UNIVERSIDADE DE TRÁS-OS-MONTES E ALTO DOURO
Detection of Borrelia burgdorferi sensu lato DNA in serum and parasitizing
ixodids of the wild boar (Sus scrofa) in northern Portugal by nested-PCR
Deteção de DNA de Borrelia burgdorferi sensu lato em soro e ixodídeos
parasitas de javali (Sus scrofa) no norte de Portugal por nested-PCR
Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde
Ana Sofia Andrade de Faria
Vila Real, 2013
UNIVERSIDADE DE TRÁS-OS-MONTES E ALTO DOURO
Detection of Borrelia burgdorferi sensu lato DNA in serum and parasitizing ixodids
of the wild boar (Sus scrofa) in northern Portugal by nested-PCR
Deteção de DNA de Borrelia burgdorferi sensu lato em soro e ixodídeos parasitas de
javali (Sus scrofa) no norte de Portugal por nested-PCR
Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde
Orientador: Invª. Doutora Maria Luísa Vieira
Grupo de Leptospirose e Borreliose de Lyme, Unidade de Microbiologia Médica, Instituto de Higiene e
Medicina Tropical, Centro de Recursos Microbiológicos, Universidade Nova de Lisboa, Lisboa, Portugal
Co-orientador: Profª. Doutora Maria das Neves Mitelo Morão de Paiva Cardoso
Departamento de Ciências Veterinárias, Escola das Ciências Agrárias e Veterinárias (CECAV),
Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal
Ana Sofia Andrade de Faria
Vila Real, 2013
Disease is very old and nothing about it has
changed. It is we who change as we learn to
recognize what was formerly imperceptible.
- John Martin Charcot, De l'expectation en medicine.
ACKNOWLEDGEMENTS/AGRADECIMENTOS
Porque há muito a quem agradecer, corro o sério risco de não agradecer a quem de
direito. Com a promessa de dar o meu melhor, porque afinal somos todos humanos,
agradeço:
À Inv.ª Doutora Maria Luísa Vieira, orientadora desta Dissertação de Mestrado,
por me ter recebido tão amavelmente no seu grupo, por todos os ensinamentos,
encorajamento e apoio que me ofereceu ao longo deste ano.
À Prof.ª Doutora Maria das Neves Paiva Cardoso, por ter aceitado co-orientar a
minha Dissertação com a maior das simpatias, pela indispensável ajuda e preocupação
que sempre demonstrou.
Ao Prof. Doutor João Cabral, por todo o apoio e tempo gentilmente cedido, tanto
com o trabalho que foi, como com o que virá.
À Prof.ª Doutora Madalena Vieira-Pinto, por toda a ajuda, amizade e
conhecimentos partilhados ao longo deste período, mas acima de tudo pela
oportunidade única que foi este projeto e sem a qual este trabalho não existiria.
À Catarina Coelho, pela simpatia e amizade que lhe são tão naturais e por toda
ajuda imprescindível, o que fez com que o trabalho de campo fosse muito mais
divertido do que alguma vez pensei que seria.
Às minhas queridas Teresa Carreira e Mónica Nunes, do Grupo de Leptospirose e
Borreliose de Lyme do IHMT, que desde o primeiro dia me trataram como uma
pequena grande amiga, e em tudo contribuíram para o sucesso da minha passagem pelo
IHMT, tanto a nível pessoal como profissional.
À Hélia Gonçalves, à Octávia Veloso e à Rita Bastos, pela colaboração,
entreajuda e acima de tudo pela amizade, sem as quais este trabalho nunca teria chegado
a bom porto.
Aos caçadores e organizadores das montarias, que nos possibilitaram e ajudaram
na recolha de amostras.
À Ana, à Cristina, à Jaqueline e ao Luís, porque este Mestrado sem eles nunca
teria sido a mesma coisa.
Ao meu irmão, à Tita, ao Vitó e ao Varão, porque muito me aturaram enquanto eu
reclamava das horas passadas no Pedrinhas a processar amostras.
Aos meus pais, que tudo fizeram para que eu aqui chegasse.
Obrigada por tudo.
ABSTRACT
Detection of Borrelia burgdorferi sensu lato DNA in serum and parasitizing ixodids
of the wild boar (Sus scrofa) in northern Portugal by nested-PCR
Lyme Borreliosis (LB) is the most common tick-borne zoonosis in the northern
hemisphere. This disease is caused by spirochetes of the B. burgdorferi s.l. complex and
because of its difficult laboratory diagnostic and broad range of clinical presentations,
it’s still underdiagnosed in Portugal.
Vertebrates like mice, deer and passerine birds play an essential role in the
transmission of the LB agents, either as reservoir hosts for the spirochete or as hosts for
infected ticks to feed on. The goal of this study was to determine the role of the wild
boar (Sus Scrofa) in the epidemiological cycle of LB in the Trás-os-Montes region,
northern Portugal.
To perform this objective, 91 sera and 239 ticks collected from vegetation and 107
wild boars (and one fox) shot in this region during the hunting season of 2011/2012.
A total of 205 samples (114 female ticks and 91 sera from captured animals) were
subjected to DNA extraction and nested-PCR amplification. Borrelia DNA was
detected for the first time in wild boar sera and the sequencing showed 93-99% identity
with B. afzelii in three of the four positive serum samples (4 +/91).
The molecular results obtained in this study emphasize the potential role (?) of the
wild boar as a reservoir host for the agent of LB and host for feeding ticks, as well as
the importance of sensitive techniques like the nested-PCR for a successful DNA
detection. Last but not least, these results are relevant in a way that they contribute for a
better understanding and knowledge of B. burgdorferi s.l. epidemiology in the wild
animals of the country.
Keywords: B. burgdorferi s.l., Lyme Borreliosis, zoonosis, wild boar, molecular
diagnosis, epidemiology.
v
vi
RESUMO
A Borreliose de Lyme (BL) é a zoonose transmitida por carraças mais frequente
no hemisfério norte. Esta doença é causada por espiroquetas do complexo B.
burgdorferi s.l. e por causa do seu difícil diagnóstico laboratorial e amplo número de
manifestações clínicas é ainda subdiagnosticada em Portugal.
Vertebrados como os ratos, veados e aves passeriformes têm um papel essencial
na transmissão dos agentes da BL, tanto como reservatórios para as espiroquetas como
hospedeiros onde as carraças infetadas se alimentam. O objetivo deste estudo foi
averiguar qual o papel do javali (Sus scrofa) no ciclo epidemiológico da BL, tendo
como área de estudo a região de Trás-os-Montes, norte de Portugal.
Para concretizar este objetivo, foram colectados 91 soros e 239 carraças a partir da
vegetação e de uma amostra populacional de 107 javalis (e uma raposa) abatidos em
montarias na região durante a época venatória de 2011/2012.
Um total de 205 amostras (114 carraças fêmeas e 91 soros de animais capturados)
foram submetidas a extração de DNA e amplificação por nested-PCR. O DNA de
Borrelia foi detetado pela primeira vez em soro de javali e a sequenciação demonstrou
93-99% de homologia com B. afzelii em três das quatro amostras de soro positivas
(4+/91).
Os resultados moleculares obtidos neste estudo sugerem o papel potencial (?) do
javali como reservatório para o agente da BL e hospedeiro para carraças, assim como a
importância de técnicas sensíveis como o nested-PCR para uma deteção de DNA bem
sucedida. Por último, mas não menos importante, estes resultados são relevantes na
medida em que contribuem para uma melhor compreensão e conhecimento da
epidemiologia de B. burgdorferi s.l. na fauna silvática do país.
Palavras-chave: B. burgdorferi s.l., Borreliose de Lyme, zoonose, javali, diagnóstico
molecular, epidemiologia.
vii
viii
TABLE OF CONTENTS
Acknowledgements/Agradecimentos .................................................................. iii
Abstract ............................................................................................................... v
Resumo .............................................................................................................. vii
List of Figures ..................................................................................................... xi
List of Tables .................................................................................................... xiii
List of Abbreviations ......................................................................................... xv
1
Introduction ................................................................................................. 1
2
Objectives ................................................................................................... 3
3
Review Of The Literature ............................................................................ 5
3.1
Lyme Borreliosis - Historical Perspective ............................................. 5
3.2
Epidemiology ....................................................................................... 6
3.3
Taxonomy and Geographical Distribution ............................................ 7
3.4
Etiological Agent Characterization - Borrelia burgdorferi sensu lato.... 8
3.5
Vector Life Cycle, Transmission and Hosts ........................................ 10
3.6
Clinical Manifestations of Lyme Borreliosis ....................................... 15
3.6.1 In humans ....................................................................................... 15
3.6.2 In animals ....................................................................................... 17
3.7
Clinical Diagnosis and Treatment ....................................................... 18
3.8
Laboratory Diagnosis ......................................................................... 19
3.8.1 Indirect methods (immunologic tests) ............................................. 19
3.8.2 Direct methods (molecular techniques) ........................................... 20
4
Material and Methods ................................................................................ 21
4.1
Study Area - Biogeographical Characterization .................................. 21
4.2
Target populations: Animals and Ticks ............................................... 22
4.3
Field work .......................................................................................... 23
ix
4.3.1 Planning/scheduling of the hunts to attend ...................................... 23
4.3.2 Implementation and adaptation of a field form previously made for
other research projects ......................................................................................... 23
4.3.3 Collection of blood from wild boars shot down during the hunts ..... 24
4.3.4 Collection of ticks from the prospected animals .............................. 25
4.3.5 Collection of ticks from vegetation ................................................. 25
4.4
Laboratory work ................................................................................. 25
4.4.1 Morphological identification of collected ticks................................ 26
4.4.2 DNA extraction from ticks and blood sera ...................................... 26
4.4.3 DNA amplification of Borrelia burgdorferi s.l. ............................... 27
5
Results ...................................................................................................... 31
5.1
Characterization of Target Population ................................................ 31
5.1.1 Sampled animals ............................................................................. 31
5.1.2 Collected ticks ................................................................................ 32
5.2
Climatic Conditions............................................................................ 34
5.3
Molecular Analysis ............................................................................ 36
6
Discussion and Final Considerations ......................................................... 39
7
Bibliographic References........................................................................... 45
8
Annexes .................................................................................................... 53
x
LIST OF FIGURES
Figure 1 - Global distribution of most B. burgdorferi s.l. complex species.
8
Figure 2 - Electron micrographs of B. burgdorferi s.l. morphology.
9
Figure 3 - Known distribution of I. ricinus in Europe.
11
Figure 4 - I. ricinus ticks.
12
Figure 5 - Life cycle of I. ricinus ticks.
13
Figure 6 (A-F) - Clinical manifestations of LB.
16
Figure 7 - Land uses in Trás-os-Montes region.
21
Figure 8 (A, B) - Distribution of temperature and precipitation in Trás-os-Montes
region.
22
Figure 9 (A, B) - Collection of blood sample and thoracic and abdominal cavities of the
wild boar.
24
Figure 10 - Schematic representation of the intergenic region 23S (rrl)-5S (rrf) of the
rRNA of B. burgdorferi s.l., targeted by nested-PCR.
27
Figure 11 - Followed hunts in the districts of Vila Real and Bragança during the hunting
season of 2011/2012.
31
Figure 12 (A, B) - Wild boar distribution by age and gender.
32
Figure 13 (A, B) - Tick distribution by genus and gender.
33
Figure 14 - Distribution of ticks collected per hunt according to genus.
34
Figure 15 - Barometric pressure values registered during the hunts.
34
Figure 16 - Chill, heat index and dew point levels registered during the hunts.
35
Figure 17 - Temperature and humidity levels registered during the hunts.
35
Figure 18 (A, B) - Sensitivity essay for the nested-PCR protocols.
36
Figure 19 - DNA amplification results for “fla” nested-PCR protocol.
37
Figure 20 - DNA amplification results for “1,2,3,4” nested-PCR protocol.
37
Annexes - Supplementary figure 1 - Original field form (1st page).
53
nd
Annexes - Supplementary figure 2 - Original field form (2 page).
54
Annexes - Supplementary figure 3 - Final field form (1st page).
55
Annexes - Supplementary figure 4 - Final field form (2nd page).
56
Annexes - Supplementary figure 5 - Morphological identification of collected ticks. 57
Annexes - Supplementary figure 6 - Ixodids’ identification form (1st page).
xi
58
Annexes - Supplementary figure 7 - Ixodids’ identification form (2nd page).
59
Annexes - Supplementary figure 8 - DNA extraction from serum samples.
60
Annexes - Supplementary figure 9 - Nested-PCR amplification.
60
Annexes - Supplementary figure 10 - Visualization of the electrophoresis gel.
61
xii
LIST OF TABLES
Table 1 - Currently known genospecies of the Borrelia burgdorferi s.l. complex.
7
Table 2 - Differential diagnoses for Lyme Borreliosis.
17
Table 3 - Mix protocols for nested-PCR.
28
Table 4 - Protocols for nested-PCR amplification cycles.
29
Table 5 - Tick distribution by gender and species.
33
Annexes - Supplementary table 1 - Primer sequences used in “1,2,3,4” and “fla” nestedPCR amplifications.
61
xiii
xiv
LIST OF ABBREVIATIONS
ACA - Acrodermatitis chronica atrophicans
B. burgdorferi s.l. - Borrelia burgdorferi sensu lato
B. burgdorferi s.s. - Borrelia burgdorferi sensu stricto
BLAST - Basic Local Alignment Search Tool
BSK - Barbour-Stoener-Kelly media
CDC - Centers for Disease Control and Prevention
CREM
-
Centre
for
Microbial
Resources
of
‘Faculdade
Tecnologia/Universidade Nova de Lisboa – FCT/UNL
CSF - Cerebrospinal fluid
DGHM - Deutsche Gesellschaft für Hygiene und Mikrobiologie
DNA - Deoxyribonucleic acid
ECDC - European Centers for Disease Control and Prevention
EDTA - Ethylenediaminetetraacetic acid
EIA - Enzyme Immunoassay
ELISA - Enzyme-Linked Immunosorbent Assay
EM - Erythema migrans
EUCALB - European Concerted Action on Lyme Borreliosis
IFA - Indirect Fluorescent Assay
INE - Instituto Nacional de Estatística
LB - Lyme Borreliosis
OSP – Outer surface protein
PCR - Polymerase Chain Reaction
RFLP-PCR - Restriction Fragment Length Polymorphism-PCR
RNA - Ribonucleic acid
xv
de
Ciências
e
rRNA - ribosomal RNA
RT-PCR - Reverse Transcriptase PCR
Salp15 - Salivary protein 15
TAE - Tris-acetate-EDTA
TROSPA - tick receptor for OspA
WB - Western Blot
xvi
1 INTRODUCTION
Lyme Borreliosis (LB), also known as Lyme disease, is a zoonosis caused by
spirochetes of the Borrelia burgdorferi sensu lato complex, which are transmissible
through the bite of infected ticks. It’s currently the most common disease transmitted by
ticks in the United States and Europe, and Portugal is no exception, where the disease is
of mandatory declaration since 1999 (Portaria n.º 1071/98 de 31 de Dezembro Diário da
República Nº 301, Série I-B, 1998; De Michelis et al., 2000; Couceiro et al., 2003). In
Europe, the tick Ixodes ricinus is the main vector of the disease.
The risk of contracting the disease is increased in individuals that live and/or work
in endemic areas for the disease, especially forest zones where the LB vector is more
abundant. Consequently, forest-associated jobs (forest guards, farmers, hunters, etc.)
and outdoor activities are considered risk factors for LB (Lindgren & Jaenson, 2006;
Hubálek, 2009). The peak of spirochete transmission takes place between late Spring
and Autumn, when the immature stages molt into nymphs and adults respectively, and
need to make a blood meal to do so. Wild animals are important hosts for ticks, and
some might even constitute reservoir hosts for these spirochetes, like small rodents,
hares, lizards, ground foraging passerines and game birds (Gern et al., 1998; EUCALB,
2013). Man is an accidental host and infection occurs through the bite of infected ticks.
Human infection is particularly favored during hunting season, where both hunters and
hunting dogs come in close contact with ticks that parasite wild animals. Therefore,
epidemiologic surveillance should be present in hunting activities in order to prevent
human infections, since LB constitutes a Public Health concern. The importance of
hunting activities in the LB transmission cycle in Portugal is still unknown, which
makes this research of extremely importance in the epidemiological context of this
zoonosis.
The first clinical case of LB described in Portugal took place in 1989 and since
then many steps have been taken with the goal of characterizing the circulating B.
burgdorferi s.l. species in Portugal. These efforts led to the first isolation of B.
lusitaniae ever in 1993 (Le Fleche et al., 1997), followed by the first human isolate of
B. lusitaniae spirochetes from a skin biopsy of a Portuguese patient in 2003 (CollaresPereira et al., 2004). Many other isolations of this and other species of the B.
burgdorferi s.l. complex have been made from the vector and animal hosts (e.g.
1
Apodemus sylvaticus and Turdus merula) of the bacteria (De Michelis et al., 2000;
Baptista et al., 2004; Lopes de Carvalho et al., 2010; Norte et al., 2012).
LB is a multi-systemic disease (but can also be present as a subclinic infection)
characterized by clinic manifestations involving the skin (erythema migrans, borrelial
limphocytoma and acrodermatitis chronica atrophicans), articulations (Lyme arthritis),
nervous system (Lyme neuroborreliosis) and less frequently, the heart (Lyme carditis).
The disease is commonly divided in three stages, and symptoms tend to start three to
thirty days after the infected tick bite, with the appearance of an expanding “bull’s eye”
shaped rash at the site of the tick bite, usually accompanied by a series of flu-like
symptoms (headaches, myalgias, fatigue, fever) (Strle & Stanek, 2009).
Diagnose of LB is essentially clinic, which isn’t always easy because not all
patients exhibit the characteristic erythema migrans, the hallmark of the disease, during
early infection. In addition, the clinical manifestations usually observed are not
pathognomonic of LB, and several differential diagnosis should be considered (Stanek
et al., 2012). The facts described and the lack of notification of the disease are
considered to be the main reasons for the status of underdiagnosed disease that
characterizes LB in Portugal (Lopes de Carvalho & Núncio, 2006).
Because of this, laboratory tests play an important role in the diagnosis of the
infection. Nevertheless, isolation of spirochetes through culture is not easy because B.
burgdorferi s.l. is a fastidious microorganism. Furthermore, serological tests show low
sensitivity in the early stages of the disease, even using a two-step approach. Falsenegative results are often observed because of the delay in immune response and falsepositive results are due to the occurrence of cross reactions. All this emphasizes the
growing need for sensitive and specific molecular techniques to perform a reliable and
accurate diagnosis. Polymerase Chain Reaction (PCR) based techniques, namely the
nested-PCR, have been shown to be very useful techniques for this purpose. These are
extremely sensitive and specific in the identification and amplification of borrelial DNA
in blood, serum, skin biopsy, synovial fluid and even sometimes in cerebrospinal fluid
samples (Schmidt, 1997; Couceiro et al., 2003).
2
2 OBJECTIVES
The main goal of this study was to detect B. burgdorferi s.l. DNA in the serum of
wild boars and ticks parasitizing them, in order to contribute to the understanding of the
eco-epidemiological role of these ungulates in the cycle of infection of this spirochete.
This was accomplished under the following specific objectives:
 To identify, through morphological and taxonomical characterization, the main
ixodid species that parasite the wild boar in the Portuguese northeastern region of
Trás-os-Montes;
 To detect and identify B. burgdorferi s.l. DNA in the blood of wild boars and
parasitizing ticks through PCR methods;
 To establish the correlation of the resulting data with possible concerns associated
to the Public Health, in particular the occupational groups connected to hunting
activities.
3
4
3 REVIEW OF THE LITERATURE
3.1 LYME BORRELIOSIS - HISTORICAL PERSPECTIVE
The name Borrelia was first used by Nicholas Swellengrebel in 1907, when he
named the genus after Amédée Borrel, whose findings about the peritrichous coat of
Borrelia gallinarum set the first basis of distinction between this genus and other
spirochetes (Wright, 2009). Despite that, documented references of spirochetes can be
found from 1873 and onward, with Dr. Otto Obermeier findings about “threads of
different lengths in constant movement among blood cells” he believed to be the causal
agent of the relapsing fever (Burgdorfer, 2001), confirmed the following year by Gregor
Münch. This author also theorized that the disease was passed on to humans by bloodsucking insects such as fleas and lice, and the theory was corroborated in 1910 by
Sergent and Foley (Burgdorfer, 2001).
The first documented description of erythema migrans appears in 1909 at the
hands of Arvid Afzelius, as he describes a skin lesion he believed to be caused by the
bite of a tick (as cited by Johnson et al., 1984). In 1975, Allan Steere named the disease
as “Lyme” after the city of Old Lyme, Connecticut, where two mothers alerted the
Connecticut State Department of Health for a peculiar outbreak of juvenile rheumatoid
arthritis (Steere et al., 1984).
In 1982, Willy Burgdorfer was the first to isolate spirochetes of the Borrelia
genus, the agent of Lyme disease, from ticks of the Ixodes scapularis species in the
USA (Burgdorfer et al., 1982).
The first isolation of the etiologic agent of Lyme disease in Europe occurred the
very next year in Switzerland (Barbour et al., 1983; Johnson et al., 1984), but the first
clinical case to be diagnosed in Portugal occurred in 1989 by David de Morais (as cited
by Lopes de Carvalho & Núncio, 2006). The status of notifiable disease was attributed
ten years later by the Portuguese Ministry of Health.
5
3.2 EPIDEMIOLOGY
Lyme Borreliosis (LB) is currently the most common tick-borne disease in
temperate regions of the northern hemisphere (Rizzoli et al., 2011; EUCALB, 2013)
such as Europe and North America, but also Asia (Wang et al., 1999; Aguero-Rosenfeld
et al., 2005; Zhang et al., 2010) and North Africa (Hubálek, 2009).
The disease is particularly prevalent in the US, where Centers for Disease Control
and Prevention statistics’ indicate 24,364 confirmed cases and 8,733 probable cases of
Lyme disease in 2011, amounting to a 7.8 incidence rate (CDC, 2012). This disease is
also prevalent in Europe, but few countries have deemed LB as a mandatory notifiable
disease, which makes it difficult to evaluate the true numbers of LB in this continent.
Approximate estimates for individual countries is probably the best way to determine
incidence rates for the disease and these numbers can go from one end of the spectrum,
with low incidence rates like the ones registered in Ireland (0.6/100,000) and the UK
(0.7/100,000), to the very other end where incredibly high incidence rates are reported
such as in Austria (300/100,000) or even in Slovenia (155/100,000) (Lindgren &
Jaenson, 2006; Stanek et al., 2011)
Despite the impressive number of cases registered in some European countries,
Portugal had only 14 reported cases during 2007 and 7 during 2008 with incidence rates
of 0.13 and 0.07 cases per 100,000 inhabitants, respectively (DGS, 2010), which
combined with studies that compare the number of reports and positive cases confirmed
by laboratory analysis (Lopes de Carvalho & Núncio, 2006) leads to the belief that the
disease is underreported in Portugal.
As far as risk populations, it is well established that people who live and/or work
in endemic regions - forest areas, for example - are at greater risk of acquiring LB
(Lindgren & Jaenson, 2006). Occupations like forestry workers, rangers, military field
personnel, farmers, gamekeepers, hunters, deer handlers and outdoor workers in general
have an especially high risk of contracting LB (Lindgren & Jaenson, 2006; Hubálek,
2009). Other outdoor activities such as orienteering, gardening or picnicking are also
considered risk factors for the disease. Epidemiology studies also report higher
seroprevalence and disease incidence rates for males, most likely due to occupation and
recreation activities. The disease has been shown to affect all age groups, though some
6
studies claim there’s a higher incidence rate in children while others reach the same
conclusion for the working age group (Lindgren & Jaenson, 2006; EUCALB, 2013).
3.3 TAXONOMY AND GEOGRAPHICAL DISTRIBUTION
The Borrelia genus is a member of the family Spirochaetaceae, which belongs to
the order Spirochaetales. Despite the large number of Borrelia species, only the B.
burgdorferi sensu lato (s.l.) complex genospecies are known to cause LB. This complex
currently comprises 19 genospecies (EUCALB, 2013). See Table 1 for further detail.
Table 1 - Currently known genospecies of the Borrelia burgdorferi s.l. complex
(adapted from EUCALB, 2013).
Genospecies
Distribution
References
B. afzelii
Europe
(Canica et al., 1993)
B. americana
USA
(Rudenko et al., 2009b)
B. andersonii
USA
(Marconi et al., 1995)
B. bavariensis
Europe
(Margos et al., 2009)
B. bissettii
Europe, USA
(Postic et al., 1998)
B. burgdorferi
Europe, USA
(Baranton et al., 1992)
B. californiensis
USA
(Postic et al., 2007)
B. carolinensis
USA
(Rudenko et al., 2009a)
B. finlandensis
Europe
(Casjens et al., 2011)
B. garinii
Europe, Asia
(Baranton et al., 1992)
B. kurtenbachii
USA
(Margos et al., 2010)
B. lusitaniae
Europe
(Le Fleche et al., 1997)
B. japonica
Japan
(Kawabata et al., 1993)
B. sinica
China
(Masuzawa et al., 2001)
B. spielmanii
Europe
(Richter et al., 2006)
B. tanukii
Japan
(Fukunaga et al., 1996)
B. turdi
Japan
(Fukunaga et al., 1996)
B. valaisiana
Europe, Asia
(Wang et al., 1997)
B. yangzte
Asia
(Chu et al., 2008)
7
From these 19 genospecies, B. afzelii, B. bavariensis, B. burgdorferi s.s., B.
garinii and B. spielmanii are the main recognized pathogens responsible for human LB
in Europe. B. bissettii, B. lusitaniae, and B. valaisiana, even though having been
detected in patients, they are not as frequently associated to the disease (Stanek et al.,
2012; EUCALB, 2013). Figure 1 illustrates the world distribution of most species of the
B. burgdorferi s.l. complex.
In Portugal, several B. burgdorferi s.l. species circulate in ticks, namely B. afzelii,
B. burgdorferi s.s., B. garinii, B. lusitaniae and B. valaisiana (De Michelis et al., 2000;
Kurtenbach et al., 2001; Baptista et al., 2004).
Figure 1 - Global distribution of most B. burgdorferi s.l. complex species. The grey
areas represent the distribution of tick vectors (Margos et al., 2011).
3.4 ETIOLOGICAL AGENT CHARACTERIZATION - Borrelia
burgdorferi SENSU LATO
Borrelia are long (8-30 μm), thin (0.2-0.5 μm diameter) spiral bacteria, each with
3 to 10 loose coils that give the cell it’s helical shape (Barbour & Hayes, 1986; Stanek
& Strle, 2003; Aguero-Rosenfeld et al., 2005; Krupka et al., 2007).
Structurally, B. burgdorferi is composed by a multilayered outer membrane
surrounding the periplasmic space and an inner compartment, the protoplasmic cylinder
8
(Figure 2). The motility of the cells is ensured by approximately seven flagella (7 to 14
in total) inserted on each extremity of the cytoplasmic membrane that limits the
protoplasmic cylinder. The flagella wrap around the cylinder, giving the cell its typical
spirochaete shape, and finally meet and overlap at the middle section of the bacteria
(Shapiro & Gerber, 2000).
Figure 2 - Electron micrographs of B. burgdorferi s.l. ultrastructural morphology and
schematic illustration of the bacterial membranes and flagella (Singh & Girschick,
2004b).
Members of the Borrelia genus are Gram negative, catalase negative bacteria that
dye well with Giemsa stain and have no polysaccharides in its outer cellular membrane
(Takayama et al., 1987; Murray et al., 2009). The cell’s membranes are made
essentially of phospholipids and atypical glycolipids. In addition, the cell expresses
several outer surface proteins (or Osp’s) (Wang et al., 1999; Krupka et al., 2007). OspA
and OspC are particularly important surface lipoproteins. Expression of the ospA gene
occurs when the bacterium colonizes the midgut of the vector and OspA binds to the
vector’s midgut epithelium through TROSPA (tick receptor for OspA). Another
function of OspA is binding to the plasminogen from the host’s blood during tick
feeding. The plasminogen is activated and helps Borrelia pass from the gut epithelium
9
to the salivary glands of the tick. During tick feeding, ospA is downregulated and the
production of OspC is initiated. In the salivary glands, OspC binds to Salp15 (or Salp15
homologues in I. ricinus). This salivary protein function as an immunosuppressant,
inhibiting CD4+ T-cell activation and making the spirochete undetectable to the host’s
immune system during the early stage of colonization (Hovius et al., 2007; Krupka et
al., 2007).
Culture of Borrelia spirochetes is difficult and generally unsuccessful. The cells
require an environment low in oxygen and have very specific nutritional needs like
amino acids or N-acetyl-D-glucosamine, thus the necessity for a complex media like the
Barbour-Stoener-Kelly media (BSK) and its derivatives (Aguero-Rosenfeld et al., 2005;
Murray et al., 2009). Incubation temperature in these media sits between 30 and 40ºC
and generation time can go from 7 to 20 hours or even longer. Microscopic observation
of Borrelia cells is possible with phase-contrast or dark field microscopy (Singh &
Girschick, 2004a).
The first spirochete to have its genome sequenced (Fraser et al., 1997), B.
burgdorferi strain B31, has a linear chromosome of 910,725 base pairs (bp) and a
G+C% content of 28.6%, as well as 21 plasmids (12 linear and 9 circular) with a
sequence total of 610,694 bp, amounting to a total of 1,521,419 bp in its genome (Fraser
et al., 1997; Casjens et al., 2000; Rosa et al., 2005; Norris, 2006). DNA identity
between different species of the B. burgdorferi s.l. complex is about 46-74% (Baranton
et al., 1992; Postic et al., 1994; Wang et al., 1999).
Particular attention should be paid to the rrn gene cluster (Schwartz et al., 1992)
existent in the genome of B. burgdorferi s.l. The unusualness of the tandem repeated
sequence of rRNA 23S and rRNA 5S makes it perfect for B. burgdorferi s.l. specific
detection, identification and typing (Postic et al., 1994; Baranton & De Martino, 2009).
3.5 VECTOR LIFE CYCLE, TRANSMISSION AND HOSTS
As the most widespread vector-borne disease in Europe (Figure 3), knowledge
about the transmission cycle of the Lyme disease agent, Borrelia burgdorferi s.l. is
imperative. In Europe, the LB agent is carried mainly by a hard tick named Ixodes
ricinus (Linnaeus, 1758). This tick of small dimensions is an obligate hematophagous
10
ecto-parasite, member of the Ixodes genus, Ixodidae family, Ixodida order (Parola &
Raoult, 2001).
Figure 3 - Known distribution of I. ricinus in Europe, September 2012 (ECDC, 2012).
The life cycle of this tick, after oviposition, can be divided into three
developmental stages – larvae, nymph and adult (Figure 4).
A blood meal has to take place every time before the tick can molt into its next
stage, except for adult females, which drop to the ground after the blood meal to lay the
eggs (Figure 5). Adult males may also partake on a blood meal, but in far smaller
quantities (Gern, 2009).
Transmission occurs through the bite of a tick infected with the agent, during the
blood meal. A questing tick uses the mechanical and chemical stimuli produced by
passing hosts to detect their presence nearby and grab them. Using the hypostome, the
tick anchors itself to the host and slices the skin with the chelicera, allowing the blood
to be then sucked by the tick (Gern, 2009).
11
Figure 4 - I. ricinus ticks (1 small square = 1mm2) (adapted from Humair & Gern,
2000).
The blood meal takes place only once each stage and the duration depends on the
current life stage, taking 2-4 days for larvae, 4-6 for nymphs and 6-10 for adults (Gern,
2009; EUCALB, 2013). If the tick is infected, the spirochetes migrate from the midgut
to the salivary glands of the tick, from where they are transmitted to the host via saliva
during the feeding, usually 2-3 days after the beginning of the meal (Humair & Gern,
2000). Nevertheless, in some cases, only 17 or 29 hours are required after tick
attachment for borrelial transmission (Kahl et al., 1998b) depending on the B.
burgdorferi s.l. species involved (EUCALB, 2013) and in certain cases, the presence of
the bacteria in the salivary glands prior to feeding may also contribute to an earlier
transmission (Lebet & Gern, 1994).
All I. ricinus stages are capable of transmitting the LB agent, but nymphs seem to
be the most prone to transmit the spirochete for several reasons. They are far more
inclined to bite humans than other forms, and are more numerous than adults – a female
adult specimen of I. ricinus can lay up to 3,000 eggs. Other motive is their diminutive
size, more likely to go unnoticed while attached to the host than adult ticks (Kurtenbach
et al., 1998; Santos-Silva et al., 2006; EUCALB, 2013).
12
Figure 5 - Life cycle of I. ricinus ticks (EUCALB, 2013).
Like most ixodids, Ixodes ticks have a three-host life cycle (Figure 5), following a
pattern of search for a host, feeding and then dropping to the ground to molt into the
next stage, therefore using three different hosts through the course of its life cycle. The
cycle usually lasts a year but can go up to three years depending on many factors,
particularly favorable climate conditions, host availability and the species of the tick
involved (Santos-Silva et al., 2006).
In Portugal, I. ricinus ticks have a heterogeneous distribution, but can be found
from North to South almost year-round (immature forms - April through June; adults September through March) in areas of deciduous woodland and mixed forest with mild
temperatures. Most importantly, I. ricinus is highly dependent of relative humidity
because of its susceptibility to desiccation when questing for hosts on the vegetation,
thus needing microhabitats with minimum amounts of 80-90% relative humidity under
which it cannot survive nor complete its life cycle (Santos-Silva et al., 2006).
13
Even though humans are not part of the Borrelia burgdorferi s.l. transmission
cycle in nature (Figure 5), Lyme Borreliosis is still the most widespread vector-borne
disease in Europe, mostly because the bacteria easily circulates between ticks and an
array of vertebrates that host such ticks. Common tick hosts are birds (passerines, game
birds and seabirds), lizards, mammals, both small (squirrels, mice, voles, shrews)
medium-sized (European badgers, hedgehogs and hares) and large (wild and domestic
ungulates, foxes, dogs) (Gern et al., 1998; Kurtenbach et al., 1998; Estrada-Peña et al.,
2005; Dsouli et al., 2006; Hasle et al., 2011)
Despite the numerous animals I. ricinus feeds on (over 300 different hosts), few
have been confirmed as having an important role in the ecology of the B. burgdorferi
s.l. complex. Small rodents are common reservoirs for B. afzelii, as well as B.
burgdorferi s.s. and B. garinii in some cases, while passerine and game birds seem to
have a distinct association with B. garinii and B. valaisiana and some lizard species
have been found as reservoirs for B. lusitaniae, although exceptions may occur (Humair
& Gern, 2000; Piesman & Gern, 2004; Dsouli et al., 2006; Gern, 2009; De Sousa et al.,
2012; Norte et al., 2012).
Larger mammals, like ungulates, present a challenge to study because of its size,
making it difficult to access/evaluate its role in the maintenance of the LB agent in
nature. The role of cervids like deer or roe deer in the ecology of this pathogen, once
thought to be incompetent reservoirs for B. burgdorferi s.l. cannot be completely
discarded in light of more recent studies (Pichon et al., 2000). Cervids still provide a
blood source for infected and non-infected ticks to co-feed, thus contributing to the
indirect maintenance of the LB agent (Richter et al., 2002; Ruiz-Fons et al., 2006). An
even greater role may be that of the wild boar (Sus scrofa), a well distributed ungulate
across Europe, as corroborating evidence about its potential as reservoir host is slowly
being uncovered. (Estrada-Peña et al., 2005; Cadenas et al., 2007; Juricová & Hubálek,
2009).
Wild boar populations, once very abundant in Portugal, suffered an enormous
decrease in numbers till the 1960’s due to the great hunting pressure and an outbreak of
classic swine fever (Fonseca, 2004, as quoted by Ferreira et al., 2009). This led to the
banning of wild boar hunting in 1967, which in turn contributed to increase of
populations levels from 1970 to the present days (Serôdio, 1985, as quoted by Ferreira
et al., 2009).
14
In Portugal, wild boars can be found across the country, with very little exceptions
(Fonseca, 2004, as quoted by Ferreira et al., 2009) and therefore, of “low concern” in
terms of species conservation (Cabral et al., 2005, as quoted by Ferreira et al., 2009).
As a consequence of these high levels in wild boar populations, management of
the species is very important to prevent them from becoming a plague that ruins crop
fields and native vegetation, one of the biggest concerns of farmers and land managers.
Hunting activities in Trás-os-Montes region are of great importance, both for the
management of wild boar populations and socioeconomic development of rural areas.
3.6 CLINICAL MANIFESTATIONS OF LYME BORRELIOSIS
3.6.1 IN HUMANS
Lyme Borreliosis can present itself either as a subclinical infection (due to the
lack of symptoms) or as a number of different clinical manifestations, depending on the
affected tissues, the particular species of the B. burgdorferi s.l. complex involved and
the duration of the infection. The disease can be described in three stages (early
localized Lyme disease, early disseminated Lyme disease and late Lyme disease) after
the infected tick bite (Hengge et al., 2003; Stanek et al., 2012).
The first stage is characterized by the appearance of a slowly expanding skin lesion
called erythema migrans (EM). EM is the result of local spreading of the spirochete
agent and therefore clinical hallmark of LB, with 70-80% of patients developing this
“bull’s eye” shaped rash (Figure 6 A, B) at the site of the tick bite between 3 to 30 days
after the bite. In addition, this phase of the illness is almost always accompanied by a
series of flu-like symptoms such as headaches, myalgia, arthralgia, fatigue, fever and
chills (Hengge et al., 2003; Steere et al., 2004; Aguero-Rosenfeld et al., 2005; Strle &
Stanek, 2009).
Multiple EM lesions may also appear, commonly associated with multiple bites or
hematogenous dissemination of spirochetes (Arnež et al., 2003).
15
Figure 6 - Clinical manifestations of LB. A, B) Erythema migrans; C) Borrelial
lymphocitoma, D) Acrodermatitis chronica atrophicans (ACA); E) Lyme arthritis;
F) Acute facial nerve palsy (adapted from Nau et al., 2009; Stanek et al., 2012).
If treatment does not occur, the infection can spread through the bloodstream and
lymphatic system in the weeks or months following the bite, causing systemic
complications in:
i) the nervous system (common manifestations of Lyme neuroborreliosis include
lymphocytic meningoradiculitis - Bannwarth’s syndrome, acute facial nerve palsy
(Figure 6 F) and lymphocytic meningitis) (Pfister & Rupprecht, 2006; Mygland et al.,
2010);
ii) skin (multiple EM, borrelial lymphocytoma (Figure 6 C)) (Müllegger, 2004);
iii) articulations (Lyme arthritis (Figure 6 E)) (Nardelli et al., 2008);
iv) heart (a wide range of clinical complications, from atrioventricular block,
pericarditis, myocarditis to the more rare cardiomyopathy, known as Lyme carditis)
(Lelovas et al., 2008).
Late Lyme disease settles in years after the tick bite, with chronic manifestations
of arthritis, Lyme neuroborreliosis and Acrodermatitis chronica atrophicans or ACA
(Figure 6 D), a dermatological condition characteristic of LB in Europe, marked by the
appearance of red or bluish-red lesions in the extremities that tend to atrophy with time
(Nadal et al., 1988; Strle & Stanek, 2009).
16
Clinical manifestations may vary from patient to patient depending on the B.
burgdorferi s.l. genospecies causing the infection. In Europe, Lyme neuroborreliosis is
most often caused B. garinii and skin complications are usually associated to B. afzelii,
but the strains of B. burgdorferi s.l. causing articulation complications are relatively
heterogeneous (B. afzelii, B. garinii and B. burgdoferi s.s.), while in U.S., B. burgdoferi
s.s. is the main responsible for cases of Lyme arthritis (Strle & Stanek, 2009).
Because of its multiple clinical manifestations, Lyme Borreliosis is often mistaken
for other pathologies, which causes the disease to be underreported in several countries.
Differential diagnoses for Lyme disease are presented in Table 2.
Table 2 - Differential diagnoses for the main clinical manifestations of Lyme
Borreliosis (adapted from Stanek et al., 2012).
Clinical
Manifestations
Differential diagnosis
Erythema migrans
Tick/insect-bite hypersensitivity reaction, bacterial cellulitis, erysipelas, erythema
multiforme, southern tick-associated rash illness (STARI), tinea, nummular eczema,
granuloma annulare, contact dermatitis, urticaria, fixed drug eruption, pityriasis rosea,
parvovirus B19 infection in children
Lyme
neuroborreliosis
Borrelial
lymphocytoma
Other causes of facial palsy, viral meningitis, mechanical radiculopathy, first episode of
relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis
Breast cancer (when lymphocytoma occurs on the breast), B-cell lymphoma,
pseudolymphoma
Lyme arthritis
Gout, pseudo-gout, septic arthritis, viral arthritis, psoriatic arthritis, HLA B27-positive
juvenile oligoarthritis, reactive arthritis in adults, sarcoid arthritis, early rheumatoid
arthritis, seronegative spondyloarthropathies
Lyme carditis
Other infectious and non-infectious causes of conduction disturbances or myopericarditis
Acrodermatitis
chronica atrophicans
Consequence of old age (old skin), chilblains, vascular insufficiency (chronic venous insuffi
ciency), superficial thrombophlebitis, hypostatic eczema, arterial obliterative disease,
acrocyanosis, livedo reticularis, lymphoedema, erythromelalgia, scleroderma lesions,
rheumatoid nodules, gout (tophi),erythema nodosum
3.6.2 IN ANIMALS
Like humans, domestic animals such as dogs, cats, cattle and horses seem to
develop clinical symptoms of LB (Krupka et al., 2007). Arthritis is the most frequent
clinical manifestation in dogs and is normally accompanied by lameness, fever,
anorexia and fatigue. Neurological dysfunctions, renal and heart involvement have also
17
been described, though less frequently (Appel et al., 1993; Skotarczak, 2002; Littman et
al., 2006).
Cats are apparently more resistant to the development of clinical symptoms than
dogs, but if clinically ill, they might exhibit lameness (Bushmich, 1994). One study has
detected high levels of the spirochete in cats with joint lameness and several others have
reported detection of antibodies against B. burgdorferi. Nevertheless, information is still
scarce to correctly assess the significance of the disease in these felines (Fritz &
Kjemtrup, 2003; EUCALB, 2013).
In horses, commonly reported clinical manifestations of LB include lameness,
swollen joints and fever. Uveitis, laminitis and encephalitis can occur, but are rare
(Bushmich, 1994; Egenvall et al., 2001; Gall & Pfister, 2006).
Evidence of infection has been detected in sheep and cattle and asymptomatic
infection is common. Symptoms, if present, are usually lameness and joint swelling
(Bushmich, 1994; Fritz & Kjemtrup, 2003; EUCALB, 2013).
Unlike domestic animals, wild animals don’t seem to develop clinical symptoms
of LB (Krupka et al., 2007; EUCALB, 2013).
3.7 CLINICAL DIAGNOSIS AND TREATMENT
Diagnosis of Lyme Borreliosis is mainly clinical, based on the recognition of
signs and symptoms (the most distinguishing being EM), the patient’s history of tick
bite or exposure, medical history, and other pertaining epidemiologic data. Clinical
diagnosis should be followed by laboratory tests to determine the presence of specific
antibodies for B. burgdorferi s.l., except in patients with early LB, because antibodies
may not be detectable at the time of sample collection and thus lead to false-negatives
(Relman, 1991; Aguero-Rosenfeld et al., 2005). In this case, a direct search of Borrelia
DNA (molecular approach) should be the chosen method to confirm diagnosis, as it is a
sensitive and incredibly specific method.
Choice of treatment for LB depends greatly on the stage and the clinical
manifestations of the disease, as well as the age of the patient (Hansmann, 2009; Nau et
al., 2009). In early LB, doxycycline is the treatment of choice (but is contraindicated for
18
pregnant women and children under eight years old), followed by amoxicillin as the
second choice (Bratton et al., 2008; Donta, 2012). Late or severe disease is treated with
parenteral antibiotics like ceftriaxone, cefotaxime or penicillin G (Weber, 2001;
Wormser et al., 2006; Girschick et al., 2009).
3.8 LABORATORY DIAGNOSIS
Laboratory tests are a very important and complementary step for the diagnosis of
LB and they can be used either for the indirect detection of anti-B. burgdorferi s.l.
antibodies (immunologic methods) or the direct search of Borrelia DNA (molecular
methods).
3.8.1 INDIRECT METHODS (IMMUNOLOGIC TESTS)
According to Centers for Disease Control and Prevention (CDC) and Deutsche
Gesellschaft für Hygiene und Mikrobiologie (DGHM) guidelines, detection of anti-B.
burgdorferi s.l. antibodies in cerebrospinal fluid (CSF) and serum should follow a “twotier protocol”. First, the biological sample is screened using sensitive tests like EnzymeLinked Immunosorbent Assay (ELISA), Indirect Fluorescent Assay (IFA) or Enzyme
Immunoassay (EIA). If the result of the screening test is positive or doubtful, a Western
blot (WB) should be used to confirm the results (Brouqui et al., 2004; Wilske et al.,
2007; Rizzoli et al., 2011; Stanek et al., 2012). WB is a much more sensitive and
specific serologic test, allowing detection of IgM and IgG antibodies (the levels of
which vary depending on the stage of the disease) against B. burgdorferi s.l antigens
(Brouqui et al., 2004). Nevertheless, serodiagnostic tests might be insensitive during the
first several weeks of infection (early disease) due to delay in immune response,
producing false-negative results. False-positive results are also a concern, as they may
be a consequence of cross reactions observed when other pathologies are present, such
as mononucleosis, infection by Treponema pallidum, and some autoimmune diseases
like rheumatoid arthritis or systemic lupus erythematosus (Magnarelli, 1995; Brouqui et
al., 2004; Bratton et al., 2008).
19
3.8.2 DIRECT METHODS (MOLECULAR TECHNIQUES)
Direct detection of Borrelia DNA is carried through Polymerase Chain Reaction
(PCR) using biological samples of blood, skin biopsy (PCR sensitivity of 50-70% in
patients with EM and ACA), CSF (10-30% in patients with acute Lyme
neuroborreliosis) or synovial fluid (50-70% in patients with Lyme arthritis) for
detection of spirochetes (both viable and not) in the early stage of disease (Wilske et al.,
2007; Bratton et al., 2008; Stanek et al., 2011).
Conventional PCR or nested-PCR can be used, but nested-PCR is more specific
and sensitive than the conventional technique because it uses two rounds of
amplification, each with one set of primers, instead of the single round and single pair
of primers involved in the classic PCR. Primers used on PCR-based assays are designed
to target a number of different borrelial DNA fragments, such as the 5S/23S rRNA
intergenic region,16S rRNA genes, flagellin and p66 chromosomal genes or the ospA
and ospB genes (Priem et al., 1997; Schmidt, 1997; Wilske et al., 2007).
There are several other PCR-based techniques that can be used in the diagnosis of
Lyme disease, like Multiplex Real-Time PCR or Reverse Transcriptase PCR (RT-PCR)
(Limbach et al., 1999; Courtney et al., 2004), but a standardized protocol has yet to be
established (Brouqui et al., 2004; Wilske et al., 2007).
Restriction Fragment Length Polymorphism - PCR (RFLP-PCR) is used for
genotyping B. burgdorferi s.l. species. The intergenic region 23S (rrl)-5S (rrf) of the
rRNA is the preferential target for borrelial DNA. PCR amplification and the resulting
amplicon is digested by an endonuclease (like MseI or DraI), creating different
restriction patterns that will allow the differentiation between Borrelia genospecies
(Postic et al., 1994).
Culture in selective media of biological samples can be used for direct detection
the LB agent, but it’s a time consuming (long generation time) and difficult task,
characterized by a low sensitivity in body fluids and therefore not routinely used in
laboratory diagnosis. Nevertheless, skin biopsy culture of EM lesions detects B.
burgdorferi s.l. in 80% of cases and is 100% specific (Wilske et al., 2007; Bratton et al.,
2008). Dark field microscopy also can be used for direct detection of B. burgdorferi s.l.
in biological samples.
20
4 MATERIAL AND METHODS
4.1 STUDY AREA - BIOGEOGRAPHICAL CHARACTERIZATION
The study took place in Trás-os-Montes, the most northeastern region of Portugal
and part of the biogeographical Carpetan-Iberian-Leonese Province (Costa et al., 1998).
In terms of land use, Trás-os-Montes is a region characterized by broad leaved
and coniferous woodland as well as a strong presence of agricultural land. Urban areas
are relatively small and scattered (Figure 7).
Figure 7 - Land uses in Trás-os-Montes region.
Potato, corn and rye are the main crops in this region and wine grapes play an
important role in the economy of the territories closest to the Douro river.
Trás-os-Montes’ vegetation includes Quercus suber (cork oak), Quercus ilex
(holm oak), Quercus faginea (portuguese oak) Quercus robur (english oak), Eucalyptus
spp., Pinus pinaster (maritime pine), Quercus pyrenaica (pyrenean oak), Pinus pinea
(stone pine) and Castanea sativa (chestnut).
21
Species of the Genista, Erica and Ulex genera, are the main representatives of
shrub vegetation, but we can also find Quercus coccifera (kermes oak), Arbutus unedo
(strawberry tree), Lavandula spp., Rubus spp and Cistus ladanifer (Baptista et al., 2004;
Godinho-Ferreira et al., 2005).
The climate of Trás-os-Montes region is essentially Mediterranean, with an
influence of the Atlantic climate. This combination creates hot and dry short summers
(Figure 8 A) and cold long winters with high levels of precipitation (over 2800 mm) that
tend to diminish with the advance into the interior of this region (Figure 8 B).
A
B
Figure 8 - A) Distribution of temperature in Trás-os-Montes region. B) Distribution of
precipitation in Trás-os-Montes region.
4.2 TARGET POPULATIONS: ANIMALS AND TICKS
The main target populations for this study were the wild boar (Sus scrofa)
specimens shot down during the hunting season of 2011/2012 and the ticks collected
from those animals.
22
4.3 FIELD WORK
The field work contemplated numerous tasks, namely:
4.3.1 PLANNING/SCHEDULING OF THE HUNTS TO ATTEND
The hunts were selected from a list of scheduled hunts to occur from October
2011 to February 2012 mainly in the districts of Vila Real and Bragança (and some in
the districts of Viseu and Guarda) according to the hunting season of 2011/2012 defined
by law (Portaria n.º 147/2011 de 7 de Abril Diário da República Nº 69, Série I, 2011).
Members of the organization of the hunts were contacted and permission was
requested to collect samples, while trying to cover as many different municipalities as
possible.
4.3.2 IMPLEMENTATION AND ADAPTATION OF A FIELD FORM
PREVIOUSLY MADE FOR OTHER RESEARCH PROJECTS
Several variables were included in the form, which fell mainly in one of two
groups – climate conditions at the time of sample collection and characteristics of the
animal from which the samples were collected.
The quantifiable climate conditions like temperature, humidity, barometric
pressure, wind speed, chill, heat index and dew point were measured with a Kestrel®
Weather Meter. The qualitative variables like visibility, precipitation, nebulosity and
wind intensity were verified according to a predefined numbered scale which ranged
from null (scale minimum) to high or very strong (scale maximum). Each of these
variables had its own scale, to better suit and facilitate measurement, as can be seen in
the annexes.
Animal characteristics included gender, age and reproductive state (should the
females be pregnant).
Other data were taken into account, namely the county and place of the hunt,
hunting area, map registry, time of death and evisceration of the animal, number of shot
23
and sampled animals, number of hunters and hounds per pack, artificial feeding and
repopulation of the wild boar, as well as field observers.
Field work conditions weren’t always favorable, which occasionally restricted the
data collection.
The field form was adapted several times to speed up the filling process during the
field work. The original and the final version of the form can be found in the annexes
(Supplementary figure 1, 2, 3 and 4).
4.3.3 COLLECTION OF BLOOD FROM WILD BOARS SHOT DOWN
DURING THE HUNTS
With the animal facing up, an incision was made from its trachea to its diaphragm,
to expose the thoracic cavity, allowing the collection of the blood pooled there from the
shot into a properly labeled test tube with a disposable plastic pipette (Figure 9 A). If
there was no blood in the thoracic cavity, the incision was extended to expose the
abdominal cavity and the blood sample was collected from there (Figure 9 B). Blood
from the chest cavity was preferred because it was less likely to have been contaminated
by the contents of the digestive system organs that may have been ruptured by the shot,
as opposed to that of the abdominal cavity, where the contents of intestines and stomach
would have likely leaked into and mixed with the blood.
Figure 9 - A) Collection of blood sample. B) Thoracic and abdominal cavities of the
wild boar (source: Catarina Coelho).
24
If the animal happened to have completely bled out before being brought in for
sample collection, the heart was cut open - either on site or back at the necropsy room at
UTAD’s Veterinary Hospital - and blood or blood clots were collected to a tube for later
processing.
The blood was centrifuged for 15 minutes at 3,000 rpm to extract the serum, no
longer than 4-6 hours after collecting the samples to ensure the quality of the biological
material. The serum was stored at -20ºC for later use.
4.3.4 COLLECTION OF TICKS FROM THE PROSPECTED ANIMALS
Either after or before collecting the blood samples, the animals were searched for
ticks, which were collected with disinfected metal tweezers into labeled test tubes. Ticks
were most easily found behind the ears, close to the groin and in the tail of the animal.
Upon arrival at UTAD’s laboratories after the hunt, the ticks were frozen at -20ºC.
4.3.5 COLLECTION OF TICKS FROM VEGETATION
In addition to the ticks collected from the shot animals, the study also
contemplated the collection of specimens from the vegetation. The “dragging” method
was the chosen technique for this procedure, which consists in dragging a one meter
square white terry cloth towel or flannel sheet attached to a pole on one side and rope
attached to both ends of the pole. The rope is pulled as the user walks and the towel is
dragged behind on the ground or vegetation, allowing the ticks to leap and attach to the
fabric.
4.4 LABORATORY WORK
The laboratory work was carried out at the Laboratory of Leptospirosis and Lyme
Borreliosis (Unit of Medical Microbiology), Instituto de Higiene e Medicina Tropical,
and supported by CREM (Centre for Microbial Resources) of Faculdade de Ciências e
Tecnologia, Universidade Nova de Lisboa, between the months of April and July.
25
4.4.1 MORPHOLOGICAL IDENTIFICATION OF COLLECTED TICKS
The collected ticks were one by one examined and identified based on their
morphological characteristics with the aid of multiple dichotomous keys and a Motic ®
SMZ168 Stereo Zoom Microscope (Supplementary figure 5). A form was created to
help identify and catalog the ticks collected from each animal by species, sex and stage,
based on the observed morphological characteristics (Supplementary figure 6 and 7).
4.4.2 DNA EXTRACTION FROM TICKS AND BLOOD SERA
4.4.2.1 DNA EXTRACTION FROM TICKS
The female ticks were subjected to DNA extraction using a “rapidSTRIPE Tick
DNA Extraction kit” commercialized by Analytik Jena AG® and metallic beads as a
homogenizer.
A second extraction protocol (the protocol used in the Laboratory of Leptospirosis
and Lyme Borreliosis at IHMT) was used to extract DNA from the ticks, a method
previously described by Guy & Stanek, 1991 named alkaline hydrolysis in which the
tick is placed in a 1.5 mL safelock eppendorf previously filled with 500 μL (500 μL for
adult ticks, 100 μL for lavae and nimphs) of a 1:20 ammonia solution. A sterile 20 μL
micropipette tip inside a 1000 μL one is then used like a pestle to macerate the tick. The
closed eppendorfs are boiled at 100°C for 20 min. Afterwards, they are opened and left
in the thermoblock to reduce the volume down to half. The extracted DNA is stored at
−20°C.
Three negative controls were included, using ultrapure water in substitute of the
biological samples in both procedures and all steps were done in a vertical laminar flow
bench, using appropriate micropipettes destined for molecular techniques only, filtered
tips and sterilized material to ensure a contamination-free environment.
4.4.2.2 DNA EXTRACTION FROM BLOOD SERA
DNA extraction was done using two very similar commercial kits, the “Gentra
Puregene® Blood Kit for DNA Purification from Body Fluid” by QIAGEN ® and the
“DNA Purification Protocol for 100 μL Body Fluid” from Citogene ® (Supplementary
26
figure 8). As in the DNA tick extraction protocols, three negative controls were
prepared using ultrapure water and the same measures were taken to ensure the quality
and prevent contamination of the biological material.
4.4.3 DNA AMPLIFICATION OF Borrelia burgdorferi S.L.
Both the DNA (templates) obtained from the tick and blood sera extractions and
the respective negative controls were afterwards subjected to nested-PCR for Borrelia
DNA amplification (Supplementary figure 9). Two different protocols were used, one
targeting the intergenic space 23S (rrl)-5S (rrf) of the ribosomal RNA of B. burgdorferi
s.l. and the other targeting the fla gene, encoding the flagellum protein (flagellin).
The first protocol (to facilitate comprehension, this protocol shall from here on
now be referred to as “1,2,3,4” nested-PCR) uses specific primers for the intergenic
region between the rrf gene (5S) and the rrl gene (23S) of Borrelia burgdorferi s.l.,
producing a first amplification fragment of 380 bp and a second one of 230 bp (Figure
10).
Figure 10 - Schematic representation of the intergenic region 23S (rrl)-5S (rrf) of the
rRNA of B. burgdorferi s.l., targeted by nested-PCR (original scheme).
The second protocol targets the flagellin encoding fla gene (from here on now to
be referred to as “fla” nested-PCR), according to the method described by Wodecka et
al., 2010, and produces a first amplification fragment of 774 bp and a second one of 604
27
bp. The “primers” sequences for both protocols can be found in the annexes
(Supplementary table 1).
Each mix was prepared according to the protocols presented in Table 1.
Table 3 - Mix protocols for “1,2,3,4” and “fla” nested-PCR.
1st PCR Amplification Mix (25
2nd PCR Amplification Mix (25
μL)
μL)
Mix protocols for the “1,2,3,4” nested-PCR
14.925 μL ultrapure H2O
14.925 μL ultrapure H2O
2.5 μL buffer (10x)
2.5 μL buffer (10x)
1.25 μL MgCl2 (50mM)
1.25 μL MgCl2 (50mM)
0.2 μL dNTP’s (100mM)
0.2 μL dNTP’s (100mM)
0.5 μL primer 1 (23SN1) (10μM)
0.5 μL primer 3 (23SN2) (10μM)
0.5 μL primer 2 (23SC1) (10μM)
0.5 μL primer 4 (5SCB) (10μM)
0.125 μL Taq DNA polymerase 0.125 μL Taq DNA polymerase
(5U/μL)
(5U/μL)
5 μL DNA template
5 μL 1st PCR product
Mix protocols for the “fla” nested-PCR
13.8 μL ultrapure H2O
15.3 μL ultrapure H2O
2.5 μL buffer (10x)
2.5 μL buffer (10x)
0.75 μL MgCl2 (50mM)
0.75 μL MgCl2 (50mM)
0.25 μL dNTP’s (100mM)
0.25 μL dNTP’s (100mM)
2.5 μL primer fla1 (132f) (10μM)
2.5 μL primer fla3 (220f) (10μM)
2.5 μL primer fla2 (905r) (10μM)
2.5 μL primer fla4 (823r) (10μM)
0.2 μL Taq DNA Polymerase
0.2 μL Taq DNA Polymerase
(5U/μL)
(5U/μL)
2.5 μL DNA template
1 μL 1st PCR product
Manufacturer
Bioline®
(Primers
synthesized by
Eurofins
MWG Operon)
Bioline®
(Primers
synthesized by
Eurofins
MWG Operon)
Aside from the biological samples, two negative controls were prepared for the
“1,2,3,4” and the “fla” nested-PCR’s, one for each amplification, using ultrapure water
instead of the DNA template, as well as a positive control, in which the DNA template
was substituted by DNA of a culture of B. burgdorferi sensu stricto strain B31
(3.57x107 cells/mL). All steps were done in a separate vertical laminar flow bench using
a different set of micropipettes, for PCR use-only as well filtered tips and sterilized
material to ensure a contamination-free environment. The amplifications for each
nested-PCR were performed in a Bio-Rad® MyCycler Thermal Cycler according to the
amplification conditions described in Table 4.
28
Table 4 - Protocols for “1,2,3,4” and “fla” nested-PCR amplification cycles.
1st PCR Amplification Conditions
2nd PCR Amplification Conditions
Stages of
Stages of
T (ºC)
Time
Cycles
T (ºC)
Time
Cycles
amplification
amplification
Conditions protocol for the amplification cycles of the “1,2,3,4” nested-PCR
Initial Denaturation
94.5
1 min
1
Initial Denaturation
94.5
1 min
1
Denaturation
Annealing
Elongation
94
52
72
30 sec
30 sec
1 min
25
Denaturation
Annealing
Elongation
94
55
72
30 sec
30 sec
1 min
Final Elongation
72
5 min
1
Final Elongation
72
5 min
Conditions protocol for the amplification cycles of the “fla” nested-PCR
Initial Denaturation
94
10 min
1
Initial Denaturation
94
10 min
40
1
1
Denaturation
Annealing
Elongation
94
50
72
30 sec
45 sec
1 min
40
Denaturation
Annealing
Elongation
94
54
72
30 sec
45 sec
1 min
40
Final Elongation
72
7 min
1
Final Elongation
72
7 min
1
Electrophoresis was performed using a 1.5% agarose gel (Bioline ®) in 1x TAE
buffer with 2 μL of RedSafe DNA Stain (INtRON Biotechnology®). 2 μL of Orange G
Loading Buffer were added to 6 μL of each final amplified product (respective negative
and positive controls included) and loaded into the wells. 6 μL of molecular weight
marker “HyperLadder IV”, (Bioline ®) were loaded into the last well. The
electrophoresis ran at 120 V for 40 minutes and the gel was then observed and
photographed under ultraviolet radiation with the aid of the Wealtec® “Dolphin-Doc
Plus Gel Image system” (Supplementary figure 10). The remaining amplified products
were stored at -20ºC for later use.
The amplified products of samples positive for Borrelia DNA were directly
purified and sequenced on both strands by Portuguese company StabVida®, using the
same primers as those used for the Borrelia burgdorferi s.l. amplification.
The sequencing results were analyzed by BLAST (Basic Local Alignment Search
Tool) and compared with GenBank’s B. burgdorferi s.l. reference strains in order to
determine the percentage of existing identity.
29
30
5 RESULTS
This study followed 23 wild boar hunts in 18 municipalities from the districts of
Vila Real (7), Bragança (9), Viseu (1) and Guarda (1), covering a grand total area of
11304,3 km2 [municipalities’ areas according to (INE, 2011)]. In some counties,
collection of samples could be made two or three times. Represented in Figure 11 are
the hunts that took place in Vila Real and Bragança.
Figure 11 - Followed hunts in the districts of Vila Real and Bragança during the hunting
season of 2011/2012.
5.1 CHARACTERIZATION OF TARGET POPULATION
5.1.1 SAMPLED ANIMALS
A total of hundred and seven wild boars (Sus Scrofa) (nw=107) were considered in
this study, all of them with respective age and sex data except for two. Because the
opportunity presented itself, one fox (Vulpes vulpes) was also included in the study
(nf=1). A total of hundred and eight (Na=108) animals was enrolled in this study.
31
Ninety one blood samples (Nb=91) were collected from the prospected wild boars
and fox. Only ninety one blood samples were collected from a universe of one hundred
and eight animals included in the study because most animals had already bled out
before arriving at the site of sample collection.
Based on the field information collected during the hunts, the age distribution
between wild boars is of 39 adults, 37 subadults and 29 juveniles, with two animals of
unknown age due to lack of information (Figure 12 A).
Gender wise, 71 out of 107 wild boars were female and 34 were male, with only
two animals of unknown sex. It was also observed that of all the females included in
this study, 14 of them were pregnant (Figure 12 B).
Wild Boar Distribution by
Age (nw=107)
A
B
Wild Boar Distribution by Gender
(nw=107)
2 (1.87%)
29
(27.10%)
14
(19.72%)
39
(36.45%)
34 Males
(31.78%)
71
Females
(66.36%)
57
(80.28%)
37
(34.58%)
2 (1.87%)
Adult
Sub Adult
Juvenile
Unknown
Unknown
Pregnant Females
Males
Non Pregnant Females
Figure 12 - Wild boar distribution A) by age; B) by gender.
As for the fox included in the study, it wasn’t possible to collect any data
pertaining age or sex.
5.1.2 COLLECTED TICKS
Two hundred and thirty eight ticks (nt=238) were collected from twenty five
prospected animals (twenty four wild boars and one fox) and one tick was collected
from the vegetation (nv=1), performing a total of two hundred and thirty nine collected
ticks (Nt=239) (Figure 13).
32
A
B
Tick Distribution by Genus (Nt=239)
2
(0.84%)
206
(86.19%)
31
(12.97%)
Distribution of Ticks by Gender and
Genus (Nt=239)
Rhipicephalus
1
(50%)
1
(50%)
Dermacentor
Ixodes
0 25 50 75 100 125 150 175 200 225
Ixodes
Dermacentor
Rhipicephalus
Rhipicephalus (vegetation)
Females (Engorged)
Females
Males
Total
Figure 13 - Tick distribution A) according to genus representation; B) by gender and
genus.
Of the two hundred and thirty nine collected ticks, two hundred and six belong to
the Dermacentor genus, thirty one ticks were identified as members of Ixodes genus and
the other two as members of the Rhipicephalus genus. Two species of Dermacentor
ticks were identified, D. marginatus and D. reticulatus, with a distribution of one
hundred and ninety eight (nDm = 198) and five (nDr = 5) respectively, and three (nD? = 3)
specimens where visualization of species’ distinctive morphological characteristics was
inconclusive. The Ixodes genus was represented single-handedly by I. ricinus specimens
and the same thing happened with R. sanguineus, the only species of the Rhipicephalus
genus identified among the collected ticks (Table 5). As for the four ticks collected from
the fox, they were all I. ricinus specimens.
Table 5 - Tick distribution by gender and species [*Tick captured from the vegetation;

four (4) of the 31 ticks were collected from a fox: one (1) male and three (3)
females, two of which were engorged].
Males
Females
- Of which
were
engorged
Tick total
Ixodes
(I.
ricinus)
Dermacentor
D.
marginatus
D.
reticulatus
Unidentified
specimens
15
16
6
107
91
52
1
4
1
31
198
5
33
Totals
Total
Rhipicephalus
(R.
sanguineus)
1
2
2
109
97
55
1
1*
0
125
114
61
3
206
2
239
Sex ratio was well balanced in ticks, with 125 males and 114 females, of which 61
were engorged.
The Ixodes specimens were all collected early on during the first and third hunt,
while ticks of the Dermancentor genus could be collected throughout the entire hunting
season (Figure 14).
29-10-2011
05-11-2011
12-11-2011
19-11-2011
26-11-2011
03-12-2011
03-12-2011
10-12-2011
17-12-2011
17-12-2011
07-01-2012
15-01-2012
21-01-2012
22-01-2012
28-01-2012
28-01-2012
04-02-2012
04-02-2012
11-02-2012
18-02-2012
18-02-2012
25-02-2012
No. of ticks
Ticks collected from animals on each hunt
35
30
25
20
15
10
5
0
Hunts 2011/2012
Ixodes
Dermacentor
Rhipicephalus
Figure 14 - Distribution of ticks collected per hunt according to genus.
5.2 CLIMATIC CONDITIONS
As mentioned previously, data pertaining climate condition at the time of sample
collection were registered in the field records. Visibility was overall very good with
moderate nebulosity, only two records of light rain and mostly low to moderate wind.
Barometric pressure variation
1040
1020
1000
980
960
940
920
900
880
29-10-2011
05-11-2011
12-11-2011
19-11-2011
26-11-2011
03-12-2011
03-12-2011
10-12-2011
17-12-2011
17-12-2011
07-01-2012
15-01-2012
21-01-2012
22-01-2012
28-01-2012
28-01-2012
04-02-2012
04-02-2012
11-02-2012
18-02-2012
18-02-2012
25-02-2012
Barometric pressure values
(hPa)
The quantitative measurements are represented in Figures 15, 16 and 17.
Hunts 2011/2012
Barometric pressure (hPa)
Linear (Barometric pressure (hPa))
Figure 15 - Barometric pressure values registered during the hunts.
34
Chill, Heat Index and Dew Point variation
25
Chill (°C.m/s)
20
15
Heat Index
(%.°C)
10
5
Dew point
(°C)
0
-5
Linear (Chill
(°C.m/s))
29-10-2011
05-11-2011
12-11-2011
19-11-2011
26-11-2011
03-12-2011
03-12-2011
10-12-2011
17-12-2011
17-12-2011
07-01-2012
15-01-2012
21-01-2012
22-01-2012
28-01-2012
28-01-2012
04-02-2012
04-02-2012
11-02-2012
18-02-2012
18-02-2012
25-02-2012
-10
Hunts 2011/2012
Linear (Heat
Index (%.°C))
Linear (Dew
point (°C))
Figure 16 - Chill, heat index and dew point levels registered during the hunts.
Temperature and Humidity variation
60
Humidity (%)
50
40
Temperature (ºC)
30
20
Linear (Humidity
(%))
10
29-10-2011
05-11-2011
12-11-2011
19-11-2011
26-11-2011
03-12-2011
03-12-2011
10-12-2011
17-12-2011
17-12-2011
07-01-2012
15-01-2012
21-01-2012
22-01-2012
28-01-2012
28-01-2012
04-02-2012
04-02-2012
11-02-2012
18-02-2012
18-02-2012
25-02-2012
0
Linear
(Temperature
(ºC))
Hunts 2011/2012
Figure 17 - Temperature and humidity levels registered during the hunts.
With the exception of barometric pressure, all other climatic parameters registered
significant decrease as the hunting season progressed.
35
5.3 MOLECULAR ANALYSIS
Prior to B. burgdorferi s.l. DNA search in the sera and tick samples, a test was
conducted to evaluate the sensitivity of the two nested-PCR protocol (“1,2,3,4” and
“fla”) using serial dilutions from cultures (108 cells/ml) of B. burgdorferi s.s., strain B31
and B. garinii, strain Pbi.
Figure 18 - A) Sensitivity essay for the “fla primers” nested-PCR protocol using serial
dilutions of a 108 cells/ml culture of Pbi strain (B. garinii) – 604 bp amplicon.
Lane 1 and 14 - 100 to 1000 bp DNA ladder; Lanes 2 to 9 – Serial dilutions (lane
9 shows that the limit detection is 101 cells/ml); B) Sensitivity essay for the
“1,2,3,4 primers” nested-PCR protocol using serial dilutions of a 108 cells/ml
culture of B31 strain (B. burgdorferi s.s) - amplicons of 380 bp and 230 bp (1 st
and 2nd PCR amplification), respectively. Lane 1 - 100 to 1000 bp DNA ladder;
Lanes 2 to 8 – Serial dilutions (lane 8 shows that the limit detection is 10 2
cells/ml).
The sensitivity threshold for the “1,2,3,4 primers” protocol was 102 cells/ml
(Figure 18 B) a detection limit lower than that of the “fla primers” protocol, which is
able to detect up to 101 cells/ml (Figure 18 A), thus making the “fla” protocol the most
sensitive of the two for detection of B. burgdorferi s.l. DNA in the samples.
After testing the sensitivity, the extraction products of sera and the female ticks
were subjected to amplification using both nested-PCR protocols. No positive results
for presence of B. burgdorferi s.l. DNA were found in the tick samples. As for the sera,
four of the 91 analyzed samples (4,396%) were positive for the presence of B.
burgdorferi s.l. DNA (samples from wild boars 26, 30 and 31 were positive with both
36
protocols while sample 27 was positive with the “1,2,3,4” protocol). These results can
be observed in Figures 19 and 20.
Figure 19 - DNA amplification results for “fla” nested-PCR protocol of serum samples
(lanes 2 - 5); negative extraction controls (lanes 6 and 7); positive control (lane 8);
negative control for 1st PCR cycle (lane 10); negative control for 2nd PCR cycle
(lane 11). Samples 26, 30 and 31 are positive for B. burgdorferi s.l. DNA (lanes 2,
4 and 5, respectively). 100 to 1000 bp DNA ladder (lanes 1 and 9).
Figure 20 - DNA amplification results for “1,2,3,4” nested-PCR protocol of serum
samples. 100 bp DNA ladder (lane 1); sera samples (lanes 2-14); negative
extraction controls (lanes 15-17); positive control (lane 18); negative control for
1st PCR round (lane 19); negative control for 2 nd PCR round (lane 20). Samples
26, 27, 30 and 31 are positive for B. burgdorferi s.l. DNA (lanes 10, 11, 13 and
14, respectively).
37
Samples 26 and 27 were collected in Montalegre (homonymous county), both
from subadult females, while samples 30 and 31 came from Vilarinho de Cotas (county
of Alijó), the first being an adult male and the last was an adult female.
The set of amplicons were directly purified and sequenced on both strands by a
Portuguese company StabVida ®, using the same primers as those used for Borrelia
burgdorferi s.l. amplification. One of these samples (no. 27) did not produce sequencing
results.
The sequences obtained were compared with those of the GenBank for Borrelia
burgdorferi s.l. complex, using the BLAST and revealed a 93 to 99% identity with
Borrelia afzelii.
38
6 DISCUSSION AND FINAL CONSIDERATIONS
In recent years, the interest on Lyme Borreliosis has increased significantly in
Portugal, much due to the high prevalence and increasing incidence of the disease
across Europe but also to the multitude of different vertebrates that serve as hosts for
infected ticks and reservoirs for the LB agent itself.
For this reason, the present study had as its main goal to contribute to a better
understanding of the role of wild animals, in this case the wild boar (Sus scrofa), in the
epidemiology of the disease, the circulating B. burgdorferi s.l. genospecies among such
hosts as well as the circulating tick species and their part on the transmission of the
spirochetes to hosts and vice-versa.
Considering the nature of this study, it’s important to underline the difficulties
faced in terms of data and sample collection. Not all contacted hunting organizers
accepted to collaborate with this study or authorized sample collection, which
immediately during the scheduling of hunts to attend narrowed down our choices. Field
conditions weren’t always ideal (rain, very low temperatures and lack of daylight).
Some hunters were in a rush to take their shot wild boars home or would not allow for
the animal to be cut open to collect blood, which sometimes hindered the collection of
ticks and blood from the animals as well information about their sex and age. Also,
details about the number of hunters, dog packs and map of hunting area weren’t always
available.
In the geographical context, the objective was to cover as many counties in the
districts of Vila Real and Bragança (Trás-os-Montes region). However, due to the
difficulties described above not all were sampled, but the overall covered area is still
representative of the area initially predicted for this study.
Distribution of sampled wild boars was equitable regarding to age of the
prospected animals but not gender, where females were more than half of the sampled
animals and males little more than a quarter of that same population.
Tick population was consistent in terms of age with the collection solely of adult
forms while no other life stages were spotted on the wild boars or during the application
39
of the “dragging” technique. As for the sex ratio, the number of collected female and
male specimens was fairly similar on both cases.
Most of the collected ticks from the wild boars belonged to the Dermacentor
genus (D. reticulatus and D. marginatus), few specimens were Ixodes ticks (I. ricinus)
and only one was a Rhipicephalus tick (R. sanguineus). Considering the climatic data
obtained during the hunts and what is known about tick seasonality and the influence of
climate conditions on tick survival and activity (Lindgren & Jaenson, 2006; SantosSilva et al., 2006; EUCALB, 2013) it’s possible that the weather conditions, particularly
temperature and humidity, might be at least partially responsible for the distribution of
species verified.
I. ricinus needs microhabitat humidity levels of at least 80-90% to survive
(Lindgren & Jaenson, 2006; Santos-Silva et al., 2006). During the hunts, humidity
levels not only never reach 60%, but also experience a continued decrease throughout
the hunting season. Considering these facts, one can reason that such low levels of
relative humidity might have affected the microhabitat humidity of I. ricinus,
undermining the survival of this tick’s population, effectively lowering its questing
activity during the winter season and leading to the low number of captured specimens
registered.
Dermacentor ticks (D. marginatus and D. reticulatus), which are some of the
most commonly found ticks in Portugal (Santos-Silva et al., 2011), are not as dependent
of humidity as I. ricinus, which might be the reason why they have thrived where Ixodes
ticks did not, justifying the high number of specimens collected during the hunting
season (Santos-Silva et al., 2006).
Rhipicephalus ticks are too not as dependent of humidity levels as I. ricinus, but
unlike the Dermacentor ticks, whose adults forms are more active during autumn and
winter months, Rhipicephalus ticks are better adapted to higher temperatures and
therefore more active and abundant during spring and summer, which might explain
why nearly no ticks of this genus were collected during the hunting season (SantosSilva et al., 2006).
The reason why no tick life stages other than adults were found might be
connected to the seasonality of each stage. Generally, immature stages are more likely
to be found during the warmer months, when they are more active (summer months for
40
Rhipicephalus and spring/summer for Ixodes and Dermacentor) while adults are more
active in less warmer months (mainly autumn but also winter for Ixodes and
Dermacentor) with the exception of Rhipicephalus, which could explain the
documented results.
Because the main vector of B. burgdorferi s.l. in Europe is I. ricinus, it might not
be unreasonable to assume that the absence of larger numbers I. ricinus ticks in the
sampled population might have had a negative effect in the detection of the LB agent,
lowering the probability of finding spirochetes of B. burgdorferi s.l. complex in the tick
population.
The molecular analysis performed by nested-PCR, one protocol targeting the
rRNA intergenic region 23S(rrl)-5S(rrf) and the other targeting the flagellin gene were
used for detection of B. burgdorferi s.l. DNA in tick and serum samples. Because adult
male ticks seldom partake on blood meals, only the female ticks were used for detection
of borrelial DNA.
Nested-PCR of the serum samples yielded four positive results, despite only three
having been positively identified as B. afzelii (considering the percentage of identity
obtained for this species), while nested-PCR of ticks yielded no positive results.
Many factors may have contributed for this result, among them freezing and
unfreezing of ticks during identification as well as storage time of ticks and sera, which
could have led to degradation of the samples and any trace of spirochetes. A reduced
concentration of spirochetes in the samples may also be responsible. Nested-PCR is an
extremely sensitive technique but even using the most sensitive protocols like the “fla”
nested-PCR protocol, which detects up to 101 cells/ml, we cannot assure a 100%
reliable detection. The “fla” protocol could not detect the presence of spirochetes in
serum sample 27 while the “1,2,3,4” protocol, less sensible than the first one (detects up
to 102 cells/ml), effectively could. This might have to do first with the low
concentration of spirochetes in the sample, which in turn might then not be contained at
all in the necessary volume retrieved from the sample to use as DNA template for the
first cycle of PCR amplification, translating into a lack of amplification.
Visualization of electrophoresis gels can represent a problem, as confirmation
relies on the human eye for detection of amplification bands that if they are weak
enough, might easily pass as an artifact or not be detectable at all without further
41
adjustment of the images’ contrast and brightness, as was the case of samples 30 and 31
using the “1,2,3,4” protocol, which required a continuous increase and decrease of
contrast of the image in the Dolphin-Doc Plus Gel Image system program and second
visual confirmation of a laboratory colleague to ascertain the positivity of those two
samples (Schmidt, 1997).
Another possibility to factor in is the inhibition of DNA amplification due to
substances present in the serum samples or the blood contained in engorged ticks (Gern
et al., 1998; Kahl et al., 1998a). To this effect, DNA purification extraction kits and
protocols were used to minimize the problem.
Last, but not least important, the detection of spirochetes in the serum samples
does not mean that we are in the presence of an active infection, because a PCR positive
detection signals only the presence of the spirochete, not whether it was viable or not at
the time of blood sample collection (Kahl et al., 1998a).
One of the positive PCR samples (no. 27) did not produce sequencing results and
while we cannot be completely sure of the reason why, it’s admissible that the low
concentration detected (about 102 cells/ml) is in the detection limit of the used primers
and that might justify why this sample could not be sequenced.
Despite the potential problems that may have contributed to a lack of more
standout results, three of the four positive samples of wild boar sera were positive for B.
burgdorferi s.l. and all three displayed very high levels of identity consistent with the
presence of Borrelia afzelii DNA, a species known to be usually associated with small
mammals, particularly rodents, and previously detected in Portugal on questing ticks
(Kurtenbach et al., 2001; Baptista et al., 2004).
The positive identification of B. afzelii in these wild boar sera is a reinforcement
and perhaps a step towards confirmation of a previously known suspicion, namely that
the wild boar is a potential reservoir host for B. burgdorferi s.l., as suggested in studies
developed in Spain, Switzerland and Czech Republic. The first two studies used blood
meal analysis for the identification of Borrelia DNA and host DNA using a reverse line
blot technique. According to the authors, the results suggest a potential correlation
42
between B. afzelii and the wild boar as a reservoir host (Estrada-Peña et al., 2005;
Cadenas et al., 2007).
The Czech Republic study detected the presence of IgG antibodies anti-Borrelia
burgdorferi s.l. (indirect hemagglutination assay) in wild boar sera. (Juricová &
Hubálek, 2009). Nevertheless, the detection of IgG antibodies can merely serve as an
indicator of contact with the LB agent, it does not determine whether a titre represents a
current or past infection.
The validation of the role of the wild boar as a reservoir for B. afzelii would
require, among other things, the detection of anti-Borrelia IgM antibodies in the sera,
for confirmation of infection. Techniques like Indirect Immunofluorescence Assay
(IFA) and Western Blot (WB) are the standard procedures used in the immunological
detection of anti-Borrelia antibodies, being the last (WB) the confirmation test. This
approach was considered while idealizing the study, but logistic difficulties, including
additional costs, did not allow its realization. However, it is an important step in
determining the wild boar as reservoir host for B. afzelii and because of that, it will be
performed as soon as possible.
The information gathered by this study raises an alarming red flag in terms of
Public Health. When the wild boar is taken home by the hunter, infected ticks feeding
on the animal before it was shot will start to detach from the carcass and drop to the
ground in search of a new host. If an infected tick had latched to the wild boar at least
two days earlier, odds are the spirochetes present in the midgut have already migrated to
the salivary glands of the tick and the probability of transmission is massively
increased. Because the hunter will probably be handling the carcass to separate skin,
bones and organs and prepare the meat for freezing, it is very likely that some of the
ticks attached to the animal’s skin might attach themselves to this human host and
transmit the spirochetes almost immediately. Also, fallen ticks present in the vehicle
may constitute a danger to other users of the transportation because most of these
hunters use their family car to transport the dead animal.
Another danger connected to hunting activities is posed by the gamekeeper's dogs
to their owners and family, because they can contact directly with questing ticks and the
ticks attached to the wild boar. In addition, the geographic mobility of these dog packs
43
is great during the hunting season, which greatly increases the dissemination of
potentially infected ticks, and therefore the LB agent.
Taking into consideration the achieved results and its analysis, it’s admissible to
say that the objectives proposed in this study were fulfilled because:
 The main ixodids species parasitizing wild boars were identified as being
Dermacentor marginatus, Ixodes ricinus, Dermacentor reticulatus, Dermacentor
sp. and Rhipicephalus sanguineus;
 DNA of B. burgdorferi s.l, particularly B. afzelii, was detected in three of four
positive wild boar sera analyzed by nested-PCR despite no positive results in the
analyzed female ticks;
 The direct contact of hunters and gamekeepers with animals that can serve both as
hosts for the spirochetes and potentially infected ticks put them at greater risk of
infection than any other group, because feeding ticks that change hosts before
completing the feeding (as is the case of ticks feeding on these shot wild boars)
are much more likely to transmit the LB spirochete to humans.
44
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8 ANNEXES
Supplementary figure 1 - Original field form (1st page).
53
Supplementary figure 2 - Original field form (2nd page).
54
Supplementary figure 3 - Final field form (1st page).
55
Supplementary figure 4 - Final field form (2nd page).
56
Supplementary figure 5 - Morphological identification of collected ticks.
57
Supplementary figure 6 - Ixodids’ identification form (1st page).
58
Supplementary figure 7 - Ixodids’ identification form (2nd page).
59
Supplementary figure 8 - DNA extraction from serum samples.
Supplementary figure 9 - Nested-PCR amplification.
60
Supplementary table 1 - Primer sequences used in “1,2,3,4” and “fla” nested-PCR
amplifications.
Primer
denomination
Dimension of
amplified product
(bp)
Primers sequences for the “1,2,3,4” nested-PCR (Protocol 1)
23SN1 (1st PCR)
Nucleotide sequence
5’-ACCATAGACTCTTATTACTTTGAC-3’
23SC1 (1 PCR)
5’-TAAGCTGACTAATACTAATTACCC-3’
23SN2 (2nd PCR)
5’-ACCATAGACTCTTATTACTTTGACCA-3’
st
nd
5SCB (2 PCR)
5’-biotin-GAGAGTAGGTTATTGCCAGGG-3’
380 bp
230 bp
Primers sequences for the “fla” nested-PCR (Protocol 2)
132f (1st PCR)
5’-TGGTATGGGAGTTTCTGG-3’
905r (1 PCR)
5’-TCTGTCATTGTAGCATCTTT-3’
220f (2nd PCR)
5’-CAGACAACAGAGGGAAAT-3’
st
nd
823r (2 PCR)
5’-TCAAGTCTATTTTGGAAAGCACC-3’
Supplementary figure 10 - Visualization of the electrophoresis gel.
61
774 bp
604 bp