Bacillus larvae: its Cultivation in vitro and its Growth in vivo

Bailey, L.; Lee, D. C.

doi:10.1099/00221287-29-4-711

Cited by 43 publications

(26 citation statements)

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“…AFB affects only the larvae younger than 2 days by ingesting food contaminated with spores of P. larvae [1,2,12,16]. The spores germinate soon after they enter the larval gut, and the vegetative rods proliferate in the tissues of larvae before pupation [1,2,12].…”

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confidence: 99%

Sporicidal Activities of Disinfectants on Paenibacillus larvae.

Okayama¹,

Sumitomo²,

Nakajima³

et al. 1997

The Journal of Veterinary Medical Science

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ABSTRACT. Sporicidal activities of glutaraldehyde, sodium hypochlorite, povidone iodine, ethylene oxide gas, chlorhexidine gluconate, and didecyl dimethylammonium chloride on wet and dry spores of Paenibacillus larvae (basonym: Bacillus larvae) were evaluated for control of honeybee American foulbrood. Glutaraldehyde was found to have a strong and rapid effect on both the wet and the dry spores among the disinfectants tested. -KEY WORDS: disinfectant, Paenibacillus larvae, sporicidal activity.J. Vet. Med. Sci. 59(10): 953-954, 1997 and mixed in J-agar for counting the colonies grown from the viable spores after 7-day incubation. The results were expressed as the means of 2 experiments. The Association of Official Analytical Chemists (AOAC) Handbook describes the preparation of dry spores for the standard method of sporicidal test, because the spores in nature are usually dried and adhere to other materials. For the present study, we prepared the dry spores according to the AOAC method [14]. Briefly, penicylinders (6 mm in diameter, 10 mm in length, Fisher Scientific Co., U.S.A.) of porcelain were soaked in the wet spore for 15 min, and subsequently dried in a vacuum desiccator at room temperature for 24 hr. The numbers of the dry spores adhering to the penicylinder were determined to be approximately 10 4 by counting the colonies grown from the spores resuspended in saline. The dry spores were dipped in the solution of disinfectants and incubated at room temperature without agitation. At different time of incubation, the samples were transferred to saline for counting the number of viable spores as described above. The results were expressed as the means of 5 experiments.In cases of the chlorhexidine gluconate and didecyl dimethylammonium chloride, the samples, the spores exposed to the disinfectants, were diluted sufficiently before mixing in J-agar for avoiding errors caused by the clumping of the spores [5]. In the case of EO gas, the dry spores were fumigated in a chamber (UDONO, Tokyo, Japan) at 60°C under 90% relative humidity.The sporicidal activities of disinfectants tested in this study are summarized in Fig. 1. The glutaral showed the most remarkable sporicidal activity on the spores of P. larvae, especially the dry spores were more susceptible than the wet spores. No viable spore was detected in the wet and the dry spores after exposure to 2% glutaral in 20 and 5 min, respectively. Similar results have already been described for B. anthracis and B. pumilis [13,15]. These results suggest that the concentrations of 2% or more of glutaral are recommended for the practical use.Similarly, the dry spores were more susceptible to NaOCl than the wet spores. No viable cell was detected in the dry spores after 40-min exposure to NaOCl at every concentrations tested, whereas many viable cells still remained in the wet spores at the concentrations below 0.0125% AvCl. On the basis of the data, NaOCl containing Paenibacillus larvae (basonym: Bacillus larvae) is the causative agent of American foulbrood (AFB), wh...

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confidence: 99%

Sporicidal Activities of Disinfectants on Paenibacillus larvae.

Okayama¹,

Sumitomo²,

Nakajima³

et al. 1997

The Journal of Veterinary Medical Science

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“…2). Although we used larval extracts, we can assume that larvae would have a good resistance against an infection in vivo because data from the literature (Woodrow and Holst, 1942;Bailey and Lee, 1962;Bamrick, 1967) show that young larvae (two-, three-and fourday old) have increasing resistance with increasing age. There are different explanations Figure 3.…”

Section: Discussionmentioning

confidence: 99%

A substance in honey bee larvae inhibits the growth of Paenibacillus larvae larvae

Wedenig

Riessberger-Gallé

2003

Apidologie

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-Paenibacillus larvae larvae, a Gram-positive and spore-forming bacterium, is the cause of American foulbrood. We investigated the resistance of larvae of different ages from different colonies against P. larvae larvae. We prepared ethanol-water-extracts from two, three, four and five day old larvae and also larvae in two capped stages to test the ability of the homogenates to inhibit the growth of P. larvae larvae in vitro. There were age and colony dependent differences in the inhibiting potentials of larvae. The results suggest that the concentration of the inhibiting substance(s) in the extract might be responsible for the known different degree of resistance against P. larvae larvae in vivo. Our results further show that the extracts of the two-, three-and four-day-old larvae have the ability to inhibit the growth of P. larvae larvae very well, while starting around day five the inhibiting effect is gradually reduced.Apis mellifera carnica / Paenibacillus larvae larvae / honeybee larvae / age-related resistance /

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“…The nature of the spore germination signals has been studied widely in Bacilli and Clostridia (21,27,28,(44)(45)(46)(47)(48)(49)(50), but the triggers for P. larvae spore germination have not been identified. In order to cultivate P. larvae, specialized media have been produced (1,31,(51)(52)(53). Under the best conditions, Ͻ10% of P. larvae spores plated on complex laboratory media germinate (54).…”

Section: Discussionmentioning

confidence: 99%

“…The spore pellet was washed five times with water and stored at 4°C (23). Spore preparations were Ͼ90% pure as determined by microscopic observation of Schaeffer-Fulton-stained samples (30 (31)(32)(33). A defined medium was prepared as described previously (34).…”

Section: Methodsmentioning

confidence: 99%

Requirements for In Vitro Germination of Paenibacillus larvae Spores

Alvarado

Phui

Elekonich

et al. 2012

Journal of Bacteriology

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Paenibacillus larvae is the causative agent of American foulbrood (AFB), a disease affecting honey bee larvae. First-and secondinstar larvae become infected when they ingest food contaminated with P. larvae spores. The spores then germinate into vegetative cells that proliferate in the midgut of the honey bee. Although AFB affects honey bees only in the larval stage, P. larvae spores can be distributed throughout the hive. Because spore germination is critical for AFB establishment, we analyzed the requirements for P. larvae spore germination in vitro. We found that P. larvae spores germinated only in response to L-tyrosine plus uric acid under physiologic pH and temperature conditions. This suggests that the simultaneous presence of these signals is necessary for spore germination in vivo. Furthermore, the germination profiles of environmentally derived spores were identical to those of spores from a biochemically typed strain. Because L-tyrosine and uric acid are the only required germinants in vitro, we screened amino acid and purine analogs for their ability to act as antagonists of P. larvae spore germination. Indole and phenol, the side chains of tyrosine and tryptophan, strongly inhibited P. larvae spore germination. Methylation of the N-1 (but not the C-3) position of indole eliminated its ability to inhibit germination. Identification of the activators and inhibitors of P. larvae spore germination provides a basis for developing new tools to control AFB.A merican foulbrood (AFB) is a bacterial disease of honey bees that kills the developing larvae (1, 2). Paenibacillus larvae spores are the infectious agents for AFB, but it is the vegetative cells that cause disease (3, 4). In 2005, a survey of almond-pollinating bee colonies indicated that 4% of colonies had a significant AFB load (5). Once a beekeeping operation is contaminated, the bacterial spores are not easily removed (6). Although autoclaving and high concentrations of chemical disinfectants effectively kill the spores, these treatments are not viable for the beekeeping industry (7). Traditionally, Terramycin and other antibiotics have been used for the treatment and prevention of AFB. However, antibiotic treatment is ineffective in the spore stage of P. larvae, and overuse of the antibiotics leads to resistant strains (8, 9). Presently, the only accepted practice for controlling the spread of AFB is burning both the infected colonies and beekeeping equipment (3, 6).AFB occurs when first-or second-instar larvae (within 48 h after the egg hatches) ingest food that is contaminated with the P. larvae spores (10). Twelve hours after ingestion, P. larvae spores germinate and the new vegetative cells start to proliferate inside the larval gut (11). Several days postinfection, extreme bacteremia causes the death of the honey bee larvae (12-14). After the nutrient levels of the honey bees are depleted, P. larvae cells stop dividing and then sporulate. As a result, billions of spores are found in the dead remains of each bee larva (15, 16). Within the col...

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Bacillus larvae: its Cultivation in vitro and its Growth in vivo

Cited by 43 publications

References 4 publications

Sporicidal Activities of Disinfectants on Paenibacillus larvae.

Sporicidal Activities of Disinfectants on Paenibacillus larvae.

A substance in honey bee larvae inhibits the growth of Paenibacillus larvae larvae

Requirements for In Vitro Germination of Paenibacillus larvae Spores

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