Wills, M1., Hudson, B2., Shafren, D1., Mitrovic, A2., Barry, R1.

1The University of Newcastle, Newcastle, NSW; 2The University of Sydney, Sydney, NSW

PCR: DNA was purified from 68 tick isolates. PCR set up using primers designed against

conserved regions of Flagellin, OspA, and 16S rRNA genes as listed in Table 1. All 68

isolates were tested with Osp A primers, 20 returned amplicons of 463bp. Of this 20 5 were

chosen for confirmation with Flagellin and 16S rRNA primers. All 5 amplified correct sized

DNA. Control PCR were set up using control strains BBss (B31), B. garinii (NBS-16) and B.

afzelii (ACA-1). Negative controls with no DNA were added in each run. Annealing temp

was 45OC with 50 cycles of amplification. Results are presented in Fig 1-3.

Primer name- [region of gene targeted] Primer Sequence

Osp A1 [ OspA amino acid 70 to 79] 5’ AC AAT GGA TCT GGA GTA CTT GAA GGC GT 3’

Osp A2 [Osp A amino acid 218 to 224] 5’ CA GGC ACT TCA ACT TTA AC 3’

F-7 [flagellin base 594-base 614] 5’ CTC TGG TGA GGG AGC TCA AAC 3’

F-3 [flagellin base 823-base 842] 5’ GTA CTA TTC TTT ATA GAT TC 3’

DD02 [16S rRNA base 1105-base 1123] 5’ CCC TCA CTA AAC ATA CCT 3’

DD06 [16S rRNA base 1452-base 1472 5’ ATC TGT TAC CAG CAT GTA AT 3’

Table 1: Primer sequences

DNA Sequencing: Due to prohibitive cost of sequencing in 1993 only 2 Osp A amplicons

were sequenced from PCR of isolate 51 and 83. Sequence of Osp A amplicon from isolate

81 showed complete homology with BBss B31, whereas Osp A amplicon from tick isolate 83

had 5 bases that differed to B31, 3 of which resulted in amino acid substitutions. Osp A base

substitutions at position 419, 438, 457, 610 and 666. Two changes (438 and 666) were

silent mutations not affecting the amino acid sequence of the protein. The three amino acid

changes were:

1. base number 419: Isoleucine Threonine

2. base number 457: Lysine Glutamic Acid

3. base number 610: Glycine Arginine

Immunological Profile: Isolate 17 was characterised with 7 monoclonal antibodies

and compared with BBss (B31), B. garinii (NBS-16) and B. afzelii (ACA-1). Results are

presented in Table 2. Antigenic profile of isolate 17 most closely resembles B. garinii.

Reacting with 5/6 MAb against BBsl and did not react with relapsing fever borrelia Mab.

MAb Antigens recognised B. burgdorferi

sensu stricto

B. garinii B. afzelii Isolate 17

H9274 Flagellin of all Borrelia sp. + + + +

H604 Flagella of BBss + + + +

H5332 C-terminal end of Osp A of BBss + + + +

H3TS Osp A of BBss + + - +

L32 Osp A of BBss + + + +

H6831 C-terminal end of Osp B of BBss + - - -

H4824 Vmp33 outer membrane protein of

B. hermsii

- - - -

Table 2: Immunological profile of control BBsl and isolate 17.

Conclusions: • Molecular and immunological characterisation of 3 Australian tick isolates is consistent

with a species belonging to the BBsl complex that cause LB.

• PCR results of 65 isolates suggest a BBsl species, full characterisation required.

• Isolates were cultured from Ixodes holocyclus (paralysis tick) and Haemophysalis

longicornis (bush tick).

• Further research is required to fully characterise isolates, identify endemic regions,

identify arthropod vectors, ascertain what percentage of ticks are infected and address

public health policy.

Sero-prevalence study of BBsl IgG antibodies Aim: Investigate patients with Lyme-like illness for evidence of IgG Antibodies

againts 3 pathogenic BBsl species.

Method: • 2,105 patients participated from each state of Australia, mostly eastern seaboard

• Data collected between 1991-97-Inclusion criteria included:

1. History of tick bite

2. Clinical symptoms consistent with LB

3. Seropositive westernblot-IgG against Flagellin B &/or Osp A , Osp B

• Assumptions made:

1. Australian LB pathogen antigenically similar to Nthn hemisphere LB

2. 3 known genospecies used as source of antigen

• BBss (B31), B. garinii (NBS-16), B. afzelii (ACA-1)

• Both positive and negative serum controls were used in each western blot in addition

to MAb for Flagellin B, Osp A and Osp B.

• Band intensity qualitative grading up to 3+.

Results: Flagellin B antigen • Flagellin B, major filament protein of

Borrelia

• 41kDa antigen

• Genus specific-epitopes are similar

across Borrelia sp.

• Marker of early infection

• Flagellin antibodies may cross react

with other spirochetes

• WB results (Fig 5)

• BBss- 56% +ve IgG Ab

• B. garinii- 51% +ve IgG Ab

• B. afzelii- 47% IgG Ab

656

107 57

148 68

159

99

0

200

400

600

800

1000

1200

1400

Flagellin +ve

Nu

mb

er

po

sit

ive

Total number +ve 1294-62%

4% Ba+Bg

5% Ba

51% Ba+

9% Ba+BBss

11% Bg+BBss

12% BBss

8% Bg

} 51%

3 sp.

}24%

2 sp.

}25%

1 sp.

Figure 5: Western blot results of IgG Flagellin

Tick Isolates:

Aim: Investigate if Australian ticks harbour BBsl species and characterise isolates.

Method: Ticks were collected between 1989-93 from two regions, that had high numbers

of patients presenting with a clinical Lyme-like illness following a tick bite. The first region,

mid north coast of NSW (MNC), north of Newcastle to Port Macquarie, the other region was

the Hunter region and Central Coast (HCC) located between Sydney and Newcastle.

Engorged female ticks and nymphs were obtained from domestic animals and cattle. Tick

cultures were set up in BSK medium and checked twice a week by dark field microscopy for

up to 7 weeks.

Results: Of 168 cultures 74 had weakly motile spiral shaped organisms that were

comparable in size and shape to BBsl at

concentrations of 102-103/ml (Fig 1). 10 tick derived cultures were chosen for

purification by alternating filtering and

antimicrobial treatment (2 fold progressive

increases in concentration of phosphomycin,

rifampacin, nalidixic acid, 5-fluorouracil,

kanamycin sulphate and ciprofoxicin). This

resulted in 5 pure isolates which were

investigated by PCR and monoclonal antibodies.

Figure 1: Darkfield photomicrograph (400x) of 9 spirochete isolates (a-i) from Australian

ticks compared with control BBss (B31).

Figure 3: 16S rRNA PCR amplicons of tick isolates and BBss B31

368bp 248bp

Figure 2: Flagellin PCR amplicons of tick isolates and BBss B31

Figure 1: Osp A PCR amplicons of tick isolates and control BBss B31

463bp

463bp

Results: Osp A • Osp A has a role in adhesion in tick

midgut

• 31kDa antigen

• Species specific epitopes-epitopes

differ across Borrelia species

• Does not cross react

• Marker of late disease

• WB results (Fig 6)

• B.garinii 18% +ve

• B. afzelii 13% +ve

• BBss 7%+ve

61

151

225

29 60

37 24

0

100

200

300

400

500

600

700

Osp A +ve

Nu

mb

er

po

sit

ive

} 11% BBss

26% Afz

38% Gar

75% 1 species

5% BBss+ Afz

10% Gar + Afz 21% 2 species present

6% Gar + BBss

}

4% 3 species

present

4% Afz+Gar+BBss

Total Number positive 587 28%

Figure 6: WB results for IgG Osp A

Results: Osp B • Role of Osp B is adhesion to and

colonisation of tick midgut

• 34kDa antigen

• Species specific epitopes

• WB results (Fig 7)

• BBss 9% +ve n=1974

• B. afzelii 27% n=358

– limited supply of Mab

• B.garinii not tested

–Mab not available

150

73

25

0

50

100

150

200

250

300

Osp B +ve

Nu

mb

er

po

sit

ive

Total Number positive 248

61% Burg alone

29% Afzelii alone

10% 2 species present

} 90% 1 species present

* B. garinii not tested

Figure 7: WB results for IgG Osp B

Possible Endemic Regions:

Northern Beaches, Sydney: 70%

of patients tested +ve Flagellin B

&/or OspA (n=544/773)

Central Coast, NSW: 80% of

patients tested +ve Flagellin

&/or Osp A (n=101/126)

Figure 8: Location of patients returning +ve IgG WB to Flagellin B &/or OspA. Pins

represent either antigen and colours represent Borrelia species that antibodies

recognised (refer to legend).

• Fastest growing arthropod borne infectious disease in the northern hemisphere.

• Caused by a spirochete bacterium and the pathogenic species are collectively called

Borrelia burgdorferi sensu lato (BBsl) and currently include 23 species.

• Syndrome of LB was described as a form of juvenile arthritis in the town of Lyme ,

Connecticut USA in 1976.

• In USA the main pathogenic species is Borrelia burgdorferi sensu stricto (BBss) and in

Europe there are 2 main pathogenic species B. garinii and B. afzelii .

• Clinical symptoms are broad and include: neurological; arthritic; cardiac, dermatological;

psychiatric; fatigue and often start with flu like illness.

• Diagnosis is difficult in non-endemic area as symptoms overlap with other conditions

• Co-infections complicate the symptomology as ticks transfer multiple pathogens.

• Treated early with antimicrobials usually results in a full recovery.

• If treatment delayed, disseminated LB can lead to a multi-systemic infection, requiring

complex treatment of longer duration and results in high morbidity and mortality

• First case of Australian patient with Lyme like clinical symptoms reported in 1982 in the

Hunter Valley NSW, further cases reported in 1986 from South Coast & Central Coast

NSW (Stewart et al., 1982; McCrossin, 1986; Lawrence et al., 1986).

• We will present data that suggests spiral bacterium isolates from Australian ticks are

consistent with a BBsl species. Serology data will also be presented that indicates

presence of IgG antibodies as detected by western blot (WB).against BBsl antigens.

• Both highly specific BBsl antigens OspA and OspB are detected as well as a genus

specific antigen Flagellin which has been shown to cross react.

Lyme borreliosis (LB), also known as Lyme disease;

Conclusions: • Serology data is consistent with exposure to BBsl in patients following a tick bite.

• Research required in the epidemiology and to identify endemic areas (Fig 8).

• Development of diagnostic tests targeting Australian endemic species will aid

epidemiology.

Acknowledgement: Photograph of Ixodes holocyclus tick by Virginia Bear, National Parks and Wildlife Service

References:

1.Stewart, A., Glass, J., Patel, A., Watt G, Cripps, A. & Clancy R. (1982) Lyme arthritis in the Hunter Valley MJA 1: 139

2.McCrossin, I. (1986) Lyme disease on the NSW south coast. MJA, 144: 724-725

3.Lawrence, R.H., Bradbury, R & Cullen, J.S. (1986) Lyme disease on the NSW central coast. MJA, 145: 364