1. Case Study Pathogenic Bacteriology 2009
Helen Luu, Maria Navarrete,
& Allen Chiu

2. Case Summary
A 12 year old female from Connecticut developed a fever that lasted for several days. She also developed a large rash on her back. She had recently been walking through tall grass in an area where there was horseback riding.

3. Key Information Pointing to Diagnosis
Location: Connecticut near New York
History: Roaming in tall grass where horses roamed
Symptoms: Fever & Rash
Rash has bulls-eye shape

4. The Diagnosis for Case # 38
Lyme borreliosis
Organism: Borrelia burgdorferi
Classic bulls-eye shaped rash (erythema chronicum migrans)
Flu-like symptoms
Fever
Played in tall grass area
Horses had been in tall grass area
Ticks can be found on horses

5. Diagnosis of Lyme Disease
High risk of Lyme disease in Connecticut area

6. Classification,Gram Stain Results, and Microscopic Appearance of Borrelia burgdorferi
Gram-negative spirochaete
Order Spirochaetales
Family Spirochaetaceae
Seven periplasmic flagella at each cell end and overlap each other in the central region of the cell
A multilayer outer envelope surrounds a protoplasmic cylinder
Cylinder consists of a peptidoglycan layer, cytoplasmic membrane, and the enclosed cytoplasmic contents.

7. Microscopic Appearance of Borrelia burgdorferi
Stains well with Giemsa and Warthin-Starry
Visible by darkfield or phase-contrast microscopy
Electron Micrograph
Darkfield Microscopy

8. Diagnosis/Isolation/Identification of Borrelia burgdorferi
Lyme disease is identified by flu-like symptoms and the classic bulls-eye
rash (erythema migrans).
Center of rash clears as
it grows causing the bulls-
eye appearance
Erythema migrans does not
occur in all cases of
Lyme disease

9. Diagnosis/Isolation/Identification of Borrelia burgdorferi without erythema migrans
Lyme disease can occur without erythema migrans
Flu-like symptoms with joint aches are a sign of Lyme disease
Serological testing for IgG and IgM antibodies
are used to verify diagnosis in
later stages of disease
Sensitive testing with ELISA (enzyme-linked
immunosorbent assay or IFA (indirect
fluorescent antibody) followed by a more
specific Western blot
Not accurate due to antibodies forming 2-4
or 4-6 weeks after erythema migrans and
antibodies remaining from previous
infections of Lyme disease
Western blot

10. Isolation Media
Borrelia burgdorferi is commonly isolated on two types of media
Modified Kelly-Pettenkofer (MKP)
Barbour-Stoenner-Kelly II (BSK-II)
Kelly media is complex media designed for the complex nutritional needs of Borrelia spp.
Rich media includes rabbit serum, serum albumins, and gelatine

11. Diseases and Pathogenesis of Disease Caused by Borrelia burgdorferi
Lyme borreliosis
Organism Borrelia burgdorferi found in bloodstream
3 Stages of Lyme Disease (if left untreated)
Stage 1: Early Localized (1 - 4 weeks)
Rash (erythema migrans)
Flu-like symptoms
Lack of energy
Headache and stiff neck
Fever and Chills
Muscle and joint pain
Swollen lymph nodes

12. 2nd Stage of Lyme Disease
Stage 2: Early Disseminated (1 – 4 months)
Can affect the skin, joints, nervous system, and heart if not treated.
Skin problems including expanding rash at bite site as well as additional rashes and/or swelling
Joint problems including redness, swelling, and pain
Early nervous system problems including numbness in the arms and legs caused by nerve inflammation
Heart problems such as irregular heartbeats (arrhythmia)

13. 3rd Stage of Lyme Disease
Stage 3: Late Persistent
Joint problems such as early arthritis (especially in the knees)
Late nervous system problems such as pain, weakness, and numbness in the arms and legs due to bacteria spreading to the nerves and spinal cord
Can include headaches, fatigue, and problems with vision, hearing, memory, concentration, and thinking
Paralysis of nerves in the face (Bell’s Palsy)
Inflammation of the brain (encephalitis) and tissues surrounding the brain (meningitis) and spinal cord
Heart problems such as inflammation of structures surrounding the heart (pericarditis)

14. Pathogenesis of Lyme Disease
Lyme disease is spread
through tick bites
Bacteria spreads to salivary
glands of infected tick
Usually takes 24 – 36 hours for the bacteria to invade after tick’s initial bite
Borrelia burgdorferi is invasive and attacks the tissue

15. Ticks are the vector for Lyme Disease
Tick species Ixodes spp. (deer ticks) help spread the disease
Ixodes scapularis in Connecticut

16. Tick Life Cycle
Ixodes scalpularis can live on horses in its nymph or adult form
Ticks in nymph form are smaller and harder to see
Smaller tick bites may not be felt

17. Therapy, Prevention and Prognosis of Patient Infected with Borrelia burgdorferi
Antibiotics are given depending on the stage of the disease and body areas affected
Early illnesses are usually treated by medicines taken orally
Doxycycline
A single dose of doxycycline within 72 hours of a tick bite can decrease chances of Lyme disease by 87%.
Amoxicillin
Later illness requires intravenous drugs
Ceftriaxone
Penicillin G
Pain-relief and anti-arthritic medication may be also be used
Nonsteroidal anti-inflammatory drugs (NSAIDs)
Ibuprofen
Swollen joints can be reduced by removing fluid from them

18. Prevention of Lyme disease
Avoiding tick bites
Use insect repellent such as DEET
Cover up your body using long pants, long sleeves, and hats especially in wooded areas (Ticks are easier to spot on light clothes)
Learn where ticks and their hosts are commonly found and avoid those areas
Check for and remove ticks
Check for symptoms of
Lyme disease
No vaccine since Feb 2002
Due to uncertainty of effectiveness
and lack of demand

19. Primary Research Article Contributing to the Understanding of the Disease caused by Borrelia burgdorferi
Livengood, Jill A., Gilmore Jr, Robert D., 2006, Invasion of human neuronal and glial cells by an infectious strain of Borrelia burgdorferi, Microbes and Infection, vol 8:
Borrelia burgdorferi’s ability to attach and invade to human neuroglial and cortical neuronal cells was examined due to its role in late disorders such as neuroborreliosis
Materials
An infectious and a non-infectious strain of B. burgdorferi was grown on BSK-II medium.
Different human neural cells were grown
Human umbilical vein endothelial cells (HUVECs)
Human cortical neuronal cell line (HCN-2)
2 different human neuroglial cell lines (HS-683, H4)

20. Primary Research Article Methods
Cell-association assay
B. burgdorferi was stained one color, while human cells were stained a second color.
B. burgdorferi cells were then added to the human cells and incubated at 35° C with 5% CO2 for 20 hours.
Gentamicin protection assay
Used to show bacteria was internalized and viable
Gentacmicin was added after incubation to kill all extracellular bacteria
Cell monolayers were then collected after incubation and grown on BSK-II to estimate number of B. burgdoferi that were internalized
Cell viability assay
Trypan blue staining was used to test for human cell viability after infection
Trypan blue is not absorbed by a live cell

21. Primary Research Article Results
Cell-association assay
Human cells were dyed
red while B. burgdorferi
cells were dyed green.
Internalized spirochaete
is seen as yellow due to
the yellow and green combination

22. Primary Research Article Results
Gentamicin protection assay
Gentamicin does not readily penetrate mammalian host cells leaving internalized spirochaetes alive.
All cell lines tested for positive spirochaete growth, which shows that internalized Borrelia is still viable

23. Primary Research Article Results
Cell viability assay
No observable adverse effects on mammalian cells when compared with uninfected controls

24. Primary Research Article
Discussion & Conclusion
Borrelia burgdorferi is an invasive organism that can attack human neural cells.
It can stay within a neural cell without being detected and remain viable.
Can be incubated with human neural cells showing that CNS is a site where B. burgdorferi can be disseminated.
Leads to a greater understanding of invasive mechanism of B. burgdorferi

25. Take Home Message
Disease Lyme borreliosis involves the organism Borrelia burgdorferi
Typical early symptoms are erythema migrans, flu-like syptoms and joint pains.
Late symptoms include meningitis, arthritis, numbness, paralysis, and heart problems
Pathogen is Borrelia burgdorferi
Diagnostics include symptom analysis & serologic testing with ELISA, IFA, and Western Blot (can also be seen using Giemsa stain and darkfield microscopy
Therapy is based on antibiotics
Prognosis is dependent on what stage of Lyme disease and what areas are affect
Early antibiotic treatment causes a fast and complete recovery
Late treatment may require a long time to get better
Prevention is avoiding tick bites
Insect repellent
Covering up areas that ticks can bite
Avoiding high tick infestation areas
Transmission is via deer tick bites.
Threat is to younger children and older people.

26. References
Feder Jr., Henry M., Micha Abeles, Megan Bernstein, Diane Whitaker-Worth, and Jane M. Grant-kels. "Diagnosis, treatment, and prognosis of erythema migrans and Lyme arthritis." Clinics in Dermatology 24 (2006):
Johnson, Russell C., George P. Schmid, Fred W. Hyde, A. G. Steigerwalt, and Don J. Brenner. "Borrelia burgdorferi sp. nov.: Etiologic Agent of Lyme Disease." International Journal of Systemic Bacteriology 34 (1994):
Leboffe, Michael J., and Burton E. Pierce. A Photographic Atlas for the Microbiology Laboratory. Not Avail, 2004.
Livengood, Jill A., and Robert D. Gilmore Jr. "Invasion of human neuronal and glial cells by an infectious strain of Borrelia burgdorferi." Microbes and Infection 8 (2006):
"Lyme Disease Causes, Symptoms, Diagnosis, and Treatment on WebMD.com." WebMD Arthritis and Joint Pain Center: Symptoms, Causes, Tests, and Treatments. 11 Mar <
"Lyme Disease." Information on symptoms of menopause in women and men as well as advice on treatments. Health Information for all sexual ages. Menopause,Andropause. 11 Mar <
"Lyme Disease Symptoms, Causes, Treatment, Diagnosis and Prevention on MedicineNet.com." 11 Mar <
Mahon, Connie R., George Manuselis, and Donald C. Lehman. Textbook of Diagnostic Microbiology. Philadelphia: Saunders, 2006.
McQueen, Nancy. "Spirochaetales." California State University, Los Angeles