Acid-fast Bacteria Research Articles

First Isolation ofSegniliparus rugosusfrom a Patient with Radiologic Features Similar to Non-Tuberculous Mycobacteriosis

In 2005, a group of mycolic acid-containing bacteria was characterized as belonging to a novel genus, Segniliparus with species Segniliparus rugosus and S. rotundus. We report a case of the S. rugosus isolated from a 54-year-old woman with radiologic features mimicking that of non-tuberculous mycobacteriosis (NTM). When the patient first visited our hospital, an acid-fast bacteria (AFB) smear tested positive and Mycobacterium tuberculosis polymerase chain reaction (TB PCR) was negative in the bronchoalveolar lavage sample. After 2 months, the growing colonies were reported as NTM, but could not be identified because they had died. One year after the initial visit, induced sputum samples showed the same results, positive AFB smear and negative TB PCR. At this point, the growing colonies were identified as S. rugosus. Therefore, we should consider Segniliparus genus as a differential diagnosis for AFB in respiratory specimens in addition to the genus Mycobacterium.

Antimicrobial Activities of Seed Extracts of Mango (<i>Mangifera indica</i> L.)

Mangifera indica L. is a species of mango in the Anacardiaceae family. It is found in the wild in tropical regions and cultivated varieties have been introduced to other warm regions of the world. This present study aimed to investigate the in vitro antimicrobial activities of methanol and ethanol extracts of mango seed against 25 representatives gram positive, gram negative, acid fast bacteria and fungi. Mango fruit seed were extracted by Soxhlet using methanol and ethanol as solvents. The extracts were tested against the microorganisms using disc diffusion method at different concentrations: 5 mg/mL, 3.75 mg/mL, 3.125 mg/mL, 2.5 mg/mL, 1.875 mg/mL and 1.25 mg/mL). In vitro antibacterial activities of methanol and ethanol extracts of mango bulb showed inhibitions to tested organisms with variable inhibition zones. Except one organism (Rhodococcus equi), no resistance among the tested strains was shown. The mean zone of inhibition produced ranged between 5 mmand18 mmwith18 mm/Mycobacterium smegmatis showed the highest zone of inhibition. In most test strains comparable zones of inhibitions were noted for both methanol and ethanol extract. Candida albicans and Aspergilllus niger were both inhibited by the extracts. The methanol and ethanol extracts of mango seed showed good inhibitory effects against almost all tested strains. The inhibition zones produced by mango extract were less than those produced by standard positive control drug. This could be due to low diffusion rate of mango extract in agarose medium, a thing needed to be further investigated. The products are potential new antimicrobial therapy in the ethnopharmacology domain.

Antimycobacterials from natural sources: ancient times, antibiotic era and novel scaffolds.

Mycobacteria are a group of aerobic, non-motile, acid fast bacteria that have a characteristic cell wall composed of a mycolyl-arabinogalactan-peptidoglycan complex. They display different phenotypic attributes in their growth, color and biochemistry. Tuberculosis (TB) is defined as the infection with Mycobacterium tuberculosis complex and was declared a global health emergency principally because of the appearance of multidrug-resistant strains and the associated risk of infection in immune-compromised population. There is an urgent clinical need for novel, potent and safe anti-TB drugs. Natural products have been used since antiquity for treating diverse complaints and novel pharmacophores are discovered every year. Two of the most potent used antimycobacterials, the rifamycins and streptomycin, were first detected in Streptomyces bacteria. Plants are also the source of an exquisite variety of antimicrobials that can lead to useful therapeutics in the future. In this review, natural preparations used since antiquity for treating tuberculosis are described, together with a rapid view of the 20th century antibiotic development against TB. Finally a summary of the most potent recent natural antimycobacterials is displayed.

A Case of Lung Infection Complicated by Pneumothorax Caused by <i>Mycobacterium marinum </i>

Mycobacterium marinum is a waterborne mycobacterium that commonly infects fish and amphibians worldwide, but transmission to humans can occasionally occur, typically as a granulomatous skin infection following minor hand trauma. Infection involving the lungs is very rare. We herein describe a case of M. marinum-associated pneumonia and pneumothorax. In August 2008, an 81-year-old man was admitted to a hospital for detailed examination of weight loss and an abnormal shadow on chest imaging. Based on a sputum test, nontuberculous mycobacteriosis caused by M. marinum was diagnosed. At that time, the blood chemistry revealed no respiratory symptoms or inflammatory findings, and the patient was treated on an outpatient basis with erythromycin and an expectorant. In late November 2008, sputum and coughing were observed. Furthermore, the patient developed a fever and chest pain that increased while breathing and he visited the emergency outpatient unit of our hospital on December 1. Hypoxemia, bilateral pneumonia, and right pneumothorax were observed, and a chest tube was inserted into the right thoracic cavity. Results of an acid-fast bacteria smear from the sputum and pleural effusion were positive, and M. marinum was identified on culture. The patient was diagnosed as having a lung infection complicated by pneumothorax caused by M. marinum. The lung infection was ameliorated with clarithromycin, rifampicin and ethambutol. However, no decreased in the air leaking from the chest tube was noted and inflation of the lung was incomplete. The department of respiratory surgery therefore performed thoracoplasty and lung cerclage. Subsequently, the air leak subsided, allowing removal of the chest tube and the patient was discharged.

Direct Drug Susceptibility Testing of Mycobacterium tuberculosis for Rapid Detection of Multidrug Resistance Using the Bactec MGIT 960 System: a Multicenter Study

Conventional indirect drug susceptibility testing of Mycobacterium tuberculosis with liquid medium is well established and offers time-saving and reliable results. This multicenter study was carried out to evaluate if drug susceptibility testing (DST) can be successfully carried out directly from processed smear-positive specimens (direct DST) and if this approach could offer substantial time savings. Sputum specimens were digested, decontaminated, and concentrated by the laboratory routine procedure and were inoculated in Bactec MGIT 960 as well as Lowenstein-Jensen (LJ) medium for primary isolation. All the processed specimens which were acid-fast bacterium (AFB) smear positive were used for setting up direct DST for isoniazid (INH) and rifampin (RIF). After the antimicrobial mixture of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA) was added, the tubes were entered in the MGIT 960 instrument using the 21-day protocol (Bactec 960 pyrazinamide [PZA] protocol). Results obtained by direct DST were compared with those obtained by indirect DST to establish accuracy and time savings by this approach. Of a total of 360 AFB smear-positive sputum specimens set up for direct DST at four sites in three different countries, 307 (85%) specimens yielded reportable results. Average reporting time for direct DST was 11 days (range, 10 to 12 days). The average time savings by direct DST compared to indirect DST, which included time to isolate a culture and perform DST, was 8 days (range, 6 to 9 days). When results of direct DST were compared with those of indirect DST, there was 95.1% concordance with INH and 96.1% with rifampin. These findings indicate that direct DST with the Bactec MGIT 960 system offers further time savings and is a quick method to reliably detect multidrug resistance (MDR) cases.

Rapid Identification of Mycobacteria and Drug-Resistant Mycobacterium tuberculosis by Use of a Single Multiplex PCR and DNA Sequencing

Tuberculosis (TB) remains a significant global health problem for which rapid diagnosis is critical to both treatment and control. This report describes a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampin(r)), isoniazid(r), and pyrazinamide(r) TB. Additionally, the same multiplex reaction amplifies a specific 16S rRNA gene target for rapid identification of M. avium complex (MAC) and a region of the heat shock protein 65 gene (hsp65) for further DNA sequencing-based confirmation or identification of other mycobacterial species. Comparison of preliminary results generated with MID-DRS versus culture-based methods for a total of 188 bacterial isolates demonstrated MID-DRS sensitivity and specificity as 100% and 96.8% for MTBC identification; 100% and 98.3% for MAC identification; 97.4% and 98.7% for rifampin(r) TB identification; 60.6% and 100% for isoniazid(r) TB identification; and 75.0% and 98.1% for pyrazinamide(r) TB identification. The performance of the MID-DRS was also tested on acid-fast-bacterium (AFB)-positive clinical specimens, resulting in sensitivity and specificity of 100% and 78.6% for detection of MTBC and 100% and 97.8% for detection of MAC. In conclusion, use of the MID-DRS reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as 2 days. Since all targets needed for completing the assay are included in a single PCR amplification step, assay costs, preparation time, and risks due to user errors are also reduced.

Genitourinary and Pulmonary Multidrug ResistantMycobacterium tuberculosisInfection in an Asian Elephant (Elephas maximus)

A female Asian elephant (Elephas maximus) developed vaginal and trunk discharge. Cultures were positive for pan-susceptible Mycobacterium tuberculosis. Isoniazid and pyrazinamide were given rectally and monitored by serum levels. After being trained at 10 mo to accept oral dosing, treatment was changed and rifampin was added. Oral medications were administered for another 10 mo. A year after completion of therapy, the vaginal discharge increased and cultures yielded M. tuberculosis, resistant to isoniazid and rifampin. Treatment with oral ethambutol, pyrazinamide, and enrofloxacin and intramuscular amikacin was initiated. Although followup cultures became negative, adverse reactions to medications precluded treatment completion. Due to public health concerns related to multidrug resistant M. tuberculosis (MDR-TB), the elephant was euthanized. Postmortem smears from the lung, peribronchial, and abdominal lymph nodes yielded acid-fast bacteria, although cultures were negative. This case highlights important considerations in the treatment of M. tuberculosis in animals and the need for a consistent approach to diagnosis, treatment, and follow-up.

Multifocal Infection of Mycobacterium Other Than Tuberculosis Mimicking a Soft Tissue Tumor of the Extremity

Mycobacterium other than tuberculosis infections rarely develop in healthy individuals, but direct inoculation such as contaminated acupuncture can cause mycobacteriosis even in an immunocompetent host. A 56-year-old woman gradually developed pain on the anterior aspect of the left knee and the distal thigh after hiking without trauma. She received acupuncture for 3 consecutive days on the bilateral knees at the suprapatellar and infrapopliteal areas. After acupuncture, mild localized heat and painful swelling developed around the knees bilaterally. Magnetic resonance imaging (MRI) demonstrated soft tissue masses with a lobulated contour at the prefemoral fat between the suprapatellar pouch and the distal femur and at the proximal tibia behind the knee joint capsule. Fibromatosis, pigmented villonodular synovitis, and soft tissue sarcoma were considered. On pathologic examination, multiple granulomas with lymphoplasmatic infiltration were evident, and acid-fast bacteria staining revealed acid-fast bacilli. A mycobacterial culture confirmed Mycobacterium other than tuberculosis infection, and a polymerase chain reaction-fragment length polymorphism assay identified the isolates as Mycobacterium abscessus. After treatment with appropriate antibiotics, the patient had no evidence of disease and reported no pain during activities of daily living. Acupuncture is growing in prominence in Europe and the United States, and the number of reports on complications increases with its widespread use. Although the risk to an individual patient is difficult to determine, acupuncture may cause serious complications in patients with coagulopathy, heart valve disease, and immune deficiency. In addition, direct inoculation such as contaminated acupuncture can cause mycobacteriosis even in an immunocompetent host.

An investigation on the antibacterial, cytotoxic, and antibiofilm efficacy of starch-stabilized silver nanoparticles

The increased emergence of drug resistant microbes creates a major challenge to the scientific community for successful development of effective therapeutics. The antimicrobial activities of silver ions are well known, but limited information is available on the effects of green silver-nanoparticles (AgNPs) on human pathogens. In this article, we evaluated the antibacterial activity of starch-stabilized AgNPs against a panel of human pathogens commonly associated with air, water and food borne infections. The shape and size distribution of AgNPs were characterized by transmission electron microscopy. We showed that AgNPs were more effective against Gram-positive and Gram-negative pathogens as compared with acid-fast bacteria. AgNPs were not cytotoxic to macrophages at the bactericidal concentration and can augment intracellular killing potential of macrophages. Furthermore, we showed that AgNPs disrupt biofilm formation and exhibit better antibacterial activity compared to human cationic antimicrobial peptide LL-37. In summary, our data suggest AgNPs as a promising template for the design of novel antibacterial agents. From the Clinical EditorIn this fundamental study, the significance of starch-stabilized AgNP-s is studied as antimicrobial agents on a variety of human pathogens. AgNP-s are expected to become universally utilized agents in our efforts to prevent and fight infections.

Mycobacterial disease in a population of 339 cats in Great Britain: II. Histopathology of 225 cases, and treatment and outcome of 184 cases

This study investigated 339 cases of feline mycobacterial infection, with histopathology findings from 225 cases, and treatment and outcome information from 184 cases. Tissue samples from cats with cutaneous lesions or suspicious masses at exploratory laparotomy were submitted to the Veterinary Laboratories Agency for mycobacterial culture over a 4-year period to December 2008. The study reviewed the files for information about histopathology, treatment and outcome, and blindly reviewed histopathological changes (including staining for acid-fast bacteria [AFB]) in a sub-set of 45 cases. When a cat is suspected of having a mycobacterial infection, accurate identification of the species involved helps to determine possible treatment options and prognosis. The study confirmed that histopathology and the presence of AFB are useful tools in the recognition of mycobacterial infection. Unfortunately, they did little to help determine the species of mycobacteria involved. The study identified a group of cats that were negative for AFB at the primary laboratory, but from which mycobacteria could be cultured; commonly Mycobacterium bovis or Mycobacterium microti. The study also identified a group of cats which where culture negative, despite typical signs of mycobacterial infection and positive AFB staining. Many cases responded favourably to treatment (56% of the cases where information was available), and many cats gained complete remission (42%). However, relapses were common (64%) and often followed by pulmonary and/or systemic spread that may have resulted from treatment with short courses of single drugs. This study shows that the diagnosis and treatment of feline mycobacteriosis is complex and challenging.

Mycobacterium salmoniphilum infection in farmed Atlantic salmon, Salmo salar L.

Multiple greyish-white visceral nodules containing abundant rapidly growing and acid-fast bacteria, subsequently identified as Mycobacterium salmoniphilum, were detected in moribund and newly dead market-sized fish during a period of increased mortality in an Atlantic salmon, Salmo salar, farm in western Norway. Isolates cultured from diseased fish were phenotypically consistent with Mycobacterium sp. previously isolated from Atlantic salmon [MT 1890 (= NCIMB13533), MT1892, MT1900 and MT1901] in the Shetland Isles, Scotland. Partial sequences of 16S rDNA, ribosomal RNA internal transcribed spacer (ITS1), 65-kDa heat-shock protein (Hsp65) and β subunit of RNA polymerase (rpoB) revealed 97-99% similarity with M.salmoniphilum type strain ATCC 13758(T) . The source of infection was not confirmed. Koch's postulates were fulfilled following experimental challenge of Atlantic salmon with field isolate NVI6598 (FJ616988). Mortality was recorded in experimentally infected fish; however, the infection remained subclinical in the majority of affected fish over the 131-day challenge period.

A case report of pulmonary coinfection of Strongyloides stercoralis and Pneumocystis jiroveci

A case of pulmonary coinfection by Strongyloides stercoralis and Pneumocystis jiroveci has been detected in an AIDS patient treated in the Respiratory Intensive Care Unit of the Muñiz Hospital. At diagnosis, the patient presented cough with mucopurulent expectoration, dyspnea, fever, bilateral pulmonary infiltrates on the chest X-ray, negative bacilloscopy for acid fast bacteria and a CD4+ T lymphocytes count of 52 cells/μL. The microbiological diagnosis was achieved by microscopic observation of the respiratory secretions obtained by bronchoalveolar lavage, while the wet mount examination revealed rhabditiform and filariform larvae of the nematode and foamy exudates, pathognomonic of the pulmonary pneumocystosis. It was the unique case of this association among about 3 000 samples performed in our laboratory in the last 10 years and diagnosed by microscopy. Other complementary stains (a rapid modification of Grocott, Kinyoun and Giemsa) were applied to the smears after the diagnosis of mycotic and parasitary infections achieved by fresh microscopy. Both physicians and microbiologists should take into account the possible coexistence of respiratory pathogens in immunocompromised patients, such as those with AIDS.

Usefulness of the variable numbers of tandem repeats (VNTR) analysis for complex infections of Mycobacterium avium and Mycobacterium intracellulare

The bacilli which were isolated from a patient suspected of the mixed infections with Mycobacterium avium and Mycobacterium intracellulare, were analyzed. The genotypes of M. avium in the sedimented fractions of treated sputum and in some colonies isolated from Ogawa medium were compared by the Variable Numbers of Tandem Repeats (VNTR). A woman, aged 57. Mycobacterial species isolated from some colonies by culture in 2004 and 2006 and from the treated sputum in 2006, were determined by DNA sequencing analysis of the 16S rRNA gene. Also, by using VNTR, the genotype of mycobacteria was analyzed. [Results] (1) The colony isolated from Ogawa medium in 2004 was monoclonal M. avium. (2) By VNTR analyses of specimens in 2006, multiple acid-fast bacteria were found in the sputum sediment and in isolated bacteria from Ogawa medium. (3) By analyses of 16S rRNA DNA sequence, M. avium and M. intracellulare were found in the colonies isolated from the sputum sediment and the Ogawa medium in 2006. (4) The same VNTR patterns were obtained in M. avium in 2004 and 2006 when single colony was analyzed. (5) From the showerhead and culvert of the bathroom in the patient's house, M. avium was not detected. By VNTR analyses, it was considered that the mixed infections of M. avium and M. intracellulare had been generated during treatment in this case. Therefore, in the case of suspected complex infection, VNTR analysis would be a useful genotyping method in M. avium complex infection.

Development and maintenance of a specific pathogen-free (SPF) zebrafish research facility for Pseudoloma neurophilia

Pseudoloma neurophilia (Microsporidia) is very common in zebrafish Danio rerio research facilities. A new zebrafish facility has been established at the Sinnhuber Aquatic Resource Laboratory (SARL), Oregon State University, Corvallis, OR, U.S.A., and this was an opportunity to establish a specific pathogen-free (SPF) colony of zebrafish for this microsporidium. Progeny from 9 zebrafish lines (n=2203) were initially transferred to the SARL facility in 2007 following PCR screening of broodstock and a subpopulation of progeny (258 of 1000 fish from each family). Screening of fish for P. neurophilia within the facility was conducted as follows: (1) Moribund or dead fish were examined by histology. (2) Each line was regenerated on a 4 mo rotation, and a subsample of each of these major propagations (60 fry, in pools of 10) was PCR-screened at 10 d post hatch. (3) Adult fish (approximately 1 yr old) from each line were euthanized; 20 fish were examined by histology and the brains of another 60 fish (in pools of 5) were screened by PCR. (4) This screening was replicated on sentinel fish held in 4 tanks receiving effluent water from all tanks in the facility (20 fish per tank). (5) Four-month old fish (n=760) from a toxicology study conducted within the laboratory were examined by histology. To date, we have evaluated 2800 fish by PCR and 1222 fish by histology without detecting P. neurophilia. Thus, we have established 9 lines of zebrafish SPF for P. neurophilia. However, 26 fish exhibited mycobacteriosis, with acid-fast bacteria present in tissue sections, and 49 other fish had incidental lesions.

Mycobacterium mucogenicum blood stream infections after hematopoietic cell transplantation (HCT).

e19649 Background: M. mucogenicum is a rapid growing, nontuberculous acid-fast bacterium that causes catheter related bloodstream infection (CRBI) in immunocompromised hosts. Methods: Analysis of i...

Bacteremic pneumonia caused by Nocardia veterana in an HIV-infected patient

Disseminated Nocardia veterana infection has rarely been reported. We describe the first reported case of N. veterana bacteremic pneumonia in an HIV-infected patient. The isolate was confirmed by 16S rRNA sequencing analysis. The patient initially responded well to trimethoprim-sulfamethoxazole treatment (minimum inhibitory concentration 0.25 μg/ml), but died of ventilator-associated pneumonia.

A Rare Case of Infectious Colitis with Ulcers in the Cecum Caused by Mycobacterium Gordonae

A 69-year-old female complained of persistent abdominal pain, and annular ulcers and ulcer scars were detected endoscopically in the cecum. Pathological findings included caseous granulomas with some Langhans giant cells, and Ziehl-Neelsen staining was negative. Mycobacterium gordonae (M. gordonae) was identified by the DNA-DNA hybridization method and culture (Ogawa medium) of biopsy samples from ulcerous cecal lesions. After 6 months of antibiotic therapy, ulcerous cecal lesions were healed, and no acid-fast bacteria were detected by culture of biopsy samples from scar tissue. We believe this is the first report of M. gordonae infection in the alimentary tract.

Patients in whom active tuberculosis was diagnosed after admission to a Japanese university hospital from 2005 through 2007

To identify problems in early diagnosis of tuberculosis and to design countermeasures against the disease, we examined the status of active tuberculosis among patients admitted to a university hospital that did not have an isolation ward for tuberculosis. Between 2005 and 2007, we analyzed demographic characteristics, disease type, chest radiologic findings, and the process leading to diagnosis. Active tuberculosis was diagnosed after admission in 55 patients (34 males and 21 females): pulmonary tuberculosis, 26; tuberculous pleuritis, 13; tuberculous meningitis, 6; miliary tuberculosis, 4; tuberculous pericarditis, 3; lymph-node tuberculosis, 2; and tracheal and bronchial tuberculosis, 1. Although radiographic examinations provided abundant information, chest radiography showed normal findings in 7 patients (12.7%). Computed tomographic scanning was useful for detailed evaluation of abnormalities. Twenty patients (36.4%) were given diagnoses at departments other than ours (Department of Pulmonary Medicine). Numbers of days between hospital admission and diagnosis of tuberculosis (50th percentile/80th percentile) were 8.0/37.8 for miliary tuberculosis, 8.0/8.0 for tracheal and bronchial tuberculosis, 7.5/17.8 for tuberculous pleuritis, 7.0/8.8 for tuberculous pericarditis, 6.0/15.6 for pulmonary tuberculosis, 3.5/4.4 for lymph-node tuberculosis, and 1/1 for tuberculous meningitis. Early diagnosis of tuberculosis requires adherence to the following precautions. Tuberculosis should be suspected in any patient with respiratory symptoms. Sputum tests for acid-fast bacteria should be performed at least three times initially. If findings on chest X-ray films are equivocal, high-resolution computed tomography should be performed to confirm details of shadows and to detect minimal pulmonary shadows or cavitary lesions. Physicians from all specialties should be repeatedly informed about the risk of tuberculosis and should include tuberculosis in the differential diagnosis in patients suspected to have pulmonary diseases.

Nested PCR on semen samples for the detection of Mycobacterium avium subsp paratuberculosis

Mycobacterium paratuberculosis (MAP) is a gram positive, acid-fast bacterium and cause of Johne’s disease in some animals. The important signs of this disease in bovine are diarrhea, weight loss, bowel inflammation, fever and reduce of milk production. The symptoms of this disease are very similar to Crohn' s disease in humans. The aim of this study was to use nested-PCR as an accurate and fast method to trace MAP in bull semen. Semen samples from 112 bulls were collected and DNA was extracted. Then, nested-PCR was performed by specific primers for IS900 gene of MAP. The PCR products with 230 bp length were estimated as a positive. The frequency of MAP in semen samples were 12.50%. The results were showed nested-PCR is a good procedure with high efficiency for detection of intracellular bacteria such as M. paratuberculosis in bull' s semen samples. Thus, despite this abundance more attention to this disease in bulls to identify MAP quickly is essential.

Mycobacterium liflandiiOutbreak in a Research Colony ofXenopus (Silurana) tropicalisFrogs

A research colony of Xenopus (Silurana) tropicalis frogs presented with nodular and ulcerative skin lesions. Additional consistent gross findings included splenomegaly with multiple tan-yellow nodular foci in the spleen and liver of diseased frogs. Copious acid-fast positive bacteria were present in touch impression smears of spleen, skin, and livers of diseased frogs. Histologically, necrotizing and granulomatous dermatitis, splenitis, and hepatitis with numerous acid-fast bacilli were consistently present, indicative of systemic mycobacteriosis. Infrequently, granulomatous inflammation was noted in the lungs, pancreas, coelomic membranes, and rarely reproductive organs. Ultrastructurally, both extracellular bacilli and intracellular bacilli within macrophages were identified. Frogs in the affected room were systematically depopulated, and control measures were initiated. Cultured mycobacteria from affected organs were identified and genetically characterized as Mycobacterium liflandii by polymerase chain reaction amplification of the enoyl reductase domain and specific variable numbers of tandem repeats. In recent years, M. liflandii has had a devastating impact on research frog colonies throughout the United States. This detailed report with ultrastructural description of M. liflandii aids in further understanding of this serious disease in frogs.