Analysis of tryptophan-rich region in Clostridium septicum alpha-toxin involved with binding to glycosylphosphatidylinositol-anchored proteins.

Clostridium septicum alpha-toxin has a unique tryptophan-rich region ((302)NGYSEWDWKWV(312)) that consists of 11 amino acid residues near the C-terminus. Using mutant toxins, the contribution of individual amino acids in the tryptophan-rich region to cytotoxicity and binding to glycosylphosphatidylinositol (GPI)-anchored proteins was examined. For retention of maximum cytotoxic activity, W307 and W311 are essential residues and residue 309 has to be hydrophobic and possess an aromatic side chain, such as tryptophan or phenylalanine. When residue 308, which lies between tryptophans (W307 and W309) is changed from an acidic to a basic amino acid, the cytotoxic activity of the mutant is reduced to less than that of the wild type. It was shown by a toxin overlay assay that the cytotoxic activity of each mutant toxin correlates closely with affinity to GPI-anchored proteins. These findings indicate that the WDW_W sequence in the tryptophan-rich region plays an important role in the cytotoxic mechanism of alpha-toxin, especially in the binding to GPI-anchored proteins as cell receptors.

Mycobacterium bovis infection at the interface between domestic and wild animals in Zambia.

BACKGROUND: In Zambia, the presence of bovine tuberculosis in both wild and domestic animals has long been acknowledged and mutual transmission between them has been predicted without any direct evidence. Elucidation of the circulating Mycobacterium bovis strains at wild and domestic animals interphase area in Zambia, where bovine tuberculosis was diagnosed in wildlife seemed to be important. RESULTS: A PCR identified 15 and 37 M. bovis isolates from lechwe and cattle, respectively. Spoligotype analysis revealed that M. bovis strains from lechwe and cattle in Kafue basin clustered into a major node SB0120, where isolates outside the Kafue basin clustered into different nodes of SB0131 and SB0948. The comparatively higher variety of strains in cattle compared to lechwe elucidated by Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats analyses are consistent with cattle being the probable source of M. bovis in wild and domestic animals interphase area in Zambia. CONCLUSIONS: These results provide strong evidence of M. bovis strains transfer between cattle and lechwe, with the latter having developed into a sylvatic reservoir host.

Field observations of fish species susceptible to epizootic ulcerative syndrome in the Zambezi River basin in Sesheke District of Zambia.

A field investigation was conducted in the Sesheke District of Zambia along the Zambezi River to determine the fish species susceptible to epizootic ulcerative syndrome (EUS), a newly confirmed disease in Southern Africa. A total of 2,132 fishes were inspected for gross EUS-like lesions, of which 188 (8.82%; 95% CI=7.67-10.1%) were found with typical characteristic lesions of EUS. Of these 188 samples, 156 were found to have mycotic granulomas on histopathological analysis, representing 83.0% (95% CI=76.7-87.9%) of the initially identified in the laboratory through gross examination. The following 16 species of fish were examined and found with EUS lesions; Clarias ngamensis, Clarias gariepinus, Barbus poechii, Tilapia sparrmanii, Serranochromis angusticeps, Brycinus lateralis, Micralestes acutidens, Sargochromis carlottae, Hydrocynus vittatus, Phryngochromis acuticeps, Schilbe intermedius, Hepsetus odoe, Labeo lunatus, Oreochromis andersonii, Barbus unitaeniatus, and Barbus paludinosus. T. sparrmanii did not show any lesions, while the Clarias species were found to be the most afflicted with EUS. These results could be useful to fish farmers and organizations interested in improving aquaculture in the area.

Risk mapping and eco-anthropogenic assessment of anthrax in the upper Zambezi basin.

In Zambia, anthrax has emerged as a serious disease decimating humans, livestock and wildlife with devastating effects on eco-tourism resulting in the destabilization of major pristine wildlife sanctuaries. Consequently, the thrust of this study was to establish the spatial distribution of anthrax and determine ecological drivers of its recurrence, maintenance and epidemiological linkage to anthropogenic activities. Environmental and biological samples were collected within the livestock production and conservation areas (n = 80). Each sample was serially tested for Bacillus anthracis positivity through blood agar culture and Gram stain technique, and then confirmation by multiplex polymerase chain reaction (MPCR). Questionnaires (n = 113) were conducted at independently distinct villages in terms of space and time. Most respondents showed that animals that died from anthrax were not properly disposed off. More likely than not, poverty being the main driver for anthrax carcass dressing and meat distribution contributed to environmental contamination with anthrax spores in areas where the animals subsequently died resulting in further environmental contamination, which is the major source of primary infection for livestock and wildlife. From the samples, 15 pure isolates of anthrax were obtained which were spatially distributed across four districts. Twelve, biologically plausible variables were found to be highly significant on multivariable logistic regression analysis model for questionnaires which included herd size (odds = 10.46; P = 0.005; CI 8.8-16), carcass disposal method (odds = 6.9; P = 0.001; CI = 3.4-9.8), access to veterinary services (odds = 10.87; P = 0.004; CI = 4.8-15.9) and management system (odds = 2.57; P = 0.001; CI = 1.3-7.5). In summary, the majority (78.7%) of anthrax outbreaks were observed in areas with low veterinary services (χ2  = 8.6162, P = 0.013) within the newly created districts of Nalolo, Mwandi and Luampa.

Relationship between Clostridium septicum alpha-toxin activity and binding to erythrocyte membranes.

The activity of Clostridium septicum alpha-toxin was determined in erythrocytes of various animals, with sensitivities observed in the order of mouse, rat, canine, equine, rabbit, chicken, bovine, swine and ovine. Temperature and protease treatment affected the sensitivity of erythrocytes to alpha-toxin. Proteinase K treatment decreased the sensitivity of murine, canine, equine and bovine erythrocytes, but ovine erythrocytes did not change the sensitivity to alpha-toxin activity. On the other hand, the activity of alpha-toxin on swine erythrocytes increased after treatment with proteinase K, trypsin, chymotrypsin or lysyl endopeptidase. Toxin overlay assay showed that alpha-toxin bound to erythrocyte membrane proteins with a molecular mass of 30 to 45-kDa in mouse, equine, bovine, swine and chicken, whereas in rat erythrocyte membranes the toxin reacted with 100-kDa protein. The treatment of murine and swine erythrocyte membranes with phosphatidylinositol-specific phospholipase C resulted in liberation of the toxin-binding protein from the individual membranes in a native state. These results show that alpha-toxin associates with specific erythrocyte membrane proteins in any animal species, and are subsets of glycosylphosphatidylinositol-anchored proteins in various animal species. These results may reflect distinct characteristics of the hemolytic activity of alpha-toxin in response to various erythrocytes.

Genome Sequence of a Bacillus anthracis Outbreak Strain from Zambia, 2011.

In August 2011, an anthrax outbreak occurred among Hippopotamus amphibius hippopotamuses and humans in Zambia. Here, we report the draft genome sequence of the Bacillus anthracis outbreak strain CZC5, isolated from tissues of H. amphibius hippopotamuses that had died in the outbreak area.

Human-animal anthrax outbreak in the Luangwa valley of Zambia in 2011.

There has been a reduction of incidences of anthrax in the developed countries but it is still a public health problem in the developing countries where communities live in interface areas with wildlife. An outbreak of anthrax in Hippopotamus amphibious was observed in Zambia. Following the death of hippopotamuses, suspected human cases were reported. The objective of this study was to isolate and confirm Bacillus anthracis and to determine the antimicrobial susceptibility for the management of the disease. Of the specimens collected, 29.4% (95% confidence interval [CI], 11.4-56.0) were from humans, 42.1% (95% CI, 21.1-66.0) were from hippopotamuses and 20.0% (95% CI, 6.61-44.3) from the soil were found to be positive were for B. anthracis. An antimicrobial susceptibility test revealed that all the isolates were found to be sensitive to the recommended antibiotics. The disease control was achieved by case management and by explaining to the communities that they should avoid contact with animals that die from unknown causes.

Cytotoxicity of Clostridium septicum alpha-toxin: its oligomerization in detergent resistant membranes of mammalian cells.

Alpha-toxin is an important agent of the virulence of Clostridium septicum. We examined cytotoxicity for alpha-toxin to various mammalian cells with recombinant toxin fused with a histidine-tag at the amino-terminal. The recombinant toxin retained the activity indistinguishable from the native form. Mammalian nucleated cells examined in this study are more sensitive to the protoxin than to the trypsinized toxin, except RAW 264.7 and P3U1 cells of myeloid lineage. Cellular proteins of various molecular sizes interacted with the toxin. The size and SDS-PAGE pattern of the proteins were different among cell lines but they were liberated from the cells by the treatment with phosphatidylinositol-specific phospholipase C. The toxin appeared to target and utilize detergent resistant membranes (DRMs) for binding and subsequent oligomerization. In discontinuous sucrose density gradient, we demonstrated by immunoblotting that the toxin bound to DRMs contained in L929 cells and caused the oligomer formation. Furthermore, cholesterol depletion with cholesterol-interacting agents reduced toxin oligomerization and lowered cytotoxicity of the toxin towards cells. These results suggest that alpha-toxin preferentially exploits DRMs for oligomerization.