Isolation of Pseudobutyrivibrio ruminis and Pseudobutyrivibrio xylanivorans from rumen of Creole goats fed native forage diet.

We isolated and identified functional groups of bacteria in the rumen of Creole goats involved in ruminal fermentation of native forage shrubs. The functional bacterial groups were evaluated by comparing the total viable, total anaerobic, cellulolytic, hemicellulolytic, and amylolytic bacterial counts in the samples taken from fistulated goats fed native forage diet (Atriplex lampa and Prosopis flexuosa). Alfalfa hay and corn were used as control diet. The roll tubes method increased the possibility of isolating and 16S rDNA gene sequencing allowed definitive identification of bacterial species involved in the ruminal fermentation. The starch and fiber contents of the diets influenced the number of total anaerobic bacteria and fibrolytic and amylolytic functional groups. Pseudobutyrivibrio ruminis and Pseudobutyrivibrio xylanivorans were the main species isolated and identified. The identification of bacterial strains involved in the rumen fermentation helps to explain the ability of these animals to digest fiber plant cell wall contained in native forage species.

Intracellular trafficking of Brucella abortus in J774 macrophages.

Brucella abortus is a facultative intracellular bacterium capable of surviving inside professional and nonprofessional phagocytes. The microorganism remains in membrane-bound compartments that in several cell types resemble modified endoplasmic reticulum structures. To monitor the intracellular transport of B. abortus in macrophages, the kinetics of fusion of phagosomes with preformed lysosomes labeled with colloidal gold particles was observed by electron microscopy. The results indicated that phagosomes containing live B. abortus were reluctant to fuse with lysosomes. Furthermore, newly endocytosed material was not incorporated into these phagosomes. These observations indicate that the bacteria strongly affect the normal maturation process of macrophage phagosomes. However, after overnight incubation, a significant percentage of the microorganisms were found in large phagosomes containing gold particles, resembling phagolysosomes. Most of the Brucella bacteria present in phagolysosomes were not morphologically altered, suggesting that they can also resist the harsh conditions prevalent in this compartment. About 50% colocalization of B. abortus with LysoSensor, a weak base that accumulates in acidic compartments, was observed, indicating that the B. abortus bacteria do not prevent phagosome acidification. In contrast to what has been described for HeLa cells, only a minor percentage of the microorganisms were found in compartments labeled with monodansylcadaverine, a marker for autophagosomes, and with DiOC6 (3,3'-dihexyloxacarbocyanine iodide), a marker for the endoplasmic reticulum. These results indicate that B. abortus bacteria alter phagosome maturation in macrophages. However, acidification does occur in these phagosomes, and some of them can eventually mature to phagolysosomes.

A phorbol ester-binding protein is required downstream of Rab5 in endosome fusion.

Previous observations indicate that a zinc and phorbol ester binding factor is necessary for endosome fusion. To further characterize the role of this factor in the process, we used an in vitro endosome fusion assay supplemented with recombinant Rab5 proteins. Both zinc depletion and addition of calphostin C, an inhibitor of protein kinase C, inhibited endosome fusion in the presence of active Rab5. Addition of the phorbol ester PMA (phorbol 12-myristate 13-acetate) reversed the inhibition of endosome fusion caused by a Rab5 negative mutant. Moreover, PMA stimulated fusion in the presence of Rab5 immunodepleted cytosol. These results suggest that the phorbol ester binding protein is acting downstream of Rab5 in endosome fusion.

Calcium- and zinc-binding proteins in intracellular transport.

The complex mechanism of intracellular transport is regulated by free calcium in different manners. Calcium binding proteins regulate several aspects of the vesicle fusion mechanism mediated by NSF (N-ethylmaleimide sensitive fusion factor). At least in some regulated exocytosis, calcium-binding proteins are the trigger for fusion downstream of NSF, Still, calcium-binding proteins, such as annexins, may be part of a different fusion mechanism mediating some specific transport steps or working in parallel to the NSF-dependent fusion process. Calcium is not the only ion necessary for the function of factors involved in vesicular transport. A zinc requirement has been also proposed. One of the zinc-dependent factors is probably a protein with a cysteine-rich region that coordinates zinc and binds phorbol esters. Although protein kinase C is the more prominent family of proteins carrying this domain, the factor necessary for transport does not appear to function as a kinase.

[Type D and A Clostridium botulinum in necropsy samples from bovines with "mal de Aguapey"]. / Clostridium botulinum tipos D y A en materiales de necropsia de bovinos afectados por el mal de Aguapey.

Bacteriological studies were carried out on several necropsy samples from five animals whose deaths had been attributed to bovine botulism. This disease, regionally called Mal de Aguapey, enzootically affects animals from a wide area of the north-east of Argentina (Province of Corrientes) with a bovine population estimated at near to 2,500,000. Either C. botulinum type D, its toxin or both were identified in all animal samples, alternatively in contents of rumen, jejunum, ileum, caecum and in samples of spleen, liver and kidney (Table 1). C. botulinum type A was isolated respectively from the liver and the kidney of two animals. Cultures of 100 soil samples taken in the enzootic area were positive only for C. botulinum type A (3%). These results enlarge and confirm previous findings and lend support to the botulinic etiology of the Mal de Aguapey.

Clostridium botulinum tipos D y A en materiales de necropsia de bovinos afectados por el mal de Aguapey. / [Type D and A Clostridium botulinum in necropsy samples from bovines with [quot ]mal de Aguapey[quot ]]

Bacteriological studies were carried out on several necropsy samples from five animals whose deaths had been attributed to bovine botulism. This disease, regionally called Mal de Aguapey, enzootically affects animals from a wide area of the north-east of Argentina (Province of Corrientes) with a bovine population estimated at near to 2,500,000. Either C. botulinum type D, its toxin or both were identified in all animal samples, alternatively in contents of rumen, jejunum, ileum, caecum and in samples of spleen, liver and kidney (Table 1). C. botulinum type A was isolated respectively from the liver and the kidney of two animals. Cultures of 100 soil samples taken in the enzootic area were positive only for C. botulinum type A (3

). These results enlarge and confirm previous findings and lend support to the botulinic etiology of the Mal de Aguapey.

[First outbreak of botulism caused by Clostridium botulinum subtype Af]. / Primer brote de botulismo por Clostridium botulinum subtipo Af.

In December 1982 an outbreak of foodborne botulism presumably produced by the ingestion of home-made pickled trout occurred in San Rafael, province of Mendoza, Argentina. The toxin detected in blood serum and feces samples of the sole affected patient was preliminarily typed as a plain type A. A strain of Clostridium botulinum was isolated from feces which, after culture by the dialysis method, produced 1 x 10(7) LD50/mouse per ml. Quantitative neutralization tests carried out at different levels of toxin concentration, showed that this toxin consists of a major type A antigenic component (about 99% of the complex) and a minor type F component, defining its identity as a subtype Af. A rabbit antiserum obtained from this toxin neutralizes A and F reference toxins. Despite the antitoxic and supportive treatment, the patient died as a consequence of the poisoning. The polyvalent antiserum administered contained A, B and E antitoxins. Death could be due to the lethal effect of the A fraction, the F fraction or to a combined effect of both toxic components of the toxin. This is the first detection of an outbreak produced by C. botulinum subtype Af, serotype described in Argentina fifteen years ago, and not detected until now in other parts of the world.(ABSTRACT TRUNCATED AT 250 WORDS)

Primer brote de botulismo por Clostridium botulinum subtipo Af. / [First outbreak of botulism caused by Clostridium botulinum subtype Af]

In December 1982 an outbreak of foodborne botulism presumably produced by the ingestion of home-made pickled trout occurred in San Rafael, province of Mendoza, Argentina. The toxin detected in blood serum and feces samples of the sole affected patient was preliminarily typed as a plain type A. A strain of Clostridium botulinum was isolated from feces which, after culture by the dialysis method, produced 1 x 10(7) LD50/mouse per ml. Quantitative neutralization tests carried out at different levels of toxin concentration, showed that this toxin consists of a major type A antigenic component (about 99

of the complex) and a minor type F component, defining its identity as a subtype Af. A rabbit antiserum obtained from this toxin neutralizes A and F reference toxins. Despite the antitoxic and supportive treatment, the patient died as a consequence of the poisoning. The polyvalent antiserum administered contained A, B and E antitoxins. Death could be due to the lethal effect of the A fraction, the F fraction or to a combined effect of both toxic components of the toxin. This is the first detection of an outbreak produced by C. botulinum subtype Af, serotype described in Argentina fifteen years ago, and not detected until now in other parts of the world.(ABSTRACT TRUNCATED AT 250 WORDS)