Risk of acquiring tick-borne infections in forestry workers from Lazio, Italy
Supplementary material
Supplementary text
Population
All 427 subjects were informed about the purpose of the study, including the reason for taking their blood
sample. All the forestry rangers were interviewed through a structured questionnaire including information about age,
place of residence, place of work, duration of employment, work-related activities, exposure to tick bites, leisure
activities, and possible clinical symptoms of tick-borne diseases. The blood donors were asked to answer the same
questionnaire only with regard to gender, age, place of residence, job title. The blood donors who worked in outdoor
environment were excluded from the study. Data were treated confidentially and analysed anonymously. The study was
conducted in full accordance with ethical principles and Italian ethical guidelines.
Serology
Specific IgG and IgM antibodies against B. burgdorferi s.l. were determined by ELISA using commercially
available monoclonal kits (recombWell Borrelia IgG and recombWell Borrelia IgM Arnika Diagnostics, Milan, Italy),
according to the manufacturer’s instructions. These tests are based on the principle of an indirect ‘sandwich’ enzyme
immunoassay and are prepared with a recombinant form of the B. burgdorferi sensu lato antigens. The IgM assay
contains OspC and p41/internal, whereas the following antigens are used to coat IgG recomWell Borrelia plates: p100,
OspC, p41/internal and p18. Positive and borderline samples were further assayed using commercial WB kits (Borrelia
“MIQ” Virablot Test kit IgG/IgM - Arnika Diagnostics, Milan, Italy) to confirm seropositivity. These kits employ B.
afzelii (Pko) and B. burgdorferi sensu strictu strains. Samples were considered positive only if they were confirmed by
WB. The IgG blot is positive if ≥ 2 bands of the following are present: p83/100, p58, p43/45, p39, 34, 31, p30, 29,
OspC/25, p21/22, Osp 17/21, 19, p14/18; the IgM blot is positive if ≥ 1 band of the following is present: p39, OspC/25,
Osp17/21, p41 or if ≥ 2 bands of the following are present: p83/100, p58, p43/45, 34, 31, p30, 29, p21/22, 19, p14/18.
An additional B. burgdorferi IgM Western blot assay, the recomLine Borrelia IgM (Mikrogen, Neuried, DE), was then
used as second confirmatory test due to its more restrictive interpretation criteria. This IgM assay assigns different
scores to antigens (p100=5; VlsE=5; p58=4; p41=1; p39=4; OspA=5; OspC=8; p18=5) and results positive for total
score >7.
Antibodies against TBE virus were determined by a commercially available enzyme-immuno-assayFSME IgG
(FSME IgG Immunozym, Progen Biotech GmbH, Heidelberg), according to the manufacturer’s instructions. Wells in
the ELISA test strips are coated with inactivated TBE virus. Sera with a positive or borderline result in IgG ELISA were
also confirmed by HI assay at pH 6.4 with goose erythrocytes, according to Clarke and Casals (1), using arctonextracted antigen from mouse brain infected with TBE virus (Italian strain ir968) (2).
Statistical analysis
Preliminarily the mean age (and age distribution) of the exposed and control group was checked, and there
were no significant differences (Table S1). Mean ages were compared with Student's t-test for independent samples and
the chi-square test (χ2) was used to analyze differences in the distributions by sex and age.
Univariate logistic regression was employed to estimate the risk of seroprevalence of antibodies against B.
burgdorferi in the 145 forestry rangers, by sex, age, environmental features of the area of residence (urban, rural,
presence of insects, garden, cesspool, nearby stream), cohabitation with pets at risk, hours and type of recreational
activities (shooting, fishing, gardening, cattle breeding, trekking, farming, camping, road motorbike), indoor-outdoor
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Risk of acquiring tick-borne infections in forestry workers from Lazio, Italy
Supplementary material
and urban-rural gradient of work environment, use of individual protective devices, history of tick bites. Crude and
adjusted (for sex and age) odds ratios (ORs) with 95% confidence intervals (CI) - computed on the assumption of a
Poisson distribution of the cases - were calculated for these variables.
Indoor-outdoor and urban-rural gradients of work environment were compared among tasks, firstly among the
145 exposed subjects (positive and negative), then only in the 10 positive exposed ones.
A final logistic regression model was chosen to evaluate the relationship in the 145 exposed subjects between
the independent variable (seroprevalence of antibodies against B. burgdorferi) and the following explanatory variables,
assuming they were independent of each other: tick bites (x1), urban-rural gradient of work environment (x2), lack of
individual protective devices (x3), sex (x4), taking females as the reference category, and age class (x5), with the first
class (35 years) as reference category. The functional expression for the regression model is:
y = ln (P/1-P) = α + β1*x1+ β2*x2 + β3*x3 + β4*x4 + β5*x5
The univariate logistic regression of B. burgdorferi seroprevalence showed not statistically significant
increased risks for most of the considered variables (p>0.05) among the 145 forestry rangers (data not shown).
A detailed description of the occupational histories for the 10 positive subjects is reported in Table S2. Two of
them reported presence of insects and 6 of a garden. More represented leisure activities were gardening (5 cases),
trekking (3 cases), and farming (2 cases). The commonest individual protection devices were overalls (5 cases), caps (3
cases) and boots (4 cases). Two subjects referred a history of tick bites.
References
1
Clarke DH, Casals J (1958) Techniques for haemagglutination and haemagglutination-inhibition with artrhopodborne viruses. Am J Trop Med Hyg 7:561-573.
2
Venturi G, Mel R, Marchi A, Mancuso S, Russino F, Pra GD, Papa N, Bertiato G, Fiorentini C, Ciufolini MG
(2006) Humoral immunity and correlation between ELISA, hemagglutination inhibition, and neutralization tests
after vaccination against tick-borne encephalitis virus in children. J Virol Methods 134:136-9.
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Risk of acquiring tick-borne infections in forestry workers from Lazio, Italy
Supplementary material
Table S1. Main characteristics of 145 exposed and 282 control subjects
Exposed (%)
Controls (%)
Males
121 (83.4)
240 (85.1)
Females
24 (16.6)
42 (14.9)
Sex
χ2 test: p=0.654
Mean age (yrs)
mean (±SD)
41.0 (±7.8)
40.4 (±9.7)
95% CI
39.7-42.3
39.3-41.6
Student’s t test: p=0.555
Age bracket (yrs)a
≤35
41 (28.5)
94 (33.5)
36-45
61 (42.4)
99 (35.2)
>45
42 (29.2)
88 (31.3)
χ test: p=0.337
2
Total
a
145 (100.0)
282 (100.0)
Missing for one exposed and one control; age bracket defined with cut-off at 33rd percentile.
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Risk of acquiring tick-borne infections in forestry workers from Lazio, Italy
Supplementary material
Table S2. Description of the occupational history for 10 exposed forestry rangers with confirmed seropositivity
to B. burgdorferi s.l. IgG or IgM antibodies
Worker
Agea
1
2
37
42
3
33
4
36
5
53
6
38
7
8
9
47
34
35
10
33
Genderb
Present job
Work
environmentc
Female
Male
Male
Guard
Station master
Patrol
Male
Male
Guard
Chief
inspector
PG PS police
officer
Station master
Guard
Rural
Rural
Urban and
rural
Rural
Rural
Male
Male
Male
Male
Female
Supervisor
Urban and
rural
Rural
Rural
Urban and
rural
Mean age: 38.8 ± 6.6
Gender: 2 females (20.0%), 8 males (80.0%)
c
Work environment: rural (66.7%), urban and rural (33.3%)
d
Mean duration of present job: 12.3 ± 10.5
e
NA, not answered question
a
b
4
Duration of
present job
(years)d
4
20
8
4
33
8
27
6
6
7
Previous jobe
Clerk
NA
Haulier
Duration of
previous
job (years)
10
NA
3
Clerk
NA
2
NA
Athlete
NA
Workman
Farmer
NA
NA
2
10
NA
NA