Thermodependence of growth and enzymatic activities implicated in pathogenicity of twoErwinia carotovorasubspecies (Pectobacteriumspp.)

Erwinia carotovora subsp. atroseptica and Erwinia carotovora subsp. carotovora can cause substantial damage to economically important plant crops and stored products. The occurrence of the disease and the scale of the damage are temperature dependent. Disease development consists first of active multiplication of the bacteria in the infection area and then production of numerous extracellular enzymes. We investigated the effects of various temperatures on these two steps. We assayed the specific growth rate and the pectate lyase and protease activities for eight strains belonging to E. carotovora subsp. atroseptica and E. carotovora subsp. carotovora in vitro. The temperature effect on growth rate and on pectate lyase activity is different for the two subspecies, but protease activity appears to be similarly thermoregulated. Our results are in agreement with ecological data implicating E. carotovora subsp. atroseptica in disease when the temperature is below 20 °C. The optimal temperature for pathogenicity appears to be different from the optimal growth temperature but seems to be a compromise between this temperature and temperatures at which lytic activities are maximal.Key words: temperature, Pectobacterium atrosepticum, Pectobacterium carotovorum, growth, pectate lyases, proteases.

Cytochemical Investigation of the Antagonistic Interaction Between a Microsphaeropsis sp. (Isolate P130A) and Venturia inaequalis

In an attempt to better understand the mode of action of the antagonistic fungus Microsphaeropsis sp., the interaction between this fungus and Venturia inaequalis was studied, using both light and electron microscopy. Cytological observations indicated that the antagonistic interaction between the two fungi likely involves a sequence of events, including (i) attachment and local penetration of Microsphaeropsis sp. into V. inaequalis hyphae; (ii) induction of host structural response at sites of potential antagonist entry; (iii) alteration of host cytoplasm; and (iv) active multiplication of antagonistic cells in pathogen hyphae, leading to host cell breakdown and release of the antagonist. The interaction was investigated further by gold cytochemistry. The use of gold-complexed β-1,4-exoglucanase and wheat germ agglutinin/ovomucoid-gold complex to localize cellulosic β-1,4-glucans and chitin monomers, respectively, resulted in regular labeling of V. inaequalis cell walls. This finding supports other studies refuting the classification of ascomycetes as only a glucan-chitin group. At an advanced state of parasitism, the labeling pattern of cellulose and chitin, which clearly showed that the level of integrity of these compounds was affected, suggested the production of cellulolytic and chitinolytic enzymes by Microsphaeropsis sp. Wall appositions formed in V. inaequalis in response to the antagonist's attack contained both cellulose and chitin. However, penetration of this newly formed material frequently succeeded. This study provides the first detailed picture of the cytological events associated with mycoparasitism in V. inaequalis.

SCREENING OF CARBOHYDRATES FOR SPORULATION OF BACILLI IN FLUID MEDIUM

Carbohydrates that are used in fermentation media were screened for sporogenic activities on Bacillus cereus, B. subtilis, and B. megaterium. Out of the 15 carbohydrates tested, d-xylose (0.5%) in nutrient broth exerted maximum sporogenic stimulation on the test organisms. In shake culture experiment with B. subtilis the sporogenic action of xylose was evident even during the active multiplication of the organism in the medium.

LATENT VIRAL INFECTION OF CELLS IN TISSUE CULTURE

Chick embryo tissues maintained for from 11 to 28 days in Hank's balanced salt solution lost their capacity to support the multiplication of psittacosis virus. The virus however infected such cells, as active multiplication of the virus occurred on the addition of beef embryo extract to this nutritionally poor medium at any period up to 28 days of cultivation in balanced salt solution. The virus remained in a state of latency for as long as 15 days in these starved cells in a non-infectious phase. These results obtained in this in vitro model system for the investigation of latent infections of cells with psittacosis virus suggest that cell nutrition as well as an alteration in the immunological defenses of the host may prove to be an important factor in the activation of latent viral infections.

NUCLEIC ACIDS AND THE PRODUCTION OF ANTIBODY BY PLASMA CELLS

A study has been made of the relationship of antibody formation and the changes in amount of the nucleic acids in rabbit lymph nodes draining areas injected with typhoid vaccine. The increase in DNA was found to parallel the increase in weight of the nodes, as might be expected from the active multiplication of cells. The peak of PNA increase occurred between the 4th and 6th days after vaccine injection when antibody formation was at its maximum. A histologic study of methyl green- and pyronine-stained sections of the nodes revealed that during the first 6 days of the experiment the cellular reaction was chiefly one of plasma cells. During the first 3 days plasmoblasts predominated; on the 5th and 6th days mature plasma cells were the prevailing cells. Most of the PNA was contained in the plasma cells. The lymphocytes began to proliferate in significant numbers on the 3rd and 4th days, and germinal centers began to appear on the 4th and 5th days. They showed their greatest activity only on the 9th day when PNA and antibody formation had passed their peaks. These results are interpreted as indicating that the plasma cell and not the lymphocyte is responsible for antibody formation.

Water, Microorganisms and Volatile Bases in Dehydrated Fish

The relations of relative humidity (R.H.) to water content in precooked, air-dehydrated fish were determined with reference to the mould, bacterial and volatile-base content of stored samples. In general it was found that visible mould developed only in samples stored at 80% or higher R.H., while bacteria increased only at 85% or higher R.H. Viable bacteria and moulds decreased slowly in samples stored at R.H. too low to support their active multiplication. Total volatile bases and trimethylamine increased in samples stored at R. H. too low for growth of either bacteria or moulds.

The effect of the addition of various materials and bacterial cultures to grass silage at the time of making on the subsequent bacterial and chemical changes

The investigations previously described (1) have demonstrated that the changes occurring in the early stages of silage fermentation result in simultaneous development of acidity and degradation of proteins. There seems little reason to doubt that the lactobacilli play an effective part in the former by virtue of their formation of lactic acid. Contributions to proteolysis are undoubtedly made by various groups of bacteria, and in the later stages of ripening the spore-forming anaerobes, if numerous, will cause extensive protein breakdown and formation of volatile acids, especially butyric acid. The growth of the latter group of bacteria is greatly affected by the pH of the silage, for at values below about 4·0 the only group capable of anything like active multiplication is the lactobacilli. In fact the A.I.V. method of making silage consists in the addition of mineral acids at the commencement in quantities sufficient to reduce the pH to approximately 3·5. The production of good silage with a minimum loss of feeding value, low proteolysis and the presence of only small quantities of butyric acid may therefore be regarded as depending on the formation of sufficient acid in the earlier stages to retard the activities of the obligate anaerobes and proteolytic bacteria in the later stages.

The Pathology of Transmissible Anaemia (Erythromyelosis) in the Fowl; its similarity to Human Haemopathies

1. Progressive anaemia was observed in 30 White Leghorn hens obtained from a poultry farm in England, where the ailment became noticeable through the yellowish-paleness of the combs and gills of the diseased hens and the heavy mortality following on the appearance of these symptoms.Only hens of one stock were affected, other breeds on the same farm did not develop the same or a similar condition.2. The anaemia was characterised by a lasting diminution in the normal number of erythrocytes, a Hb index less than 1, the preponderance of immature and deformed erythrocytes and the presence of marrow-cells of the erythrocyte series in the circulating blood. The leucocytes were normal or slightly below normal in numbers; the monocytes were diminished.3. The essential pathological lesion is an intravascular proliferation in the marrow of the stem cell of erythrocytes, which results in the blood-stream being overrun with erythroblasts and immature erythrocytes; these are rapidly destroyed in the spleen and liver, whilst the issue of normal erythrocytes in the marrow is hindered. The myeloblastic elements do not proliferate actively. These lesions explain the pathogenesis of the anaemia.4. This intensified activity of the bone-marrow, the resulting congestion and increased localised pressure, induce the production of osteoid tissue; the femur and other bones of the fowls thicken, so that the medullary canal is nearly obliterated.5. Attempts to detect microbes in the blood or isolate bacteria from the organs of anaemic hens failed. The spontaneously diseased hens all harbour a minute cestode,Davainea proglottina, in the duodenum, where the helminth causes round-celled infiltration, petechiae and thickening of the intestinal peritoneum.6. InDavainea-free fowls of the same and other breeds, the injection of blood and cellular emulsions of liver and spleen from erythromyelotic fowls caused a condition of hypercythaemia which lasted several months. This was related to congestion and hyperplasia of the red-blood-marrow and accompanied by considerable enlargement of the liver and spleen.7. Typical erythromyelosis was reproduced in one hen of the same stock from the same farm, by the intravenous inoculation of blood and tissue from spontaneously diseased fowls. In other hens only a transient type of anaemia was obtained, corresponding to the experience of other observers, more particularly Bedson and Knight (1924).The experimental results agree with the view that a proliferative micro-plasm is the specific cause of this condition.8. It is suggested that the development of this outbreak is induced by the massive infestation of the duodenum withDavainea proglottina. This irritative cestode produces a receptive condition in the bone-marrow of the fowl, which allows the microplasm to unfold its pathogenic properties. The duodenal parasite therefore plays the part of a preparatory cause. The association of cestode and microplasm affords an example of a “compound” disease.9. The microplasm is specific and multiplying solely in certain definite blood-marrow cells ofGallus domesticus. This proliferative action can be compared to that of the microbes of epitheliosis contagiosa and Rous fowl-sarcoma; the resulting cell-reaction is then similar to tumour formation in these and other tissues of the fowl.10. Since this haemopathy in fowls is characterised by specific pathological features in the erythrocyte-producing cells of the marrow, the name erythromyelosis is suggested, to mark clearly the position of the principal cellular lesion.11. This disease (erythromyelosis) has been distinguished from other ailments of fowls, in the course of which anaemia may also occur,e.g.avian tuberculosis with involvement of the marrow. The leucocythaemic type is differentiated by diagnosis of the predominant type of cell found in active multiplication in the blood and the position of the cell-proliferation in the marrow.12. Bone-marrow tumours and neoplasms of the lymphatic system occur in fowls, without causing progressive alterations in the cellular contents of the blood. The perivascular aggregations of cells in different organs may also hypertrophy, without any cytological changes being noticeable in the blood, except diminution of the numbers of erythrocytes. Such lesions are therefore distinguishable from different forms of leucocythaemia, where a persistent and progressive increase of genetically related cells is noticeable in the bloodstream and is concurrent with certain diffuse lesions of the marrow.13. The haemoblastic diseases of fowls, such as erythromyelosis, leueo-cythaemia, lymphocythaemia, differ from tumours of the blood-producing tissues, since the former are the result of a pathological disturbance of the mechanism of haemocytopoiesis, whilst the latter are the localised proliferations of cellular elements, which then may spread by a process identical to neoplastic metastasis. There is a distinction in mode and degree.14. The many resemblances between erythromyelosis and certain blood diseases in man and animals may indicate that their pathogenesis. though not identical, is comparable. A similar analogy may be applicable to fowl-leuco-cythaemia and lymphocythaemia.