Flagellin gene (flaB) as RFLP marker for the identification of Borrelia burgdorferi sensu lato genospecies isolated from the blood of patients suspected of having Borreliosis

The clinical manifestations of Lyme disease consist of three stages*:

Early infection with localized erythema migrans

Disseminated infection occured days or weeks after the early infection (affects the nervous system, heart or joints in particular)

Late or persistent infection – weeks or months after the disseminated infection

*Steere A. 2001. Lyme disease. N Engl J Med., 2: 115-125

Detection of Borrelia burgdorferi depends on the stage and clinical symptoms of the disease but standard methods of examination are serological tests **

** Steere A., Coburn J., Glickstein L. 2004. The emergence of Lyme disease. J Clin Invest, 8:1093-1101

Detection of Borrelia burgdorferi during the early infection is connected with**:

Erythema migrans

Flu-like symptoms, including headache, muscle or joint pain

Tests used for examination in the early LD:

IgM or IgG ELISA
IgM or IgG Western blot

Detection of Borrelia burgdorferi connected with the later organ system involvement **:

IgG ELISA as a standard method in case of joint, heart and nervous system problems

IgM, IgG and IgA antibody capture test for intrathecal antibody production – optional

PCR testing of joint fluid - optional

B. burgdorferi comprises at least 11 genospecies and it has been referred to as B. burgdorferi sensu lato (s.l.)

6 genospecies are found in Europe:
B. burgdorferi sensu stricto (s.s.)
B. garinii
B. afzelii
B. valaisiana
B. lusitaniae
B. bissettii

The first three genospecies are pathogenic for both animals and humans and cause different disease symptoms

The aim of the study:

Designing of quick and independent on antibody production method of detecting of B. burgdorferi DNA, especially during the early infection

Establishing of potential material for detection of B. burgdorferi DNA in every stage of borreliosis

Recognition of B. burgdorferi s.l. genospecies responsible for different variants of Lyme disease

Material

Blood and urine samples from patients suspected for borreliosis

Joint fluid from some patients reported joint pain

Blood and tissue samples (heart, kidney) of game animals (roe deer, red deer, wild boar) received from the hunters

Methods of detection and differention of B. burgdorferi s.l. genospecies

polymerase chain reaction (PCR) for detection of B. burgdorferi s.l. DNA with primers complementary for fla gene

PCR-RFLP method for differentiation of B. burgdorferi s.l. genospecies with DdeI restriction enzyme

Results of detecting of B. burgdorferi DNA in blood samples

Positive samples were obtained only from patients who had reported typical manifestations of early infection (tick bites, erythema migrans and sometimes flu-like symptomes)

Detection of B. burgdorferi DNA in urine and joint fluid samples

Positive urine samples derived from some patients who reported manifestations related to disseminated infection (mainly joint pain)

All joint fluid samples were PCR-positive

Detection of B. burgdorferi DNA in blood and organ samples from game animals

season	sample examined [N/n (%)]
	blood	kidney	heart
spring/summer	62/19 (30,6)	64/4 (6,3)	36/3 (8,3)
autumn/winter	59/12 (20,3)	61/7 (11,5)	63/9 (14,3)
total	121/31 (25,6)	125/11 (8,8)	99/12(12,1)

N – number of samples

n – PCR positive

Restriction digestion of PCR fragment of B. burgdorferi s.l. fla gene with DdeI enzyme

There are different patterns for each European B. burgdorferi s.l. genospecies

pathogenic and non-pathogenic genospecies are easy to distinguish

Conclusions:

There is possibility of using of PCR method for detecting of B. burgdorferi s.l. DNA in blood and urine samples.

Detection of B. burgdorferi in blood is related to early infection whereas detection in other saples, for exaple urine and joint fluid is connected with disseminated infection

Every available material (fluid or tissue) is good for detecting of B. burgdorferi DNA in case of human borreliosis as well as in animal samples.

Fla gene is useful marker for differentiation of B. burgdorferi genospecies which are responsible for different disease symptoms.