Phylogenetic uniqueness of Mycobacterium avium subspecies hominissuis isolated from an abnormal pulmonary bovine case.

Mycobacterium avium subspecies hominissuis (MAH) is an important cause of infection in human pulmonary and swine intestinal cases. Although MAH is isolated from environmental sources frequently, infections of other animals have rarely been analysed. Recently, we detected granulomatous inflammation in bovine lung as an abnormal postmortem inspection case. To ascertain its genetic profile, we conducted a variable numbers of tandem repeats (VNTR) analysis and genomic characterization using deep sequencing. The VNTR type was a unique profile that differed from reported genotypes, but it was assigned within a broad genotypic complex of isolates from human patients and bathrooms. Genomic comparison with 116 registered genome sequences of the subspecies revealed that the strain was separate from five major genetic population groups proposed previously. Although the infection source remains unclear, its isolation from various resources such as animal infection cases should be elucidated more extensively to reveal its genetic diversity and ecological context.

Virulence of Mycobacterium avium Subsp. hominissuis Human Isolates in an in vitro Macrophage Infection Model.

Background: Mycobacterium avium subsp. hominissuis (MAH) is an environmental opportunistic pathogen for humans and swine worldwide; in humans, the vast majority of MAH infections is due to strains belonging to specific genotypes, such as the internal transcribed spacer (ITS)-sequevars Mav-A and Mav-B that mostly cause pulmonary infections in elderly patients and severe disseminated infections in acquired immunodeficiency syndrome patients, respectively. To test whether the different types of infections in distinct patients' populations might reflect a different virulence of the infecting genotypes, MAH human isolates, genotyped by ITS sequencing and MIRU-VNTR minisatellite analysis, were studied for the capacity to infect and replicate in human macrophages in vitro. Methods: Cultures of human peripheral blood mononuclear cells and phagocytic human leukemic cell line THP-1 cells were infected with each MAH isolate and intracellular colony-forming units (CFU) were determined. Results: At 2 h after infection, i.e., immediately after cell entry, the numbers of intracellular bacteria did not differ between Mav-A and Mav-B organisms in both phagocytic cell types. At 5 days, Mav-A organisms, sharing highly related VNTR-MIRU genotypes, yielded numbers of intracellular CFUs significantly higher than Mav-B organisms in both phagocytic cell types. MIRU-VNTR-based minimum spanning tree analysis of the MAH isolates showed a divergent phylogenetic pathway of Mav-A and Mav-B organisms. Conclusion: Mav-A and Mav-B sequevars might have evolved different pathogenetic properties that might account for their association with different human infections.

Evidence of disseminated infection by Mycobacterium avium subspecies hominissuis in a pet ferret (Mustela putorius furo).

The infection caused by the zoonotic opportunistic pathogen Mycobacterium avium subsp. hominissuis (Mah) was reported for the first time in a pet ferret. Both owners were HIV-positive. Euthanasia of the pet was recommended due to medical reasons and as a preventive action. Disseminated and open tuberculosis lesions were observed in the gastrointestinal and respiratory systems of the ferret. Ecographic and radiographic surveys showed a severe generalized lymphadenopathy, strong thickening of the gastric wall and peritoneum layer. The histopathological findings revealed a disseminated, granulomatous, chronic inflammation affecting the gastrointestinal tract, lungs, lymphoid tissues (spleen, tonsils and lymph nodes) and liver. Ziehl-Neelsen staining displayed the presence of positive acid-fast bacilli within these granulomas. Bacteriology and sequencing of the isolates yielded Mah sequevar code 3. Ferrets can act as reservoirs of mycobacteria exposing their owners to the infection, which is of major concern in immunodeficient individuals, as those HIV-infected.

Outbreak of Avian Tuberculosis in Commercial Domestic Pekin Ducks ( Anas platyrhynchos domestica).

Avian tuberculosis is a contagious disease affecting various domestic and wild bird species, and is caused by Mycobacterium avium . It is reported extremely rarely in commercial poultry flocks and has not been reported in commercial domestic ducks to date, with domestic ducks reported to be moderately resistant to M. avium infection. Here, we report the outbreak of avian tuberculosis in commercial Pekin duck ( Anas platyrhynchos domestica) flocks. Postmortem and histopathologic findings included nodules presenting in the visceral organs of ducks, and granulomas with central caseous necrosis surrounded by infiltrating lymphocytes. The M. avium pathogen was isolated and further identified by Ziehl-Neelsen staining and PCR based on insert sequence IS901 and the 16S rRNA gene. We highlight that avian tuberculosis not only has economic significance for the duck industry, but also presents a potential zoonotic hazard to humans.

Detection of Mycobacterium avium subspecies in the gut associated lymphoid tissue of slaughtered rabbits.

BACKGROUND: Rabbits are susceptible to infection by different species of the genus Mycobacterium. Particularly, development of specific lesions and isolation of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis, both subspecies of the M. avium complex, has been reported in wildlife conditions. Although, rabbit meat production worldwide is 200 million tons per year, microbiological data on this source of meat is lacking and more specifically reports of mycobacterial presence in industrially reared rabbit for human consumption have not been published. To this end, we sought mycobacteria by microbiological and histopathological methods paying special attention to Mycobacterium avium subsp. paratuberculosis in rabbits from commercial rabbitries from the North East of Spain. RESULTS: M. avium subsp. paratuberculosis was not detected either by culture or PCR. However, Mycobacterium avium subsp. avium was detected in 15.15% (10/66) and Mycobacterium avium subsp. hominissuis was detected in 1.51% (1/66) of gut associated lymphoid tissue of sampled animals by PCR, whereas caecal contents were negative. 9% (6/66) of the animals presented gross lesions suggestive of lymphoid activation, 6% (4/66) presented granulomatous lesions and 3% (2/66) contained acid fast bacilli. Mycobacterial isolation from samples was not achieved, although colonies of Thermoactinomycetes sp. were identified by 16s rRNA sequencing in 6% (4/66) of sampled animals. CONCLUSIONS: Apparently healthy farmed rabbits that go to slaughter may carry M. avium subspecies in gut associated lymphoid tissue.

Avaliação da atividade antimicobacteriana da lignana diidrocubebina extraída da Piper cubeba e de seus derivados semissintéticos / Evaluation of antimycobacterial activity of dihydrocubebin lignan extracted from Piper cubeba and its semisynthetic derivatives

RESUMO: A atividade antimicobacteriana de diidrocubebina (1), uma lignana dibenzilbutanodioica obtida a partir de extrato etanólico de sementes da Piper cubeba, e seus derivados foram avaliados in vitro contra três diferentes cepas de Mycobacterium utilizando o método de microdiluição. Dentre as lignanas avaliadas 3 e 4 foram as mais ativas, exibindo valores de CIM de 62,5 µg/mL contra M. avium e M. tuberculosis, respectivamente. Os derivados 2-6 obtidos por síntese parcial possuem diferentes substituintes nos carbonos 9 e 9 ', que alteram polaridade, solubilidade e limitam as rotações livres entre C8-C8' em relação de material (1) de partida. As diferenças estruturais entre estes compostos podem fornecer informações importantes sobre a relação estrutura-atividade antimicobacteriana do esqueleto dibenzilbutanodioico, obtido a partir de fonte natural, como um possível alvo para o desenvolvimento de drogas mais potentes contra a tuberculose

ABSTRACT: Evaluation of antimycobacterial activity of dihydrocubebin lignan extracted from Piper cubeba and its semisynthetic derivatives. The antimycobacterial activity of the dihydrocubebin (1), a dibenzylbutanedioiclignan obtained from ethanolic extract of Piper cubeba seeds, and its derivatives were examined in vitro against three different strains of Mycobacterium using amicrodilution method. Among the lignans evaluated, the 3 and 4 samples were the most active ones, displaying MIC values of 62.5 µg/mL against M. avium and M. tuberculosis, respectively. The derivatives 2-6, obtained for partial synthesis, had different substituents in the carbons 9 and 9', fact thatalters the polarity, solubility and restricts the free rotations between the bonds C8-C8' in relation to the starting material (1). The structural differences among these compounds provide important information about the antimycobacterial structure-activity relationship of the dibenzylbutanodioic skeleton, obtained from natural source, such as a possible target for the development of more powerful drugs against tuberculosis

Subspecies identification and significance of 257 clinical strains of Mycobacterium avium.

Mycobacterium avium is abundant in the environment. It has four subspecies of three types: the human or porcine type, M. avium subsp. hominissuis; the bird type, including M. avium subsp. avium serotype 1 and serotype 2, 3 (also M. avium subsp. silvaticum); and the ruminant type, M. avium subsp. paratuberculosis. We determined the subspecies of 257 M. avium strains isolated from patients at the M.D. Anderson Cancer Center from 2001 to 2010 and assessed their clinical significance. An assay of multiplex PCR was used for the typing. Results showed M. avium subsp. hominissuis to be most common (n = 238, 92.6%), followed by M. avium subsp. avium serotype 1 (n = 12, 4.7%) and serotype 2, 3 (n = 7, 2.7%). No strains of M. avium subsp. paratuberculosis were found. Of the 238 patients with M. avium subsp. hominissuis, 65 (27.3%) showed evidence of definite or probable infections, mostly in the respiratory tract, whereas the rest had weak evidence of infection. The bird-type subspecies, despite being infrequently isolated, caused relatively more definite and probable infections (10 of 19 strains, 52.6%). Overall, women of 50 years of age or older were more prone to M. avium infection than younger women or men of all ages were. We therefore conclude that M. avium subsp. hominissuis is the dominant M. avium subspecies clinically, that the two bird-type subspecies do cause human infections, and that M. avium infects mainly postmenopausal women. The lack of human clinical isolation of the ruminant type subspecies may need further investigation.

Mycobacterium avium subsp. avium and subsp. hominissuis give different cytokine responses after in vitro stimulation of human blood mononuclear cells.

BACKGROUND: Mycobacterium avium is the principal etiologic agent of non-tuberculous lymphadenitis in children. It is also a known pathogen for birds and other animals. Genetic typing of M. avium isolates has led to a proposal to expand the set of subspecies to include M. avium subsp. hominissuis. Isolates associated with disease in humans belong to this subspecies. METHODOLOGY/PRINCIPAL FINDINGS: Peripheral blood mononuclear cells from six healthy blood donors were stimulated in vitro with ten isolates of M. avium avium and 11 isolates of M. avium hominissuis followed by multiplex bead array quantification of cytokines in supernatants. M. avium hominissuis isolates induced significantly more IL-10 and significantly less IL-12p70, TNF, IFN-γ and IL-17 when compared to M. avium avium isolates. All strains induced high levels of IL-17, but had very low levels of IL-12p70. CONCLUSION/SIGNIFICANCE: The strong association between M. avium subsp. hominissuis and disease in humans and the clear differences in the human immune response to M. avium subsp. hominissuis compared to M. avium subsp. avium isolates, as demonstrated in this study, suggest that genetic differences between M. avium isolates play an important role in the pathogenicity in humans.

Different immune response of pigs to Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis infection.

Mycobacterium avium subsp. avium (MAA) and Mycobacterium avium subsp. hominissuis (MAH) are the most common mycobacterial species isolated from granulomatous lesions in swine in countries with controlled bovine tuberculosis. This study is focused on the immunological aspect of MAA and MAH infection in pigs. We detected induction of humoral and cell-mediated immunity in experimentally infected pigs. Specific antibodies were analyzed in serum by ELISA and the IFN-γ release assay was used for evaluation of cell-mediated immunity. While MAA induced a significant increase of both types of immune responses, MAH-infected pigs had an unvarying level of specific antibodies and showed low cell-mediated immunity with high individual variability. The subsequent in vitro experiment confirmed the lower immunogenicity of the MAH strain in comparison to MAA. MAH-infected porcine monocyte-derived macrophages showed a weaker induction of pro-inflammatory mediators in comparison to MAA, which included mRNA for IL-1ß, TNF-α, IL-23p19, IL-18 and chemokines CCL-3, CCL-5, CXCL-8 and CXCL-10. Additionally, qualitative proteomic analysis revealed 28 proteins exclusively in MAA and 7 proteins unique to MAH. In conclusion, closely related M. avium subspecies MAA and MAH showed different capacities to stimulate the porcine immune system. From a diagnostic point of view, the IFN-γ release assay showed higher sensitivity than the detection of specific antibodies by ELISA and seems to be an effective tool for discrimination of MAA-infected pigs. In the case of MAH infection, the IFN-γ release assay could fail because of the low immunogenic capacity of the MAH strain.

Surface proteome of "Mycobacterium avium subsp. hominissuis" during the early stages of macrophage infection.

"Mycobacterium avium subsp. hominissuis" is a robust and pervasive environmental bacterium that can cause opportunistic infections in humans. The bacterium overcomes the host immune response and is capable of surviving and replicating within host macrophages. Little is known about the bacterial mechanisms that facilitate these processes, but it can be expected that surface-exposed proteins play an important role. In this study, the selective biotinylation of surface-exposed proteins, streptavidin affinity purification, and shotgun mass spectrometry were used to characterize the surface-exposed proteome of M. avium subsp. hominissuis. This analysis detected more than 100 proteins exposed at the bacterial surface of M. avium subsp. hominissuis. Comparisons of surface-exposed proteins between conditions simulating early infection identified several groups of proteins whose presence on the bacterial surface was either constitutive or appeared to be unique to specific culture conditions. This proteomic profile facilitates an improved understanding of M. avium subsp. hominissuis and how it establishes infection. Additionally, surface-exposed proteins are excellent targets for the host adaptive immune system, and their identification can inform the development of novel treatments, diagnostic tools, and vaccines for mycobacterial disease.

Isolation and identification of Mycobacterium avium subspecies silvaticum from a horse.

Routine cultivation methods are able to distinguish between isolates of the Mycobacterium avium and the Mycobacterium tuberculosis complex. However, molecular tools are needed to further identify the several subspecies in the M. avium complex, especially for the subspecies avium and silvaticum. A rapid technique using HhaI restriction digestion of a 349 bp amplification product of the 85B antigen (α-antigen) gene was used for the identification of M. avium subsp. silvaticum in a three-year-old gelding presenting with caseous, necrotizing, granulomatous lesions. The result was confirmed by sequencing of the 85B antigen gene.

Mycobacterium avium subspecies hominissuis disseminated infection in a Basset Hound dog.

In the current report, a case in Italy of disseminated Mycobacterium avium subsp. hominissuis infection in a dog from an American lineage of Basset Hounds is described. A 2-year-old intact female Basset Hound presented with persistent lymphadenopathy, lameness, and a history characterized by coccidiosis, bacterial gastroenteritis, and alopecia. Lymphadenitis, with macrophages containing a few intracytoplasmic, negative staining, Ziehl-Neelsen-positive bacilli, was detected by a popliteal fine-needle aspirate leading to the diagnosis of mycobacteriosis. Ultrasound and X-ray examinations revealed visceral and mediastinal lymphadenopathy. Because of the extent of the disease, the dog was humanely euthanized. Significant gross abnormalities, such as enlargement of the cranial mediastinal lymph nodes with encapsulated areas of caseous necrosis and generalized lymphadenopathy, were observed at necropsy. Granulomatous lesions were histopathologically detected in the liver and spleen. Ziehl-Neelsen-positive bacilli were observed in all examined lymph node, liver, spleen, lung, and bone marrow smears. Lymph nodes and liver were collected in order to pursue speciation by bacterial culture and molecular biology; multiplex polymerase chain reaction results classified the pathogen as M. avium subsp. hominissuis. Although an immune system deficiency was not investigated, anamnesis suggests that the dog was immunocompromised. Furthermore, the dog came from an American stock of Basset Hound, and for some of this breed, a predisposition to this infection has been hypothesized.

Disseminated Mycobacterium avium subspecies infection in a cat.

An 18-month-old neutered male domestic shorthair cat, domiciled in the southwest of France, was first presented having suffered for a few days from dysorexia and vomiting. Abdominal palpation revealed lymph node enlargement. Cytological examinations of a fine needle aspirate demonstrated granulomatous inflammation with many non-staining elements consistent with mycobacteria. Diagnosis was confirmed by culture and polymerase chain reaction and Mycobacterium avium subspecies was isolated. Treatment was initiated with marbofloxacin, rifampicin and cefoxitin. There was a rapid clinical improvement. The cat suddenly died 2 months later. The main hypothesis is the administration of an inappropriate combination therapy that leads to the development of mycobacterial resistance. A volvulus and acute peritonitis secondary to the significant enlargement of a mesenteric lymph node were present at necropsy. Histopathological analysis of mesenteric lymph node, liver and spleen revealed multicentric granulomatous and severely necrotic lesions with numerous Ziehl-Neelsen positive intracytoplasmic elements.

"Mycobacterium avium subsp. hominissuis" in neck lymph nodes of children and their environment examined by culture and triplex quantitative real-time PCR.

"Mycobacterium avium subsp. hominissuis" often causes cervical lymphadenitis in children; its prompt and accurate identification enables adequate therapy, tracing, and prevention. The aims of this study were to determine the causative agent of lymphadenitis using culture, PCR, and triplex quantitative real-time PCR (qPCR) methods with DNA directly isolated from tissue, as well as to identify possible sources of infection from the environment. We confirmed the diagnoses by detecting M. avium subsp. hominissuis using qPCR with DNA directly isolated from lymph node biopsy specimens of two patients. In order to trace the source of infection from the environment, a method of DNA isolation from soil and other environmental samples, such as dust, cobwebs, and compost, was developed. The triplex qPCR examination revealed the presence of M. avium subsp. hominissuis in a high proportion of the environmental samples (42.8% in the first patient's house and 47.6% in the second patient's house). Both patients were also exposed to M. avium subsp. avium, which was present due to the breeding of infected domestic hens. The high infectious dose of M. avium subsp. hominissuis or the increased susceptibility of humans to M. avium subsp. hominissuis compared to M. avium subsp. avium could be the reason why the children were infected with M. avium subsp. hominissuis.

Epidemiological investigation of a Mycobacterium avium subsp. hominissuis outbreak in swine.

Mycobacterium avium subsp. hominissuis (MAH) infection in swine may cause granulomatous lesions in lymph nodes that must undergo differential diagnosis with those caused by M. tuberculosis complex members. Moreover, MAH outbreaks can lead to severe economic losses due to condemnation of carcasses. A number of potential sources of infection for animals can usually be identified in contaminated farms. This report describes the application of several molecular characterization techniques in order to identify the possible environmental sources of MAH infection in an outbreak involving four breeding farms and six fattening farms. Molecular profiles obtained from MAH strains suggested a likely epidemiological link between clinical and environmental isolates cultured from sawdust and cooling systems from one breeding farm. These results highlight the potential risk posed by these environmental elements in the spread of infection and the need for implementation of adequate management practices in order to minimize this risk.

Comentarios clínicos acerca de las micobacteriosis pulmonares / Pulmonary mycobacterioses: clinical commentaries

Las micobacteriosis pulmonares son afecciones provocadas por micobacterias ambientales, de evolución crónica y clínicamente similares a la tuberculosis. Se analiza una serie de 26 casos asistidos en el Hospital Muñiz de la Ciudad de Buenos Aires, con una edad promedio de 59.2 años, 73.1% de sexo femenino y 80.1% de los casos de nivel socioeconómico mediano a alto. Se hallaron enfermedades predisponentes en el 88.5% de los casos (tuberculosis previa, EPOC, silicosis, reflujo gastroesofágico). Desde el punto de vista radiológico se observaron nódulos, bronquiectasias y cavidades pequeñas en 14 casos y lesiones bilaterales cavitarias extensas en 12. Se destacó la mayor frecuencia de Mycobacterium avium complex como patógeno, la mejor evolución de los pacientes con menor compromiso pulmonar y la de los afectados por M. avium en relación con otras micobacterias. Los fármacos más utilizados en el tratamiento fueron claritromicina y azitromicina, asociados a etambutol. La proporción global de curaciones fue baja (57.7%) pero superior en los casos provocados por M. avium (86.7%).

Infection with Mycobacterium avium subspecies avium in a 10 year old Freiberger mare.

In this case report a 10 year old Freiberger mare with a Mycobacterium avium subsp. avium infection is presented. This infection leads to a tuberculosis like disease with granulomatous alterations particularly of the intestines and lungs and is only sporadically reported in horses of Central Europe. Diarrhoea, mastitis and neck stiffness as well as dyspnoea and chronic cough are more specific symptoms of the infection, while weight loss, weakness and lethargy are nonspecific signs. As these clinical signs can occur in many other diseases, the diagnosis of mycobacterial infection is difficult and consists of rectum or distal colon biopsies and staining for acid-fast bacilli and bacteriological culture of granulomatous lesions. Classification of M. avium subsp. avium was achieved by PCR-RFLP. Even though an infection with Mycobacterium avium subsp. avium is rare, it belongs to the differential diagnosis of granulomatous diseases.

Mycobacteriosis in companion psittacine birds: a review.

Mycobacteriosis has long been recognized as a disease of companion psittacine birds. In this review, the signalment, clinical signs of disease, and diagnosis of mycobacteriosis in psittacine birds are presented. Although treatment of birds with mycobacteriosis is controversial, therapeutic protocols for use in psittacine birds have been developed based on protocols used in humans. Results of recent studies indicate that mycobacteriosis in psittacine birds is unlikely to represent a significant health risk for humans. However, the potential for zoonotic disease must be considered, especially if persons who are immunocompromised are in contact with an infected bird.

Two-year combination antibiotic therapy with clarithromycin, rifabutin, and clofazimine for Crohn's disease.

BACKGROUND & AIMS: Mycobacterium avium subspecies paratuberculosis has been proposed as a cause of Crohn's disease. We report a prospective, parallel, placebo-controlled, double-blind, randomized trial of 2 years of clarithromycin, rifabutin, and clofazimine in active Crohn's disease, with a further year of follow-up. METHODS: Two hundred thirteen patients were randomized to clarithromycin 750 mg/day, rifabutin 450 mg/day, clofazimine 50 mg/day or placebo, in addition to a 16-week tapering course of prednisolone. Those in remission (Crohn's Disease Activity Index