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Animal models for Mycobacterium ulcerans infections (Buruli ulcer) include guinea pigs, rats, and mice, but each has limitations in replicating the spectrum of human disease. Here, 19 adult nine-banded armadillos were inoculated intradermally with M. ulcerans. Injection sites were examined and skin samples obtained for histologic and microbiology studies. Necropsies were conducted to assess systemic involvement. In group 1 (n = 4), 2 animals developed progressive skin ulcers with undermined borders at the injection sites within 6-10 weeks. Biopsies showed features similar to human disease including extensive necrosis in the deep dermis and subcutaneous fat, mixed cellular infiltrates, and acid-fast bacilli (AFB). In group 2 (n = 15), 5 animals developed progressive skin ulcers, 3 had evanescent papulo-nodules, 3 died shortly after inoculation of unknown causes, and 4 showed no signs of infection. Lesion samples from 3 animals with progressive ulcers were culture positive for AFB. Our findings indicate that nine-banded armadillos are susceptible to M. ulcerans and may develop cutaneous lesions that closely mimic Buruli ulcer.

To determine the prevalence of antibodies to viral diseases known or suspected to be present in Pakistan, we studied 570 sera from three groups of adults; two of the groups were involved in outbreaks of hepatitis, and the third included men admitted to a hospital for evaluation of febrile illnesses. Immunoglobulin G antileptospiral antibody was found in 1 to 6% of the subjects, with the highest rate in enlisted military personnel hospitalized for febrile illness. One man in the group with febrile illness had significantly elevated immunoglobulin M antileptospiral antibody titers. However, in a group of recruits experiencing suspected non-A, non-B hepatitis, 19 (11%) of 173 had a 4-fold rise in immunoglobulin M antibody to leptospirosis. Antibody to sand fly fever viruses was found in 27 to 70%. Antibody to West Nile virus was present in 33 to 41% of subjects. Antibody reactive with Japanese encephalitis virus was present in 25%, but plaque reduction neutralization tests suggested this to be cross-reaction with West Nile virus. All 212 specimens tested for antibody to Crimean-Congo hemorrhagic fever and Hantaan viruses were negative. This study indicates that diseases known to be prevalent in other areas of southwest Asia and the Middle East are also prevalent in northern Pakistan and may impact on those traveling or working in this area.

Little is known about the prevalence of infection with hepatitis viruses in Belize, Central America. We conducted a serologic survey among members of the Belize Defence Force (BDF), which is composed of the five major ethnic groups in Belize, to estimate prevalence rates of hepatitis A, B, and C among military-aged men and women in Belize. Of approximately 600 men and women in the BDF, 492 (82%) completed a questionnaire and blood collection. Antibody to hepatitis A was found in 94%, with similar rates by age, sex, rank, and ethnicity. Antibody to hepatitis B core antigen (anti-HBc) was found in 31%. Rates of anti-HBc varied significantly among the ethnic groups with the lowest rates in Mestizo (5%) and Mayan Indians (9%), and significantly higher rates among Creoles (30%) and Garifuna (56%). Rates increased with increasing age from 28% in those 18-24 years old to 35% in those > or = 35 years old (P = 0.07, by chi-square test for trend). Hepatitis B surface antigen was found in 21 (4%) overall. Antibody to hepatitis C was found in two (0.4%). In this young healthy population, exposure to hepatitis A before the age of 18 is almost universal, while exposure to hepatitis B is related to age and ethnic origin.

In spring 1991, Belizian health officials expressed concern about a possible hepatitis outbreak in a banana farming district. A study was designed to identify cases and to address the serological prevalence of hepatitis virus markers. Three populations were studied: (i) persons meeting a clinical case definition for hepatitis; (ii) designated banana workers; and (iii) people in a random sample of households in the community. Information was collected using questionnaires and sera were collected for laboratory testing. This report presents the preliminary results of a study conducted in June 1991. Among people who met the clinical case definition, 24% of 42 tested had immunoglobulin M antibody to hepatitis B virus (HBV) core antigen (anti-HBc IgM). In the worker and household survey populations, 284 and 280 people, respectively, were tested for anti-HBc IgM. In each group, 4% were positive. HBV surface antigen was found in 37% of 43 clinical cases, 18% of workers, and 13% of people in the household survey. Among the 3 study populations, the prevalence of HBV core antibody (anti-HBc) ranged from 73% to 81%. Almost all tested persons had evidence of prior hepatitis A virus infection. Evidence of prior infection with hepatitis viruses A and B was widespread, but an aetiology could not be established for most of the clinical cases. However, the prevalence of hepatitis B markers in this population was very high compared to other reports from the Caribbean.

Tosufloxacin (A-60969 HCl), a new quinolone with broad activity against gram-positive and anaerobic organisms, was compared in vitro with other quinolones against bacterial pathogens of diarrhea. Tosufloxacin was the most active agent against Salmonella spp., Shigella spp., Campylobacter spp., Aeromonas hydrophila, and Vibrio spp. Temafloxacin (A-62254) also demonstrated good activity against these organisms.

Mycobacterium ulcerans causes chronic nectotizing ulcers of the skin and subcutaneous tissue and is a serious health problem in some tropical countries. Chemotherapy has not been effective, and the treatment of choice is extensive debridement followed by skin grafting. In spite of this, many infections are complicated by disfiguring scars, contraction deformities, and--rarely--amputation. There are other procedures that may promote healing (e.g., heat treatment, rifampicin), but none has been completely evaluated. In our study, hyperbaric oxygenation (HBO), an effective treatment for many bacterial diseases, including some mycobacterial infections, was used to treat mice with M. ulcerans-infected footpads. Three groups (40 mice/group) were treated daily with 100% oxygen by three different protocols: 2.5 ATA for 2 h; 2.5 ATA for 1.25 h, twice a day; and 2 ATA for 3.5 h. The degree of infection in the treated mice was compared weekly with 40 positive controls (infected, not treated). The HBO therapy was more effective in the group treated at 2.5 ATA for 1.25 h, twice a day. After 25 weeks, there had been two feet autoamputated and only 12 deaths among the mice, as compared to 18 feet amputated and 24 deaths in the control group. Thus, hyperbaric oxygenation has a beneficial effect in mice and, if used in conjunction with other therapeutic procedures in man, may be an effective therapeutic adjunct in treating M. ulcerans infections.

Fractions of Mycobacterium ulcerans were tested for the ability to inhibit growth of 39 bacterial strains. At protein concentrations of up to 280 mg/ml, there was no detectable effect on the growth of any of these bacterial strains.

The effectiveness of hyperbaric oxygenation (HBO), heat, and rifampin for treating mice infected with Mycobacterium ulcerans was analyzed. Four hundred mice were innouclated in the hind footpads with 10(8) organisms. The seven treatment groups (50 mice each) were HBO (2.5 ATA for 1.5 h, twice a day), rifampin (RIF) (20 mg/kg body weight/d), heat (mice maintained at 37 degrees C), and all combinations of the three treatments. The severity of infection in the treated mice was compared weekly for 20 weeks with that of infected controls. The most effective treatments were RIF/HEAT and RIF/HBO/HEAT, RIF/HBO/HEAT treatment was further evaluated to determine the effectiveness of treating mice at various stages of infection. Three hundred mice were inoculated in the hind footpads, and, as the infection progressed, they were separated into groups (50 mice/group) according to the severity of infection. The treatment groups were compared to positive controls. The effectiveness of therapy was indirectly proportional to the severity of infection.

Sperm released into the urine of adult male frogs, Rana pipiens, after intraperitoneal injection of human chorionic gonadotropin, were used to fertilize freshly ovulated eggs. Repeated spermiations from the same animal were induced with repeated injections of the hormone at intervals of four to 11 days. Individual frogs were induced to spermiate up to four times, with fertile sperm ensuing each time. Development of zygotes produced by this method did not differ from the development of ova fertilized by conventional methods.

Mycobacterium ulcerans produces an exotoxin in culture which, when inoculated into guinea pig skin, causes inflammation, necrosis, edema, and other histopathological changes resembling those in infections of humans. The toxin was resistant to heat and to alkalies and was moderately acid labile. Toxic activity was destroyed by Pronase, phospholipase, lipase, amylase, and glucosidase but not by trypsin, collagenase, cellulase, lysozyme, hyaluronidase, or neuraminidase. Toxic activity was resistant to treatment with 2-mercaptoethanol, urea, guanidine hydrochloride, p-chloromercuribenzoate, ethylenediaminetetraacetate, and sodium deoxycholate but was destroyed by sodium m-periodate and sodium dodecyl sulfate. The toxin was precipitated by a wide range of ammonium sulfate concentrations. Extraction with chlorofrom-methanol or petroleum ether destroyed its activity. Isopycnic density gradient ultracentrifugation in KBr produced a high-density lipoprotein layer with a 24-fold increase in specific activity. The results indicate that this toxin is a high-molecular-weight phospholipoprotein-polysaccharide complex.

Krieg, R. E. (Iowa State University, Ames), and W. R. Lockhart. Classification of enterobacteria based on overall similarity. J. Bacteriol. 92:1275-1280. 1966.-A numerical study was made of 53 organisms, representing 12 genera of the family Enterobacteriaceae and 4 members of the genus Aeromonas. A total of 105 features was determined for each culture. Matching coefficients were computed, and the organisms were sorted into phenetic groups by use of the "highest-link" criterion. The genera Enterobacter (Aerobacter), Escherichia, Citrobacter, Arizona, Shigella, and Salmonella formed a single, large cluster with little or no evidence of subdivisions into tribes or genera. Paracolobactrum and Klebsiella were joined to the large group as subclusters. Members of the genera Erwinia and Serratia formed separate, distinct clusters related to the large group at a lower level of similarity. At a still lower level were appended Aeromonas, Proteus, Providencia, and some individual species from other genera. These organisms were no more closely related to one another than to the larger group.