Novel and nasty Corynebacterium

Our Discovery

• A novel Corynebacterium sp.

• Associated with clinical disease and mortality in NSG and NOD SCID mice

• Highly contagious

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IDEXX BioAnalytics

A Newly Recognized Pathogen of Immunocompromised Mice

A novel Corynebacterium sp. distinct from C. bovis causes dermatitis and death in immunocompromised mice.

• Infected NOG and NSG mice develop thick skin with hyperkeratosis• Infected nude mice have slightly thick skin without scales• Testing for the bacterial disease can be accomplished by culture and PCR

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Clinical Syndrome in Infected Mice

NSG and NOG mice were used for xenograft passage

• Severely immunocompromised mice that lack functional T, B and NK lymphocytes• Noticed rough hair coats and dehydration• Colony tested negative for Corynebacterium bovis and other murine pathogens

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Diagnostic Findings

• Skin thickened & inflamed• Abundant keratin scales with bacterial colonization• Corynebacterium bovis negative (PCR & culture)

Keratin heavily colonized by Gram-positive bacterial rods

KERATIN SCALES

GRAM STAINED TISSUE

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Diagnostic Test Development

• Cultured a coryneform bacteria - Not C. bovis or mastiditis

• Confirmed as Corynebacterium by sequence analysis - 16S rRNA and rpoB genes sequenced

• Developed a specific PCR test

• Added unique spectra to the MALDI-TOF database

Detected Species Log(Score)

Corynebacterium sp. IBRM 27989.16.3C 2.206

Corynebacterium sp. 2.140

Lactobacillus reuteri DSM 20015 DSM 1.277

Rhizobium radiobacter B177 UFL 1.248

Lactobacillus aviarius spp aviarius DSM 20654 DSM 1.229

Bacteroides fraqilis MB 9009 05 THL 1.224

Corynebacterium mastitidis DSM 44354 DSM 1.190

Mix

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Infection Study Design

Sham GroupICR, nude & NOG mice: 50uL sterile PBS topically

Corynebacterium sp. Group

ICR, nude & NOG mice: typically 4x106 Corynebacterium sp. • Inoculated 50uL on dorsal cervical area • Dripped or swabbed on skin

Culture, PCR and tissue samples were taken at 4 and 6 weeks post-inoculation; halted study at 6 weeks due to declining health of mice

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Clinical Disease Reproduceability

NOG

Rough hair coats

Abundant scales

Dehydration

Thickened skin

Gram-positive rod-shaped

bacteria in lesions

ICR

Smooth hair coats

Normal hydration

No bacteria on skin

Nude

Minimal scales

Skin erythema

No dehydration

Thickened skin

Gram-positive rod-shaped

bacteria in lesions

SHAM CONTROL - NUDE INOCULATED - NUDE

Culture and PCR Summary

Corynebacterium sp TestingGroup Strain PI PeltPCR PeltCulture FecesPCR

Sham NOG 4wk 2/2- 2/2- 2/2-

Sham Nude 4wk 3/3- 3/3- 3/3-

Sham ICR 4wk 2/2- 2/2- 2/2-

Corynesp NOG 4wk 4/4+ 4/4+ 4/4+

Corynesp Nude 4wk 4/4+ 4/4+ 4/4+

Corynesp ICR 4wk 4/4- 4/4- 4/4-

Corynebacterium sp TestingGroup Strain PI PeltPCR PeltCulture FecesPCR

Sham NOG 6wk 2/2- 2/2- 2/2-

Sham Nude 6wk 3/3- 3/3- 3/3-

Sham ICR 6wk 2/2- 2/2- 2/2-

Coryne sp NOG 6wk 4/4+ 4/4+ 4/4+

Corynesp Nude 6wk 4/4+ 4/4+ 4/4+

Corynesp ICR 6wk 4/4- 4/4- 4/4-

• All inoculated nude and NOG mice became colonized

- Pelt swabs were positive by culture and PCR - Feces were positive by PCR

• Inoculated ICR mice resisted colonization• Sham-inoculated mice remained bacteria free

Corynebacterium sp. Testing

Corynebacterium sp. Testing

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Infection Studies to Reproduce Disease

ICR outbred mice

• Immunocompetent • Supports transient infection with C. bovis

Nude (Foxn1nu) mice

• Lack T lymphocytes • Develops disease from C. bovis

NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac)

• Lack T, B, NK lymphocytes; defective dendritic cell function • Develops disease from C. bovis

Study included three genotypes of female mice, 4-5 wks old

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Corynebacterium sp. Testing RecommendationsInclude Corynebacterium sp. when testing for C. bovis

Quarantine and Health Monitoring Programs • Test immunocompromised mouse colonies - Skin, fur or swab - Culture or PCR • Screen environmental samples by PCR

Diagnostic Investigations of sick Immunocompromised Animals

Bioligical Materials Screening by PCR - Implantable trumors - Cell lines

BioAnalytics© 2018 IDEXX Laboratories, Inc. All rights reserved. IBA-RE14 Coryne Presentation

Corynebacterium sp. Testing Recommendations

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BioAnalytics© 2018 IDEXX Laboratories, Inc. All rights reserved. IBA-RE14 Coryne Presentation