Appendices of the Draft Screening Assessment for Bacillus cereus (ATCC 14579) Environment Canada Health Canada July 2013

• no known homology to cry and cyt,
• containsmobile elements and putative proteins

• Tn554, AbrB (regulator hom.)
• Bc1A (spore coat determinate)

• polysaccharide capsule

• prophage feature, Similar to Bam35

• prophage feature

• lethal toxin complex pagA, lef, cya

• no known homology to cry and cyt,
• containsmobile elements and putative proteins

• insecticidal protein toxin (cry, cyt)

• emetic toxin (cereulide)

• possesses a number of transposable genes and mobile elements

• associated with periodontal isolates

• lethal toxin complex, pag, lef and cya genes

• D-glutamic acid caspsule,
• operon cap BCADE

• Aggregation phenotype

• lethal toxin complex, pag, lef and cya genes

• D-glutamic acid caspsule,
• operon cap BCADE

• Unknown function
• Cryptic plasmid

• Resistance to mercury

• exhibits characteristics of a mobile element

• transposase

• Ribozyme (catalytic RNA)

• Ribozyme (catalytic RNA)

• Self-splicing group I introns associated with IS element

Tobacco hornworm
Manduca sexta
5^th instar larvae
Sex not specified

Purpose or context:
Insect infection model to characterize the role of the iron-responsive regulator fur gene in the virulence of B. cereus.

Route of exposure:

• Injection of vegetative cells (compartment not specified.

Test conditions:

• Not specified.

Dose regimen:

• Single injection of known but unspecified doses
• Single replicate of at least 20 larvae per dose group

Controls:

• Not specified.

Duration of study:

• 48 hours.

• 569 WT
• 569 Δfur
• Both grown in LB medium with antibiotics.
• fur is a homologue of the B. subtilis fur gene identified in B. cereus.

Observation intervals:

• Not specified.

Mortality observed:

• Median lethal dose (LD₅₀) calculated by Probit analysis of mortality data (values in parentheses are 95% confidence limits).
• Wild-type LD₅₀ value = 1859 cfu (1142-2774)
• Mutant LD₅₀ value = 4932 cfu (3609-6912)

Conclusions:

• Reduced virulence for the B. cereus 569 Δfurmutant.
• The Δfur mutant constitutively expresses siderophores and accumulates iron intracellularly to a level threefold greater than the WT.

Wax moth
Galleria mellonella
Last instar larvae
Sex not specified

Purpose:
Investigation of the opportunistic properties of acrystalliferous B. thuringiensis (Bt) and B. cereus strain and the role of the plcR gene, a pleiotropic regulator of extracellular factors.

Routes of exposure:

• Force-feeding co-ingestion.
• Intrahaemocoelic injection.

Methods of administration, respectively:

• 0.5 X 25 mm needles and microinjector.
• At the base of last proleg, using a microinjector with a 1 ml hypodermic syringe and 0.45 X 12 mm needles.

Dose regimen:

• 10 μl of spore suspension per larvae for both methods.
• For the force-feedings, spores were in association with crystal toxins.

Repetitions and replications:

• No repetition on the same animal.
• 30 larvae used for each dose and for each method.

Controls:

• Force-feedings with spores (10⁶) or Cry1C toxins alone.

Duration of study:

• Casualties recorded over 7 days.

• ATCC 14579
• Spores were obtained by culturing cells in HCT medium at 30°C for 4 days, centrifuged and resuspended in 10 mL sterile distilled water.
• Spore preparations were heated for 20 min at 80°C.

Observation intervals:

• Organisms checked daily.
• After injection, organisms were kept individually in boxes containing beeswax and pollen at 25°C.

Mortality observed:

• After 2 days.
• Very low (greater than 10%) with crystals or spores alone (controls).
• ≈70% mortality caused by co-ingestion of 106  spores with a sublethal (1μg) quantity of Cry1C toxin.

Conclusions:

• Clear pattern of synergism between the spores of B. cereus and the toxin of B. thuringiensis.

Wax moth
Galleria mellonella
2^nd and 5^th instar larvae

Reared on beeswax and pollen.

Purpose:
To evaluate whether Galleria mellonella
can function as an oral infection model for human and entomo-bacterial pathogens. 

Route of exposure:

• Oral infection

Free ingestions:

• On 2^nd instar
• Mixtures of 50% pollen with 50% water containing 10⁸  spores/mL alone or along with 2 μg of Cry1C diluted in PBS at pH 7.4.
• Both preparations (2 ml) were added to 5 cm diameter plastic Petri dishes and allowed to surface dry.
• Larvae then placed in each dish and incubated at 37°C

Force feedings:

• On 5^th instar, weighing 200 mg and starved for 24 hours prior to the test.
• Used a micro injector.
• 5 × 10⁵ - 1× 10⁶ spores or vegetative cells per larva, with (2 – 3 μg) and without Cry1C toxin.

Repetitions and replications:

• No repetition on the same animal.
• 10 X 2^nd instar larvae in each dish for free ingestions.
• 20 X 5^th instar larvae for force-feedings.
• Experiments replicated 3 times.

Controls:

• Spores alone.
• Cry1C toxin alone.

Duration of study:

• 72 hours.

1 environmental isolate :

• ATCC 14579

5 other diarrheal strains:

• D6 (F4370/ 75)
• D23 (F284/78)
• D17 (1651-00)
• D19 (NvH391/ 98)
• D24 (F352/90)

4 of them were tested in force feedings:

• D19
• D23
• D6
• ATCC 14579

Spores:

• Produced in HCT medium, washed, resuspended in water, heat treated and enumerated.

Vegetative cells:

• Collected at exponential growth phase (OD600nm ≈ 1) in LB medium.

Observation intervals:

• Mortality recorded daily.

Mortality observed for free ingestion:

• 2 ± 2% for ATCC 14579 spores alone.
• 5 ± 5% for Cry1C toxin alone.
• Ranging from 12 ± 7% (D24) to 57 ± 20% (D23) for co-ingestion of Cry1C toxin.

Mortality observed for force feeding:

• 0% (D19) to 8 ± 6% (D23) without toxin.
• 10 ± 8% (D19) to 50 ± 13% (D23) in co-ingestion.

Conclusions:

• Important variation among strains was observed.
• These results demonstrate synergy.
• The low virulence of D19 (10%) was unexpected since it is known to be a highly virulent human pathogen.
• Insect mortality values did not correlate with the pathogenic potential of the bacterial strains.

Cabbage looper
Trichoplusia ni
1 to 8-day old
healthy larvae from a stock culture.

Purpose:
Pathogenicity test to characterize the non-viral cause of larvae death in a study on NPV.

Route of exposure:

• Free ingestion of contaminated diet pathogenicity test.

Method of administration

• Suspension pipetted onto the surface of freshly prepared artificial diet in a 1-oz plastic cup.
• One larvae at a time was transferred in the cup to feed.

Physical conditions:

• 25-28°C.
• 75-85% relative humidity.

Dose regimen:

• 0.1 ml of suspension
• bacteria, virus or combination used (3 test groups)
• 2.5 or 5.0 × 10⁷ cells/cup

Repetitions and replications:

• No repetition on the same larvae.
• 50 larvae per dosage.
• 2 experiments.

Controls:

• Sterile saline or water.

Duration of study:

• 12 days.

• No strain designation specified.
• Consistent isolate from dead or moribund larvae.
• Cells suspended in sterile saline.

• The cause of death and symptoms was identified as B. cereus on the basis of criteria of  A. Krieg’s key, 1970.

Parameter measured:

• Daily mortality counts.
• Corrected according to Abbott’s formula.

Range of effects:

• The highest level, 7.2 × 10⁸ cells/cup, caused 100% mortality in 11 days.
• 69 and 50% mortality occurred among larvae exposed to 3.6 and 1.8 × 10⁸ cells/cup, respectively.
• 70 to 100% died within 10 days.
• Symptoms were identical to those observed in larvae from which original isolations were found: larvae ceased to feed, showed paralysis, darkening of integument and ultimately died.
• 1-day-old larvae appeared more susceptible to the bacterium than 2 to 8-day-old larvae.
• 1-day-old cultures of B. cereus caused greater and more rapid mortality than did 2, 3 or 20-day-old cultures.

Conclusions:

• Combinations of the two pathogens resulted in slightly higher mortality than either pathogen alone, but there were no synergistic effects.
• Pathogenicity to T. ni was not associated with any demonstrable toxin.

Silkworm
5^th instar larvae
Raised from fertilized eggs in the laboratory.
Fed antibiotic-free food for 1 day.

Purpose:
Purification and identification of a soil bacteria exotoxin, sphingomyelinase C.

Route of exposure:

• Injection into the hemolymph through the dorsal surface.

Method of administration

• 27-gauge needle.

Physical conditions:

• Not specified.

Dose regimen

• 3 test groups
• 0.05 ml of an overnight culture or culture supernatant.
• Two-fold dilutions of purified sphingomyelinase.

Repetitions and replications

• 2 silkworms for each dose of culture or culture supernatant.
• 5 silkworms for each dose of the toxin.

Controls

• None specified.

Duration of study

• 24 hours.

• ATCC 14579
• 25 distinct colonies of which 16 killed silkworm
• 9 undesignated strains of Bacillus sp isolated from soil.
• Soil samples were spread onto brain-heart infusion agar plates and colonies isolated after overnight incubation at 30°C.
• Cultures were centrifugated and filtrated through a 0.22-μm filter.

Parameter measured:

• Number of silkworms alive after 24 hours.

Range of effects for soil isolates:

• Of 25 distinct isolates, 16 killed silkworms.
• 5 out of 16 culture supernatants had a killing activity against silkworms. These 5 strains were identified as Bacillusspecies (16S rRNA sequences).
• The toxin purified from isolate #11 was identified as sphingomyelinase C from B. cereus.

Range of effects for sphingomyelinase from B. cereus ATCC 14579:

• Killed silkworms with an LD₅₀ of 0.7 μg.

German cockroaches
Blattela germanica
Adult males

Purpose:
Purification and characterization of  insect toxicity of sphingomyelinase C produced by B. cereus.

Route of exposure:

• Injection into the abdomen

Method of administration

• Not specified

Physical conditions:

• Insects reared at 26 and 60% relative humidity.

Dose regimen:

• 2 μl of cell-free supernatant or solution of protein sample.
• Untreated, heat-shocked and proteinase K treated culture broths tested on cockroaches (3 groups).

Repetitions and replications:

• 5 cockroaches used for each dose.

Controls:

• Not specified.

Duration of study:

• 10 minutes.

• ATCC 14579 (99.9% rRNA sequence homology)
• Isolated from the mandibles of last instars of antlions, Myrmeleon bore.
• Produced insecticidal factors when cultured aerobically.

Parameter measured:

• Symptoms observed 10 minutes after injection.
• Minimum paralysis dose (MPD) at which at least four or five insects were paralysed.

Range of effects:

• Rapid paralysis after injection.
• MPD of 262 ± 29 ng protein/insect.
• The bacterium was able to grow even at 50°C.
• The insecticidal activity was abolished by heating at 100°C and by proteinase K treatment.

Conclusions:

• Sphingomyelinase C produced by B. cereus is able to kill insects rapidly at low doses.
• The insecticidal factors produced by B. cereus may aid the prey-capturing action of the antlions.
• The insecticidal effect of sphingomyelinase C is due to its action on the nervous system.

• Intrahemocoelic challenge

• 4 strains comprising:
• B1
• NCIB 3329

• B1 was the most pathogenic.
• NCIB 3329 was the least pathogenic

Elm bark beetles Scolytus scolytus
5^th instar larvae
Collected from infested elm logs.

Purpose:
Investigations for a biological control for the vector of Dutch elm disease, among Bacillus spp.

Test conditions:

• Larvae suspended in a suspension of cells.
• Time of suspension: 1 hour.
• Afterwards, reared on an artificial medium at 27°C .
• Dead larvae removed daily.

Dose regimen:

• 8 × 10⁵ cells/ml

Controls:

• Larvae suspended in distilled water.

Duration of the study:

• 21 days.

• B. cereus 11796

Mortality observed:

• Corrected for natural mortality: 63.6% of 40 larvae were killed.
• Control gave 17.5% mortality (corrected to 0%) in 40 larvae.

• Oral inoculation

• No strain designation provided.

• Strains isolated from diseased beetle were pathogenic.

Boll weevil
Anthonomus grandis,

Egyptian cotton leafworm
Spodoptera littoralis

Black bean aphid
Aphis fabae.

• Free ingestion method of supernatant.
• Colonies used.

• 575 strains used for A. grandis.
• 270 strains used for S. littoralis and A. fabae.

• 4 of the 575 strains were toxic for A. grandis (85 to 100% mortality).
• 5 of the 270 strains resulted in 41 to 97% mortality in A. fabae.
• No effect on S. littoralis.

• Culture serial dilutions (200 mL) added to 20 jars containing individual neonates (24-hours old)
• Final concentration 10⁴ – 10⁶ CFU mL-1.
• Presence of death events checked each day.
• Control of uninfected animals.

• BD170 EH2, an originally non hemolytic Bacillus subtilis expressing an introduced B. cereushemolysin II gene, hlyII.
• B. cereus VKM B-771.

• Animal death resulted within 8 to 16 days.
• BD170 EH2 decreased fecundity.

Route of exposure:

• Injection (compartment not specified).

Dose regimen:

• Not specified.
• Surface culture at 24 h on an agar plate.

Control:

• 4.0 cc. glucose broth.

• ATCC 21 subcultured rapidly for a few generations.
• N. R. Smith No. 156.
• Both strains subcultured rapidly for a few generations.

• Guinea pigs killed only when strains were subcultured.
• Glucose broth failed to kill the animals.

Route of exposure:

• Injection of culture filtrates (0.05 mL) intradermally in albino guinea pigs of either sex.

Control:

• Positive and negative controls included.
• Animals were observed after 6 and 24 hours.

• B-4ac used for the dermal assay.
• 24 other B. cereus strains tested
• No designation provided.

• B-4ac and 21 strains gave necrotic reactions surrounded by inflammation at the site of injection.

New Zealand white rabbits
Oryctolagus cuniculus

Ligated ileal loop

• Food poisoning experimental model.
• 6 test loops per rabbit.
• 1 negative and 1 positive control per rabbit

• 22 different strains designated

• Rapid accumulation of 3 to 20 mL of straw-colored, often bloody fluid.
• Positive responses for 19 of the 22 strains.
• Consistently positive responses for younger rabbits.
• Most of the rabbits with at least one positive loop died within 10 hours following the surgery.

• 0.05 mL of cell-free culture filtrate injected intradermally in rabbits weighing 2 to 3 kg.
• 3 hours after injection, Evans blue dye was injected into the ear vein.

• 11 strains of B. cereus
• Only one of them had the designation B. cereus B-4ac, which was known to be positive in both the ileal loop and guinea pig dermal assays.

• Blueing area representing the increase in vascular permeability ranged from 4 to over 100 mm2 for strain B-4ac.
• 9 of the 10 other strains produced a positive vascular reaction.

Dutch rabbits
Oryctolagus cuniculus

Males weighing 1.8 ± 0.2 kg.

• 0.1 or 0.3 mL injected intramuscularly into the flank.
• 0.15 mL injected subcutaneously
• Animals were killed before the infection proved fatal.
• Vegetative cells and spore suspensions used.
• Concentration ca. 102 cells/mL.

• SV1 lecithinase negative variant

• Presence of abscesses showing inflammatory response.
• Presence of nodules under the skin with necrotic fibres and fibrosis around its periphery.
• Calcification observed in 80% of the animals after 7 days.

• Injected intradermally (0.1 mL) into rabbits weighing from 2.5 to 3 kg.

• 50 of 136 strains isolated from dairy products.
• 102 positive strains for extracellular toxins.

• All 102 strains caused vascular permeability in rabbit skin (intense blueing caused by the Evans blue dye.

• 3 enterotoxins in concentrated cell-free culture filtrate.
• Ligated rabbit ileal assay.
• Preliminary results for 23 isolates.
• 4 selected strains.

• Strain isolated from an incident which caused diarrhea in 6 of 10 monkeys
• Strain isolated from raw rice which failed to produce symptoms in eight monkey feedings.
• Strain isolated from a brain abscess (2141/74, serotype 11).
• B-4ac

• Although just 11 of the strains were tested more than twice, only 2 of the 11 exhibited a greater than 50% probability of being positive on repeated testing.
• Fluid accumulation in rabbit ileal loop for the first two strains
• Severe disruption of the mucosa in the ileal mucosa for the third strain.

• In vitro retinal toxicity assay:Measure of the cytolytic release of lactate dehydrogenase (LDH) from retinal buttons treated with B. cereus toxins (600 ng/mL HBLeq).
• In vivo sterile endophtalmitis model::Intravitreal injection of pure or crude exotoxin (0.1 to 1.15 mL).
• Control samples and toxin-containing samples included.

• MGBC 145

• Retinal buttons treated with either CET or HBL became completely disaggregated into cells and cell debris and collapsed upon removal.
• Within 4 hours, all eyes receiving greater than or equal to 0.8 μg crude exotoxin exhibited marked exudate, conjunctival edema and hyperemia.
• When receiving 1-4 μg, no or little red reflex, vitreal hemorrhage, hemorrhagic chemosis of the conjunctiva, and corneal haze.
• Milder responses to low doses.

• Ileal loop fluid accumulation model.
• Purified 3 components of HBL.

• F837/76

• Caused fluid accumulation and 3 components were required together to cause maximal activity.

New Zealand white rabbits
Oryctolagus cuniculus

2 to 3 kg

• Eyes injected intravitreally with viable B. cereus(log 2.06 CFU)or cell-free supernatant.
• Surgical and absolute controls.

• MGBC145

• Intraocular inflammation and reduction in retinal responses after 3 hours.
• Retinal detachment and photoreceptor layer folding and disrupting observed after 9 hours.
• At 18 hours, eyes demonstrated maximal inflammation, including in peri-ocular tissues.
• Injection of culture supernatant produces similar results.

• Intraperitoneal (0.5 mL) and subcutaneous (0.25 mL) injections
• 4 dilutions injected in each of 12 or more mice
• Vegetative forms and spores tested.

• NRS 201
• NRS 232
• NRS 1256

• 10 to 100 times more spores were required to kill mice.
• Death occurred upon intraperitoneal injection but not subcutaneous.
• Subcutaneous injections resulted in an open necrotic lesion.

• Subcutaneous or intraperitoneal injections (0.25 mL) of a suspension (approx. 500 million bacilli per mL)
• Cultures were frequently transferred from one medium to another to increase virulence
• Groups of 4 to 8 mice

• Activated strains
• No strain designation provided.

• Acute lethal illness at high doses, almost all within 6 hours.
• The severity of the disease was clearly dose-dependant.
• The minimal dose causing 84 to 100% mortality was approx. 2.2 X 10⁷ bacilli.
• Low doses resulted in mild illness and sometimes by necrotic skin ulcers at the injection site.

• 0.5 mL of culture filtrate injected into the caudal veins of 4 adult ICR mice.
• 2 clinical isolates were used as positive controls and culture medium was used as negative control.

• 183 strains isolated from dairy products

• Of 11 isolates having strong hemolysin activity, 3 of them killed mice.

• Intravenous injection of 8 μg of purified hemolysin II

• FS-1

• Death within 2 minutes

• Vascular permeability test, intestinal necrosis reaction and mouse lethal test.

• 116 strains.
• About 13 of them designated.

• Good correlation between production of necrosis in the skin and intestinal tests and the fluid accumulation test.

Mice
Mus musculus

BALB/c strain
5-week-old females

Kept in a biosafety containment facility in groups of 5, with sterile water and food.

Purpose:
Investigation of the opportunistic properties of a B. thuringiensis mutant and B. cereus, and the role of the plcR gene.

Route of exposure:

• Nasal instillation under slight ether anaesthesia.

Method of administration:

• The spore or bacterial suspension was carefully deposited at the corner of the nostril. The mouse inhaled the inoculum by breathing.

Dose regimen:

• 50 μl of the suspension (spores or vegetative cells).

Repetitions and replications:

• No repetition.
• Groups of 5-10 mice used for each strain.

Controls:

• None specified.

Duration of study:

• Mortality observed after 24 hours.

2 B. cereus strains tested:

• ATCC 14579
• ATCC 14579 ΔplcR
• Vegetative cells were prepared from cultures in TSB at 37°C, recovered after culture for 18 h (late stationary phase) by centrifugation.
• Spore suspensions were prepared from a 10 days old culture on agar medium. They were washed and resuspended in sterile water, incubated for 1 h at 65°C to kill the vegetative forms.

Mortality observed:

• 10⁸ spores per mouse resulted in 100% mortality for both strains.
• 5 X 10⁷ spores per mouse resulted in 90% and 22% mortality, respectively.
• 10⁷ spores per mouse resulted in 90% and 0% mortality, respectively.
• 6 × 10⁶ vegetative cells per mouse resulted in 100% and 0% mortality, respectively.

Conclusions:

• ATCC 14579 possesses additional factors, not regulated by PlcR, which may potentiate its opportunistic properties.
• Rapid death of the host if large doses of vegetative or sporulated cells are used.
• The cause of death is unlikely to be due to the growth of the bacteria.

Mice
Mus musculus

BALB/c strain

Route of exposure:

• Endotrachea

• ATCC 14579

• Exposure to spores results in negligible effects
• Exposure to vegetative cells
• experiments terminated at 4h due to severity of symptoms;
• elevated pyrogenic cytokines,
• pulmonary granulocyte infiltration,
• acute phase response markers.

Monkeys
Macaca mulatta

Rhesus strain

Purpose:
Determine the usefulness of Rhesus monkeys model for enteropathogenicity of B. cereus.

Route of exposure:

• Force-feeding.

Method of administration:

• Monkeys fasted 18 hours prior to feeding.
• Given to monkeys by stomach tubes.

Dose regimen:

• 3 types of test material fed: whole cultures, sterile culture filtrates or purified precipitated toxin.
• 40 ml by monkey.
• Normal food available afterwards.

Repetitions and replications:

• 6 monkeys for each test material.
• Fluid accumulation in rabbit ileal loops and skin capillary permeability tests also performed.

Control:

• Sterile BHIG as negative.

Duration of study:

• till symptoms were observed (at least 210 min)

• B-4ac, isolated from a food poisoning outbreak.
• 6 other strains with no specified designation, isolated from the rice-associated outbreaks

Observation intervals:

• continuous

Symptoms observed:

• Diarrhea elicited by the three test materials 35-150 minutes after administration.
• Lasted 60-210 minutes.
• Control was negative.
• Considerable variation in sensitivity among test monkeys.
• Approx. 50% of the monkeys showed positive responses.
• Vomiting never observed.
• 4 of the 6 undesignated strains were positive diarrheal but negative vomiting.
• When grown on rice, B-4ac induced diarrhea in 3 of 6 monkeys but not vomiting.

Conclusions:

• Direct correlation between ability to cause fluid accumulation in rabbit ileal loops, alteration of skin capillary permeability and ability to induce diarrhea in monkeys.
• Rhesus monkeys are a suitable model.
• Diarrhea is due to synthesis and excretion of a toxin by logarithmically growing cells.

Monkeys
Macaca mulatta

Sex not specified
Young Rhesus strain of approximately 3 kg.

Purpose:
Attempt to confirm that food-associated outbreaks were caused by B. cereus and to determine the involvement of a new enterotoxigenic material.

Route of exposure:

• Force-feeding.

Method of administration:

• Contaminated rice was homogenized and concentrated.
• Organisms were also grown in liquid broth.
• Under anaesthesia, the test material was administered per os via a lubricated feeding tube.

Dose regimen:

• 30 ml of an 8-hour culture
• In food, about 1010 viable organisms.
• In broth, about 1011 organisms.
• 100 to 150 ml of the material.
• Also, ileal fluid accumulation tested with 12-15 fold concentrated filtrates.

Repetitions and replications:

• Between 4 and 24 monkeys for each combination of strain and culture medium.

Control:

• Uninoculated medium

Duration of study:

• 24 hours.

3 strains of B. cereus:

• 4810/73 (formerly strain 88) isolated from vomitus, associated in illness.
• 4433/73 isolated from meat loaf, implicated in food outbreak.
• 2532B/74 isolated from rice.

Parameter measured:

• Emetic activity: vomiting within 5 hours.
• Diarrhea: presence of watery or loose stools within 24 hours.
• Faecal examination.
• Ligated ileal assays

Range of effects for emetic activity:

• Feeding of uninoculated media had no adverse effect.
• Only cultures grown on rice could cause vomiting.
• 10 of 24 monkeys showed positive vomiting for strain4810/73.

Range of effects for diarrheic activity:

• Feeding of uninoculated media had no adverse effect.
• Largely confined to strain 4433/73.
• Chiefly, diarrhea in 6 of 10 monkeys, for rice.
• Present in both rice and broth cultures.
• Faecal examination.
• Bacteriological picture accurately reflected the quantities in the material fed.

Ligated ileal assays

• Of 13, 15 and 12 tests respectively for each strain, 2, 12 and 9 were loop positives.

Conclusions:

• A clear distinction is made between the strains causing vomiting and diarrheal.
• The difference between the activities of the 2 first strains is reinforced by the rabbit loop test.
• The range of foods involved in diarrheal outbreaks was wide whereas vomiting outbreaks have been restricted to the consumption of rice.

Monkeys
Macaca mulatta
Rhesus strain 6-8 kg

Mice
Mus musculus
ICR, strain not specified.
Sex not specified.
Weighing 20-24 g.

Purpose:
Study the correlation between emetic toxin and vacuolation factor HEp-2 produced by B. cereus isolated from an outbreak of vomiting-type food poisoning.

Tests conducted:

• Mouse lethality.
• Emetic activity to monkeys.

Route of exposure:

• Intravenous injection.
• Intragastric administration.

Method of administration:

• Details not specified for mice.
• Oral administration by catheter for monkeys.

Dose regimen:

• 0.2 ml of the test sample.
• 20 ml of the test sample.

Substances tested:

• Purified cereulide.
• Partially purified vacuolation factor.

Test conditions:

• Foods and water were provided prior to administration of test sample to monkeys.

Replication:

• Not specified.

Control:

• Tested on 2 monkeys each.
• Supernatant of B. cereus No. 35 as negative control.
• Physiological saline.

Duration of study:

• Mice observed for 60 minutes.
• Monkeys observed for 24 hours.

• B. cereus No. 35, produces enterotoxin, but no vacuole factor.
• B. cereus No. 55, isolated from the outbreak. Produces vacuolation factor but no enterotoxin.

Mouse lethal activity:

• Was not observed for 100-500 units for both substances.
• Was found for more than 1000 units of toxin.

Monkey emetic activity:

• No activity was observed for controls.
• For cereulide at 14 000 units, all 3 monkeys showed emesis within 2-4 hours.
• For partially purified factor at 30 000 units, 1 of 2 monkeys showed emesis after 6 hours.
• For partially purified factor at 36 000 units, the 2 monkeys showed emesis after 2 and 4 hours.

Significance:

• Demonstrates that the HEp-2 vacuolation factor is an emetic toxin like cereulide.
• These toxins can produce emesis in monkeys

Sheep and cattle

Young females

Route of exposure:

• Intravenous injection.

Number and condition of animals:

• 5 ewes, pregnant 90 to 110 days.
• 6 heifers, pregnant 7 months.
• All animals housed in an isolation building for 10 days prior to inoculation.

Dose regimen for ewes:

• 5.1 × 10⁵ organisms.

Dose regimen for ewes:

• 3 groups (2 animals each):
• Group 1: 8 × 10⁶ organisms.
• Group 2: 8 × 10⁵ organisms.
• Group 3: 8 X 103 organisms.

Replications:

• Duplicate sections of tissues were stained.

• No strain designation provided.
• Isolated from an aborted bovine fetus.

Symptoms observed for ewes:

• 4 aborted dead lambs between 3 to 8 days postinoculation.
• One died after having fever, tachycardia, tachypnea and central nervous system disorders.

Symptoms observed for heifers:

• Groups 1 and 2 aborted dead calves between 7 to 12 days postinoculation.
• Group 3 had normal calves at term.

Examination of lambs and calves:

• Varying degrees of autolytic change.
• Blood-tinged ascites, hydrothorax, hydropericardium and subcutaneous edema.
• The foetal membranes were hyperemic and edematous.

Bacteriological findings:

• B. cereus isolated in pure cultures from tissues of the dead ewe, lambs and calves.

Conclusion:

• Necrotic placentitis was consistent in all the abortions, indicating that the placenta is the principal site of infection.

• Purified enterotoxin.

• FM-1

• Vascular permeability in rabbits.
• Lethal to mice.
• Caused fluid accumulation in mouse ligated intestinal loop.

• Intravenous injection of purified enterotoxin.

• B. cereus 96.

• Minimum lethal dose of 300 μg per mouse.
• 70 to 80 μg  per kg caused vomiting in cats.

Conditions of the animals:

• Throughout 2 resting seasons, the rate of sick larvae carrying dermal brown lesions were 4.1 and 1.7%.
• The rates of dead larvae carrying dermal brown lesions were 2 and 0.4%.

Number of animals studied:

• For each year:
• 50/ 28 in July
• 347/ 51 in August
• 1612/ 458 in September

Control:

• None specified.

Duration of study:

• 2 years

• No strain designation given.

Symptoms observed:

• When these larvae were kept in the laboratory, many of them died within 8-45 days.
• Death rates from December to April were 45, 54, 20, 8 and 0% in the first season.
• In the second season, rates were 56, 20, 20, 20 and 0%.

Bacteriological observation:

• B. thuringiensis var. finitimus and B. cereus were isolated from these larvae, but not from the healthy larvae or dead larvae not presenting the lesions.

Conclusion:

• Decreasing virulence with the advance of the resting period may indicate that the larvae catching the disease late may be or may become more resistant to its effect.

• Isolates from an atrophied pupa.

• WGPSB-2 (MTCC 7182)

• The strain was able to infect and cause 92 and 67% mortality in second instar larvae of Anomala dimidiata and Holotrichia seticolis, respectively.

• Up to one-fifth of the population was found to exhibit symptoms of bacterial infection.

• WGPSB-2

• Of 27 bacterial isolates tested against A. dimidiata, the most highly toxic strain was identified as B. cereus.

Route of exposure:

• Injection into quarters.

Dose regimen:

• Contaminated commercial antibiotic product.

Number and condition of the animals:

• 8 dairy herds.
• Total 80 cows affected.

Study conducted:

• Two whole carcasses which died of acute mastitis were examinated.
• Selection of tissues was made on the carcasses and also on 9 cows: mammary tissue, supramammary lymph nodes, liver, spleen and kidney.

Controls:

• None specified.

Duration of study:

• Approximately one year (1974)

• None specified.
• B. cereus identified according to cases previously described (Perrin et al. 1976)

Symptoms observed:

• Some of the affected cows developed acute mastitis within 24 hours, most of them shortly after calving.

Gross examination:

• Watery blood that had failed to clot.
• Marked subcutaneous edema over the udder.
• Numerous dark red, well demarcated areas were scattered throughout the affected quarters.
• Markedly enlarged supramammary lymph nodes.
• Moderately edematous and emphysematous lungs.
• Twice the normal size, dark red and turgid spleens

Histological findings:

• Mammary glands: interstitial septa were found to be edematous, acute thrombosis of veins and lymph vessels was noted.
• Erythrocytes found in the interstitial tissue.
• Gram-stained sections revealed Gram-positive organisms in the necrotic alveoli only.
• Acute lymphadenitis in sections of supramammary lymph nodes with focal areas of necrosis and large numbers of inflammatory cells.
• Liver showed presence of centrolobular hypoxic necrosis.
• Renal tissue revealed hemoglobinemic casts in the tubules.
• Hyaline thrombi were evident in capillaries of glomerular tufts and in the corticomedullary junction.
• Lungs revealed thickened alveolar septa due to edema. Alveolar capillaries were engorged with blood and many had hyaline thrombi.

Case 1:

• Male bovine fetus, 8 months in gestation.
• Second abortion in an 8-month period in a herd of 80 Brown Swiss cows.

Case 2:

• Male bovine fetus, 8 months in gestation.
• Second abortion in an 8-month period in a herd of 150 Holstein-Friesian cows.

Case3:

• Female bovine fetus, 7 months in gestation.
• The only abortion in a 1-year period in a herd of 21 Holstein-Friesian cows.

• No strain designation provided.

• 3 case reports of abortions.
• Necropsy, microbiologic and histopathologic examinations conducted for each fetus and fetal membranes when available.

Necropsy findings:

• Atelectatic, firm and dark red lungs.
• Fibrinous pleuritis, pericarditis and peritonitis.
• Yellow liver, twice the normal size.
• Enlarged and congested lymph nodes.

Microbiological findings:

• B. cereus was the only microorganism isolated from gastric contents and tissues.

Histopathologic findings:

• Vasculitis, edema, inflammation and necrosis in the intercotyledonary space.
• Hyperplasia in spleen.
• Congested liver.

Dairy cattle
Bos taurus

Adult females

• Quarters inoculated with B. cereus.

• No strain designation provided.

• Acute mastitis developed, followed by atrophy and cessation of milk secretion.

Dairy cattle
Bos taurus

Adult females

Purpose:
Accidental occurrence of B. cereus mastitis in several herds involved in efficacy trials of a proposed “dry-cow” therapy product.

Route of exposure:

• Injection into quarters.

Dose regimen:

• Experimental product containing 500 mg of cloxacillin in peanut oil and 3% monostearate base.

Number and condition of the animals:

• 5 herds of 120, 70, 1 600, 1 500 and 1 500 milking cows, respectively.
• Deliberate injection in 151 non-lactating cows.
• Inadvertent injection in 33 lactating cows.

Study conducted:

• Sample of the foremilk from all 4 quarters was taken immediately before the last milking of the lactation period.
• Quarters were treated soon after completion of this milking.
• Teats were dipped in an iodophor teat-dip after treatment and adverse reactions were checked by owners.
• Re-sampling and culture of the samples were made.

Controls:

• None specified.

Replications:

• Double sample (2 independent samples collected aseptically with cleansing and drying of the teat prior to collection)
• Single postreatment samples taken.

Duration of study:

• Approximately 3 months.

• No strain designation provided.
• Isolated from the experimental product and from the quarters.

Symptoms observed:

• Gangrenous mastitis developed in 5 cows at calving.
• Clinical mastitis developed in 15 other infected quarters, chiefly at calving or during lactation.
• Only 26 of 184 cows and 37 of 735 quarters exposed were infected.

Culture study:

• Agreement between the double samples was excellent.
• B. cereus was recovered in 9.9% of the treated quarters, in 3.6% of quarters having another infection at the time of exposure, and in 15.5% of cultures negative at the time of exposure, when later recultured.
• Most of the isolations were made 33 to 56 days after exposure and from quarters with no clinical evidence of mastitis.

Conclusions:

• The numbers of organisms in infected quarters vary widely, often being low.
• It is postulated that the organism is chiefly in spore form and less responsive to simple cultural or treatment procedures.
• The number of organisms in each product tube was low and not all tubes were contaminated.

Dairy cattle
Bos taurus

Adult females

• 11 cows with acute mastitis between 1963 and 1973.

• None specified.

• B. cereus was isolated from 1 cow.

Holstein dairy cattle
Bos taurus

Adult females

Purpose:
Antibiotic therapy using cloxacillin as part of a herd health program.

Physical conditions:

• Well managed cows with no serious mastitis problems.
• Antibiotic program initiated in 67 cows.
  • Infusions of the antibiotic during the dry period or the lactating period, or both.
• Vaccination of 41 cows, before or after the antibiotic treatment. Subcutaneous injection of 10 ml of the bacterin.

Number of animals studied:

• 129 out of a 140 cow herd.

Control:

• None specified.

Duration of study:

• 3 months.

• No strain designation given.
• Isolate from the milk of infected cows.

Preparation of the bacterin:

• Incubation of the isolate in brain-heart infusion broth. Sediment resuspended in 0.85% saline with 0.25% formaldehyde and tested for sterility. Finally diluted to MacFarland No. 3.

Symptoms observed for infusions:

• Acute severe mastitis occurred in 62 of the 67 cows infused with cloxacillin.
• During the dry period:
  • Of 25 that were infused 11 developed severe mastitis (average 24 days later, range 2 to 94 days).
  • Post mortem examination of one of the cows revealed scarlet-colored mammary glands surrounded by gelatinous material and filled with serosanguineous fluid. Mammary lymph nodes were wet in appearance and surrounded by gelatinous material.
• During lactation:
  • All of 33 cows infused developed mastitis 1 to 30 days later (the majority within 1 to 3 days).
  • Observation of one of the cows the day after parturition revealed a very hard hind quarter that contained only serous, red fluid. The cow refused to eat and her rectal temperature was 39.5°C, feces slightly diarrheic. In the succeeding days the mammary gland became cold, black and started to slough.
• During both dry and lactating periods:
  • All 4 cows infused developed mastitis.
• 5 cows infused with cloxacillin did not develop mastitis.

Symptoms observed when given the bacterin:

• Of 21 non-vaccinated cows, 6 died suddenly and 15 survived.
• All of 41 vaccinated cows developed less severe but recurrent mastitis and showed poor milk production.

Conclusions:

• The disease usually occurs as the result of injection of B. cereus into the teat cistern when treating mastitis of other causes. Contaminated antibiotics, teat tubes, syringes and dilators have been described as the source of infection.
• Both gangrenous inflammation and acute mastitis with systemic involvement have been reported.
• The secretion was serous and frequently contained erythrocytes, fibrin and leukocytes.
• Very low numbers of B. cereus can produce profound pathogenic effects.

Dairy cattle
Bos taurus

Goat
Capra hircus

Adult females

Purpose:
Report of bovine mastitis apparently caused by B. cereus.

Physical conditions:

• Trimmed tissues from one affected animal were fixed for sectioning.
• Toxins tests with the rabbit skin vascular permeability and necrosis reaction.

Number of animals studied:

• 28 cows
• 1 goat
• Distributed on 4 farms.

Control:

• None specified.

Duration of study:

• Not specified.

• No strain designation given.
• Identified as B. cereus by colony morphology.

Symptoms observed:

• 5 rapidly fatal.
• Others ranging from gangrenous to mild.

Farm 1:

• 3 cases of very acute mastitis in one week.
• First cow died within 24 hours.
• No response to antibiotic therapy.
• Milk “port-wine” in color.
• Second animal had subnormal temperature and a swollen and cold udder. Both milk and urine were port-wine; animal died within 24 hours.
• Examination of viscera revealed deep red kidney and udder, blood in the pelvis, congested liver and large white clots and blood stained fluid in the teat cistern.
• These latter two were in late lactation.
• The third cow was newly calved and developed mastitis 2 days later.
• She had pale brown milk and recovered from antibiotic therapy.

Farm 2:

• Symptoms were mild.
• Response to therapy was poor.

Farm 3:

• One cow recumbent after milk fever suddenly developed peracute mastitis and died. Port-wine milk.
• Second case occurred in newly-calved, in the same calving box. B. cereus was recovered from the udder.

Farm 4:

• One cow died of acute mastitis the morning following a cut in the teat.

Bacteriological examination of faeces and brewer’s grains:

• Organisms present in faeces of affected and non-affected cows at levels of 10⁵-10⁶ per g.
• 10²-10³ cells of B. cereus per g recovered from well preserved brewer’s grains and 10⁴-10⁵ when spoilage had occurred.
• 7.5 × 10⁵ and 4 × 10⁸ in grains obtained from the same supplier.
• B. cereus has been isolated on 17 other occasions in pure culture from mastitic bovine milk.

Histopathological examination:

• Lesions, interstitial septa oedematous and containing erythrocytes.
• Thrombi in veins.
• Necrosis of alveolar cells.

Permeability test:

• Only one of 19 mastitic and environmental isolates showed strong toxic activity.

Conclusions:

• Possibility of brewer’s grain being the source of infection.
• The organism is more likely to establish itself when there is no pre-existing infection in the udder.

Dairy cattle
Bos taurus

Adult females

• Bovine mastitis

• 1820/77
• 1419/77
• 1414/77
• 1589/77
• 624/76

• 1820/77: Death
• 1419/77, 1414/77 and 1589/77: 2 deaths.
• 624/76: not available.

Considerations that may result in a finding of high hazard include a micro-organism that:

• Is known as a frank pathogen;
• Has irreversible adverse effects (e.g., loss of biodiversity, loss of habitat, serious disease);
• Has significant uncertainty in the identification, characterization or possible effects..

Considerations that may result in a finding of medium hazard include a micro-organism that:

• Is known as an opportunistic non-human pathogen or for which there is some evidence in the literature of pathogenicity/toxicity;
• Has some adverse but reversible or self-resolving effects.

Considerations that may result in a finding of low hazard include a micro-organism that:

• Is not known to be a non-human pathogen;
• Is well characterized and identified with no adverse ecological effects known;
• May have theoretical negative impacts for a short period but no predicted long term effect for microbial, plant and/or animal populations or ecosystems;
• Has a history of safe use over several years.

Considerations that may result in a finding of high hazard include::

• Disease in healthy humans is severe, of longer duration and/or sequelae may result;
• Disease in susceptible humans may be lethal;
• Potential for horizontal transmission/community-acquired infection;
• Lethal or severe effects in laboratory mammals at maximum hazard/challenge dose trigger multiple-dose testing.

Considerations that may result in a finding of medium hazard include:

• Case reports of human disease in the scientific literature are limited to susceptible populations or are rare, localized and rapidly self-resolving in healthy humans;
• Low potential for horizontal transmission;
• Effects at maximum hazard/challenge dose in laboratory mammals are not lethal, and are limited to invasive exposure routes (i.e., intraperitoneal, intravenous, intratracheal) or are mild and rapidly self-resolving.

Considerations that may result in a finding of low hazard include:

• No case reports of human disease in the scientific literature, or case reports associated with predisposing factors are rare and without potential for secondary transmission and any effects are mostly mild, asymptomatic, or benign.
• No adverse effects seen at maximum challenge dose in laboratory mammals by any route of exposure.

Considerations that may result in a finding of high exposure include a micro-organism for which:

• The release quantity, duration and/or frequency are high.
• The organism is likely to survive, persist, disperse proliferate and become established in the environment.
• Dispersal or transport to other environmental compartments is likely.
• The nature of release makes it likely that susceptible living organisms or ecosystems will be exposed and/or that releases will extend beyond a region or single ecosystem.
• In relation to exposed organisms, routes of exposure are permissive of toxic or pathogenic effects in susceptible organisms.

Considerations that may result in a finding of medium exposure include a micro-organism for which:

• It is released into the environment, but quantity, duration and/or frequency of release is moderate.
• It may persist in the environment, but in low numbers. 
• The potential for dispersal/transport is limited. 
• The nature of release is such that some susceptible living organisms may be exposed.
• In relation to exposed organisms, routes of exposure are not expected to favour toxic or pathogenic effects.

Considerations that may result in a finding of low exposure include a micro-organism for which:

• It is no longer in use.
• It is used in containment (no intentional release).
• The nature of release and/or the biology of the micro-organism are expected to contain the micro-organism such that susceptible populations or ecosystems are not exposed.
• Low quantity, duration and frequency of release of micro-organisms that are not expected to survive, persist, disperse or proliferate in the environment where released.