Bacillus Larvae Research Articles

Protection of Bacillus larvae from Oxygen Toxicity with Emphasis on the Role of Catalase

Sporulation of Bacillus larvae NRRL B-3650 occurred only at aeration rates lower than those used for cultivation of most Bacillus species. One possible explanation for the requirement for a low level of aeration in B. larvae is that toxic forms of oxygen such as H(2)O(2) and superoxide are involved. The superoxide dismutase levels of strain B-3650 were similar to those of Bacillus subtilis 168 during sporulation, and no NADH peroxidase was present. Catalase activity was absent during exponential growth and first appeared near the start of the stationary phase. The catalase activity was 2,700 times less than that in B. subtilis 168 at the same stage of development. Therefore, the relative deficiency of catalase (and NADH peroxidase) might be the cause of the apparent O(2) toxicity. It was postulated that B. larvae might accumulate H(2)O(2) in the medium and exhibit more than normal sensitivity to H(2)O(2). Experimental results did not verify either postulate, but the possibilities of intracellular accumulation of H(2)O(2) and unusual sensitivity to endogenous H(2)O(2) were not excluded. The catalase present in early-stationary-phase cells was soluble, heat labile, and inhibited by cyanide, azide, and hydroxylamine. An increase in catalase activity also occurred at the time of appearance of refractile spores in both B. larvae NRRL B-3650 and B. subtilis 168. The level of catalase activity in strain B-3650 was 5,400 times less than that in B. subtilis 168 at this stage. In B. larvae, this second increase occurred primarily within the developing endospore. The activity in spore extracts was particulate, heat stable, and inhibited by hydroxylamine but not by azide or cyanide. Synthesis of catalase in B. larvae was unaffected by H(2)O(2), O(2), or glucose.

Medium Promoting Sporulation of Bacillus larvae and Metabolism of Medium Components

A new medium, designated TMYGP broth, was developed that allowed the honeybee pathogen Bacillus larvae NRRL B-3650 to produce up to 5 x 10 spores per ml of culture (microscopic count). This species normally sporulates poorly, if at all, in artificial broth media. An aeration rate lower than that normally used to cultivate other Bacillus species was required for sporulation. During the exponential growth phase, acids were produced by catabolism of yeast extract components, causing a decrease in pH of the medium. Thereafter, the pH began to increase, probably because of derepression of the citric acid cycle and consumption of the acids. Only after this time did usage of glucose from the medium occur. Thus, glucose usage seems to be regulated by catabolite repression. The presence of glucose was needed for one or more of the later events of sporulation. Of many substances tested, only gluconic acid and glucosamine partially substituted for glucose as a requirement for sporulation. Pyruvate was also required for good sporulation. It was metabolized during the late-exponential phase of growth.

Gamma Radiation Inactivation ofBacillus Larvaeto Control American Foul Brood

SummaryThe radiation dose required to prevent the spread of American foul brood disease in honeybees (Apis mellifera) was investigated quantitatively. Scales from infected combs were gamma-irradiated at different doses and the number of Bacillus larvae spores surviving treatment determined. At least 99·99994% of spores were inactivated by a dose of 1 Megarad (Mrad). The decimal reduction dose for spores in scale was estimated as 0·162 ± 0·014 Mrad; the disease did not develop in hives containing heavily infected frames which had been treated with a minimum dose of 1 Mrad. The basis for selecting the radiation dose required to decontaminate infected hives is discussed. Estimates of costs indicate that substantial savings are possible if irradiation treatment replaces the destructive control method currently used.

静岡県下における<I>Bacillus larvae</I>の浸潤状況について

A macroscopic survey on abnormal maggots was conducted in all the honeybees on 3 farms with positive results. On one of these farms, however, only one abnormal maggot was found among those of 60 hives, indicating the difficulty in detecting foul brood by sampling test.A bacteriological survey on Bacillus larvae was conducted in honeybees of 20-40% of the hives on 11 farms. It showed positive results on ten of these farms. The rate of positive detection was relatively high on the farms where abnormal maggots had been found.

Bacteriophage ofBacillus Larvae

SummaryThe bacteriophage of Bacillus larvae was isolated in Czechoslovakia for the first time. Most strains of B. larvae were found to contain a temperate non-virulent phage which could be detected by a cross-test. This bacteriophage is polyvalent and may become virulent, forming plaques with various strains of B. larvae. The morphology and ultrastructure of the phage are described for the first time. This bacteriophage of B. larvae has been designated BLA.

Production of Hydrogen Sulphide byBacillus Larvae in Vitro

SummaryLaboratory cultures of Bacillus larvae, the causative agent of American foul brood disease, produced abundant hydrogen sulphide (H2S) if the medium contained added cysteine HCl or sodium thiosulphate as a source of sulphur, but little or none from an unsupplemented medium or a medium supplemented with one of several other sulphur-containing compounds, including L-cystine, methionine, and glutathione. Cysteine HCl (0·02%, w/v) added to the medium was rapidly metabolized by laboratory strains of B. larvae, and the cultures ceased to liberate H2S by 96 h. Freshly isolated strains, which sporulated well in culture, released very little or no H2S from the disulphide amino acid L-cystine during either growth or sporulation.

Volatile Acids from Honeybee Larvae Infected withBacillus Larvaeand from a Culture of the Organism

SummaryAn ether extract of honeybee larvae killed by Bacillus larvae contained several volatile organic acids, most of which were also present in an extract from an in vitro culture of the causative organism. The extracts of diseased larvae and the culture were characterized by highly odorous acids, including valeric, isovaleric, anteisovaleric, butyric and caproic acids, whereas healthy larvae were generally devoid of these compounds.

Residual Effects of Ethylene Oxide in Honey and Pollen

SummaryWorker honeybees in cages, fed on honey and pollen treated with ethylene oxide at 900 g/m3 without subsequent ventilation, did not show a higher mortality than controls. Mice fed on honey similarly treated showed neither pathological alterations in liver and kidneys, nor higher mortality, than controls. On the other hand, the addition of ethylene glycol to honey, at a concentration as low as 1·25% by weight, had a toxic action in bees, and in mice a concentration of 0·78% caused toxic damage to the liver (steatosis). The effectiveness of the ethylene oxide treatment on vegetative forms and spores of Bacillus larvae (except in dry scales), and on other micro-organisms, was confirmed.

A study on the possible utilization of immunodiffusion and immunofluorescence techniques as the diagnostic methods for American foulbrood of honeybees ( Apis mellifera)

Four types of antisera were obtained from rabbits hyperimmunized with either spores or vegetative rods from two strains of the American foulbrood pathogen, Bacillus larvae. The specificity and sensitivity of these antisera were tested with immunofluorescence and immunodiffusion methods. No cross-reactions were observed between the antisera and other different species of Bacillus or different genera of bacteria. The specificity was not found between the antisera and two strains of B. larvae although stronger fluorescent intensity was observed between the antiserum and its corresponding strain of antigen in the immunofluorescence tests. Eight samples of 1- to 2-day-old larvae, 3- to 4-day-old larvae, decayed tissue, and dry remain, collected from eight infected colonies, were tested against antisera by the immunofluorescence and the immunodiffusion methods. The results indicated that both methods are sensitive and specific for making diagnosis of field samples of American foulbrood of honey bees.

Large-Scale Fumigation with Ethylene Oxide of Honeybee Combs Contaminated with Bacillus Larvae

SummaryBrood combs contaminated with American foul brood (AFB) were fumigated with ethylene oxide (ETO) in a large heated vacuum chamber. ETO concentrations up to 435 mg/litre applied as Oxyfume 12, at temperatures as high as 54°C, did not sterilize B. larvae spores in the residues of larvae killed by AFB. Spore suspensions of an indicator organism, Bacillus subtilis, were not regularly killed by the treatments, although chemical indicator cards used in monitoring the fumigation process indicated complete sterilization.Fumigation in a small chamber at 35°C, nearly 100% RH and with an ETO concentration 450 mg/litre sterilized B. subtilis after 16 h, and B. larvae after 24 h. Indicator cards became a deep green by 24 h, showing a sufficient exposure to ETO.AFB infection in package bee colonies hived on treated combs was variable. Half the treated colonies developed fresh infection during the season and were then treated with Oxytetracycline to control the disease.

Susceptibility ofBacillus Larvaeto Ethylene Oxide and Tetracycline Hcl

SummaryIsolates of Bacillus larvae spores obtained from various geographical areas in the USA were surveyed as to susceptibility to tetracyline and to ethylene oxide (ETO). No evidence of resistance to tetracycline HCI was found, nor was there any difference in the resistance of isolates collected before and after tetracyclines were used to control bee diseases. When B. larvae spores from 28 of these sources were treated with ETO (450 mg. litre at 38°C and 50% RH), more than 99% of the spores were killed by a 60-min exposure, and only chance growth occurred after a 90-min exposure. There was no evidence of significant variations in the suceptibility of the various isolates of B. larvae to ETO.

<I>Bacillus laroae</I>の芽胞に対するエチレンオキサイドガスの殺菌作用について

<I>Bacillus laroae</I>の芽胞に対するエチレンオキサイドガスの殺菌作用について

Monitoring the Disinfection of Honeybee Combs by Ethylene Oxide

SummaryBrood combs contaminated with larvae killed by American foul brood (scales) were treated with 100 mg per litre of ethylene oxide (ETO) in a vacuum chamber. After exposure for 18 h at 27°C, the scale samples had the same odour, appearance, fluorescence, and proteolytic activity as untreated control scales. Microscopic examination of Bacillus larvae spore suspensions prepared from the scales disclosed no differences in refractility of the spores between treated and untreated samples. Culture of scale suspensions in germination medium revealed no viable spores from the treated group in concentrations as high as 107 spores per tube. However, spores from untreated scales grew in concentrations as low as 101 spores per tube.It is considered that laboratory demonstration of the effectiveness of ETO sterilization can best be made by bacteriological culture.

The effects of Penicillium waksmani growth on American foulbrood infected combs and adult honeybee activity

Penicillium waksmani was found capable of growing on the scales left by American foulbrood disease (AFB). The scale thus became loosened from the cell wall in a wax comb and was easier for the adult worker honeybees, Apis mellifera, to remove. Also, the bees were stimulated to remove the mold, and consequently, the loosened scales. This cell cleaning and removal of scale was further evidenced by a reduction in cell wall height and in the number of viable Bacillus larvae spores in the cells.

The influence of pollen on the susceptibility of honey-bee larvae to Bacillus larvae

Pollen, water, or nothing was added to the food of worker honey-bee, Apis mellifera, larvae aged 6–18 hr. Six hr later larvae from each of these three groups received, in their food, either Bacillus larvae spores in water, or water only. Pooled data from seven replicates of this procedure show that spore feeding preceded by pollen feeding resulted in a 71.17% mortality; spore feeding preceded by water feeding resulted in 94.84% mortality; and spore feeding preceded by nonexperimental feeding resulted in 92.75% mortality. Mortality in groups not receiving spores was below 11.00% in all cases. Chi-square analyses showed a highly significant reduction in mortality associated with pollen feeding.

Detection and Identification ofBacillus Larvaein a Commercial Sample of Bee-Collected Pollen

SummaryOne sample of pollen (produced in British Columbia, Canada) caused American foul brood disease in a healthy honeybee colony to which it was fed. An aqueous filtrate from the pollen contained a large number of bacterial spores (>109 per g pollen). The same pollen sample showed a proteolytic activity similar to that found in dried diseased larvae; it was also less attractive to foraging bees than was disease-free pollen.

Growth, protease formation, and sporulation of Bacillus larvae in aerated broth culture

Strains of Bacillus larvae varied in their capacity to produce spores and proteases in aerated broth culture. Addition of activated charcoal to the medium increased both products in each strain tested. The small molecular-size protease appeared first in the medium, while optimal sporulation was accompanied by release of the large molecular-size enzyme. Continued incubation usually resulted in a reduction in free spores and a disappearance of proteases.

Pathogenicity to Honey Bees of a Strain of Bacillus larvae That Does Not Reduce Nitrate12

A strain of Bacillus larvae While that does not reduce nitrate, produced typical infections of American foulbrood disease when it was injected into the head capsule of one-day-old pupae of A pis mellifera L. or was ingested with the brood food by young bee larvae. It was reisolated in pure culture from an infected pupa, and it still gave a negative nitrate reduction reaction. Like several other strains (from Montana, Nebraska, and Wyoming) that do reduce nitrate, it caused American foulbrood in young bee larvae of similar ages in the same combs.

Ultrastructure of American foulbrood disease pathogenesis in larvae of the worker honey bee, Apis mellifera

Using electron microscopy, the pathogenesis of American foulbrood disease was followed from ingestion of Bacillus larvae spores by young, susceptible honey bee larvae to death of the host and sporulation of the pathogen. Interaction between the host peritrophic membrane and B. larvae vegetatives is described. Phagocytosis was demonstrated to be a mechanism of entry of pathogen into host midgut cells. No evidence of enzymatic digestion of peritrophic membrane or host-cell microvilli was found during the initial interaction of pathogen and host midgut cell, although eventual lysis of host gut cells may have been the result of enzymatic activity. Following entry of bacteria into the hemocoel, host death resulted from systemic bacteremia.

The production and properties of Bacillus larvae proteases

Bacillus larvae, a pathogen of larval honey-bee, produces during sporulation either in the host or in culture a mixture of highly active proteases. They hydrolyse azoalbumin optimally at p H 6·6 and 60°C and retain over 80 per cent activity after 30 minutes' incubation. The proteolytic activity is inhibited by EDTA and Zn 2+ but not by crystalline soybean trypsin inhibitor or diisopropylfluorophosphoridate. The activity is restored by Co 2+ and Cu 2+ but not by Fe 2+, Mn 2+, Mg 2+, or Ca 2+. Three proteases are resolved by chromatographic and electrophoretic techniques. Two enzymes are extractable from the infected hosts and sporulated cultures and one from the vegetative cells of B. larvae. One enzyme with acidic p H optimum is found to be heat-stable while another with slightly alkaline p H optimum is heat-labile. Insensitivity to trichloroacetic acid precipitation and low sedimentation values suggest the enzymes to be of low molecular weight.