracted directly from them, while, with respect to cheese, Map was detected by culture in 2/12 (16.66%) cheeses ripened for 3-7 days, and by PCR in 2/12 (16.66%) cheeses ripened for 3-7 days and in 3/12 (25%) cheeses ripened for 45 days. Regarding individual milk samples, Map was detected by culture in 2/9 (22.22%) specimens and by PCR in 5/9 (55.55%) samples. Furthermore, Map was isolated from the intestine in 9/9 (100%) animals, from the mesenteric lymph nodes in 8/9 (88.88%) subjects, from the liver in 4/9 (44.44%) goats, from the spleen in 5/9 (55.55%) animals, while Map DNA was found in all the tissue samples analyzed.The results demonstrated the presence of paratuberculosis in a goat flock located in Garfagnana district (Tuscany, Italy).
Assuntos
Doenças das Cabras/microbiologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/microbiologia , Animais , Queijo/microbiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Doenças das Cabras/diagnóstico , Doenças das Cabras/epidemiologia , Cabras , Itália/epidemiologia , Masculino , Leite/microbiologia , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/diagnóstico , Paratuberculose/epidemiologia , Reação em Cadeia da PolimeraseRESUMO
Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease in cattle, is responsible for significant economic losses to the US dairy industry. The pathogen has also been associated with chronic human diseases like Crohn's disease, type 1 diabetes and multiple sclerosis. Determining causation requires rapid characterization and source tracking the pathogen. Here, we used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize and differentiate strains of MAP from 14 other species of Mycobacterium from bovine, human, and environmental sources. Lysates from cells disrupted by bead beating in TFA-acetonitrile solution were analyzed by MALDI-TOF. MAP strains were differentiated by mass spectral profiles that are distinct from each other and from other Mycobacterium species. Cluster analysis of spectral profiles indicates two distinct clusters, one dominated by the members of avium complex and a second group dominated by members of fortuitum and parafortuitum complexes. We believe that MALDI-TOF methods can be used to differentiate and source-track MAP strains.
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Técnicas Bacteriológicas/métodos , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Mycobacterium/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Bovinos , Análise por Conglomerados , Doença de Crohn/diagnóstico , Doença de Crohn/microbiologia , Humanos , Mycobacterium/patogenicidade , Mycobacterium avium subsp. paratuberculosis/patogenicidade , Paratuberculose/diagnóstico , Paratuberculose/microbiologia , Fatores de TempoRESUMO
Examination of samples of stool from a 61-year-old male patient, presenting with the clinical symptoms of Crohn's disease (CD), revealed massive shedding of acid fast bacilli with the morphology of Mycobacterium avium paratuberculosis (MAP), the causative agent of Johne's disease in cattle. MAP was cultured from the stool. Biotyping of the bacterium isolated from cultures of stool demonstrated, it was the Indian Bison biotype of MAP, the dominant biotype infecting livestock and humans in India. Based on this finding and because the patient was unresponsive to standard therapy used in India to treat patients with gastrointestinal inflammatory disorders, the patient was placed on a regimen of multi-antibiotic therapy, currently used to treat tuberculosis and CD. After 1 year of treatment, the patient's health was restored, concurrent with cessation of shedding of MAP in his stool. This patient is the first case shown to shed MAP from the stool who was cured of infection with antibiotics and who was concurrently cured of clinical signs of CD.
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A cohort of family members with various chronic diseases including Crohn's disease, asthma, complex regional pain syndrome, hypothyroidism, type 1 diabetes mellitus, and lymphangiomatosis and/or evidence of infection by Mycobacterium avium subsp. paratuberculosis (MAP) are described in this series of case reports. MAP was cultured from the blood of three members affected by the first five diseases and there was accompanying elevated anti-MAP IgG in two members. The patient affected by the sixth disease has a markedly elevated anti-MAP titer. The two patients affected by the first four diseases have been treated with a combination of anti-MAP antibiotics and ultraviolet blood irradiation therapy with resolution of the disease symptomatology and inability to culture MAP in post treatment blood samples. These case reports of patients with MAP infections provide supportive evidence of a pathogenic role of MAP in humans.
Assuntos
Antituberculosos/uso terapêutico , Síndromes da Dor Regional Complexa/terapia , Doença de Crohn/terapia , Mycobacterium avium subsp. paratuberculosis/efeitos dos fármacos , Mycobacterium avium subsp. paratuberculosis/efeitos da radiação , Paratuberculose/terapia , Terapia Ultravioleta/métodos , Adulto , Técnicas Bacteriológicas , Biópsia , Criança , Colonoscopia , Terapia Combinada , Síndromes da Dor Regional Complexa/diagnóstico , Síndromes da Dor Regional Complexa/microbiologia , Doença de Crohn/diagnóstico , Doença de Crohn/microbiologia , Feminino , Humanos , Masculino , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/diagnóstico , Paratuberculose/microbiologia , Paratuberculose/transmissão , Indução de Remissão , Resultado do Tratamento , Adulto JovemRESUMO
OBJECTIVES: Prior to the advent of therapies with sustained virological response rates of 94%, this study was conducted for the US Food and Drug Administration (FDA) to assess the safety and efficacy of ultraviolet blood irradiation (UVBI) for the treatment of hepatitis C virus (HCV) infection. METHODS: Nine patients received 15 UVBI treatments over the course of 22 weeks with the AVIcure Hemo-modulator, which was modified from the original Knott Hemo-irradiator. The patients' viral loads and liver function tests were obtained periodically during the study and analyzed during the course of the trial. RESULTS: At the end of the study, the overall mean reduction in HCV viral load was 21.5% (p = 0.023); on day 140, direct bilirubin declined by 41.1% (p=0.0059), aspartate aminotransferase declined by 15.2% (p=0.0069), and alanine aminotransferase declined by 19.3% (p=0.0031). The nadir of the mean and median viral load occurred on day 259, and it corresponded to a mean viral load reduction of 44.9% (p=0.0048). During the course of the study, three patients had a greater than 0.5 log reduction in viral load (patient 1, 0.56 log reduction on day 259; patient 4, 0.69 log reduction at the end of the study; patient 11, 0.91 log reduction on day 259). Two patients showed marked improvement in their concurrent psoriasis at the conclusion of the trial. CONCLUSIONS: In this study, UVBI was safe and had a beneficial effect in the treatment of HCV. This device should be studied for use in psoriasis and in infectious diseases that have few treatment options. This article describes a prospective, controlled, phase II clinical trial submitted to the FDA of this device used for the treatment of HCV infection (Investigational Device Exemption (IDE) #G030242).