Aspects oF transovarial transmission oF microorganisms in )XODES

&ARM0RZEGL.AUK†
!SPECTSOFTRANSOVARIALTRANSMISSIONOFMICROORGANISMS
IN)XODESRICINUS
!SPEKTYTRANSOWARIALNEJTRANSMISJIDROBNOUSTROJÌWU)XODESRICINUS
+RZYSZTOF0*ASIK2OBERT$7OJTYCZKA$ANUTA()DZIK
-AŒGORZATA+ÃPA*ERZY0ACHA*AN3ŒODKI
+ATEDRAI:AKŒAD-IKROBIOLOGII7YDZIAŒ&ARMACEUTYCZNYZ/DDZIAŒEM-EDYCYNY,ABORATORYJNEJW3OSNOWCU
gL’SKI5NIWERSYTET-EDYCZNYW+ATOWICACH
Abstract
Streszczenie
The widespread risk of tick-borne illnesses requires multilevel research, also pertaining to the form of the process
of pathogens dissemination among generations of ticks.
Considering the fact that the phenomenon of the transovarial transmission of microorganisms must be based on
a number of cytophysiological relations between a microorganism and a vector, it is crucial to demonstrate which
species of ticks exhibit this phenomenon and what microorganisms it concerns. The aforementioned cytophysiological relations are closely correlated with the location of
microorganisms within specified tissues, or intercellular
spaces in ticks. The research presented in this study refers
to selected aspects of the subject matter specified above.
Our analyses focused upon a species of hard ticks which is
the most important in Europe from the medical and veterinarian point of view, namely Ixodes ricinus. The analyses
were carried out on the basis of observations in the electron microscope and PCR marking of the occurrence of
specific microorganisms in mature female ticks and their
offspring.As a result of the research it was established that
Borrelia burgdorferi s. l., Anaplasma hagocytophilum,
as well as Babesia spp. can be transovarially transmitted
between generations of I. ricinus. The electron microscope analysis of embryos and larvae allows to observe
that some microorganism remain extracellular within the
midgut, whereas their considerable part is located inside
the cells of the midgut, salivary glands or Malpighian tubules.
Powszechne zagrożenie chorobami odkleszczowymi wymaga wielostronnych badań, m. in. dotyczących form
procesu rozprzestrzeniania się patogenów pomiędzy pokoleniami kleszczy. Biorąc pod uwagę fakt, że fenomen
transowarialnego rozprzestrzeniania się drobnoustrojów
musi się opierać o szereg zależności cytofizjologicznych
między drobnoustrojem a wektorem, istotne jest wykazanie u których gatunków kleszczy zjawisko to zachodzi
oraz jakich drobnoustrojów dotyczy. Wspomniane zależności cytofizjologiczne są ściśle skorelowane z lokalizacją drobnoustrojów w określonych tkankach, czy międzykomórkowych przestrzeniach kleszczy. Przedstawione
w tej pracy badania dotyczą wybranych aspektów wyżej
wspomnianej tematyki. W analizach naszych skoncentrowaliśmy się na najbardziej istotnym z medyczno-weterynaryjnego punktu widzenia gatunku kleszczy twardych
w Europie, Ixodes ricinus. Analizy zostały wykonane w
oparciu o obserwacje w mikroskopie elektronowym oraz
oznaczanie metodą PCR występowania określonych drobnoustrojów u dojrzałych samic kleszczy i ich potomstwa.
Wskutek badań ustalono że zarówno Borrelia burgdorferi
s. l., Anaplasma phagocytophilum, jak i Babesia spp. mogą
być przekazywane transowarialnie pomiędzy pokoleniami
I. ricinus. Analiza elektronowo-mikroskopowa zarodków
i larw pozwala zaobserwować, że niektóre drobnoustroje
pozostają pozakomórkowo w obrębie jelita środkowego,
natomiast spora ich część jest umieszczona wewnątrz komórek jelita, ślinianek lub cewek Malpighiego.
Key words: Anaplasma phagocytophilum, Babesia spp.,
Borrelia burgdorferi, Ixodes ricinus, transovarial transmission
Słowa kluczowe: Anaplasma phagocytophilum, Babesia spp., Borrelia burgdorferi, Ixodes ricinus, transmisja
transowarialna
Introduction
Spreading of microorganisms in ecosystems constitutes a
very broad issue. Considering the fact that man and animals
that accompany him constitutes elements of a biocenosis,
the research on the transmission of microorganisms must be
based on numerous disciplines, such as clinical microbiology, environmental microbiology, parasitology, physiology
and even ecology. Ticks are ectoparasites and at the same
time they are very significant as vectors for various microorganisms [1]. Ixodes ricinus, similarly to other hard ticks,
feeds relatively long. This feature is connected with the need
of the ticks to exhibit a number of adaptations allowing them
to maintain a long contact with their host. These adaptations
render it easier for microorganisms to get to the host organism and to begin their expansion in the initial period of infec-
COPYRIGHT‚'RUPADR!2+WIECIÊSKIEGO)33.†
tion, making ticks ideal vectors [2]. Ticks constitute a temporal habitat not only for the broadly known pathogens transmitted to hosts, but also other microorganisms are present in
their tissues – endosymbiotic ones, similarly to many other
arachnids, crustaceans, insects and nematodes [3].
All microorganism overcome immunological barriers
of the occupied organisms, and furthermore they are appropriately adapted to spread between particular specimens of
ticks, i.e. horizontally, as well as between generations, i.e.
vertically. The latter issue partly constitutes the scope of our
research presented herein.
Material and methods
Adult, fully engorged females of Ixodes ricinus were
collected from dogs in a southern district of the city of Katowice (Poland) in mid-May, during the spring peak activity of
the species. The females were placed in a breeding culture in
chambers of high air humidity. From the beginning of egglaying, individual packets were isolated and kept as separate
embryo cultures of known age. The cultures were kept at constant temperature (28 oC) and relative air humidity of 90-100%.
In such conditions the embryonic development lasted 30 days.
The embryos, larvae and ovaries of the collected females
were prepared for light and transmission electron microscope analysis. The material was fixed for 2 hours in 2.5%
glutaraldehyde solution in 0,1 M phosphate buffer (pH=7.4).
Then, following several baths in phosphate buffer, the material was fixed in osmium tetroxide solution in the same buffer. Following fixing and rinsing in the buffer, the embryos
and ovaries were dehydrated in alcohol and propylene oxide
series and then embedded in Epon 812 resin.
Serial semithin section were stained with methylene blue
and analyzed in light microscope. Ultrathin sections were
contrasted with lead citrate and uranyl acetate, and then examined in electron microscope Hitachi H500 at 75kV.
Some randomly selected embryos, originating from the
examined females, were subject to PCR analysis in order to
test them for the presence of Borrelia burgdorferi s. l., Babesia spp. and Anaplasma phagocytophilum. DNeasy Blood
set and Tissue Kit (QIAGEN) were used to isolate the bacterial DNA. The isolation was done according to the following protocols. Borrelia burgdorferi was detected with PCR
method. A fragment of fla gene, encoding flagelline, was
amplified. The isolated DNA was used in the PCR reaction
(diagnostic kit PCR-Borrelia, DNA Gdańsk). The PCR solution
contained: 21,25 μl of master mix, 2,5 μl of dNTPs nucleotide
mixture (2 mM), 0,25 μl of thermostable polymerase Hypernova, and 1,0 μl of DNA. The DNA amplification reaction was
conducted in the Personal Cycler (BIOMETRA, Germany)
– 40 cycles. The first initial denaturation was carried out at
93 ºC for 2 min. Each cycle involved: 30-s DNA denaturation at
93 ºC, 60-s addition of starters at 52 ºC, 60-s elongation of DNA
chain at 72 ºC, and 60-s final elongation at 72 ºC.
Babesia spp. was detected with PCR method. A fragment
of 18s rDNA gene was amplified. The isolated DNA was
used in the PCR reaction (diagnostic kit PCR-Babesia, DNA
Gdańsk). The PCR solution contained: 21,25 μl of master
mix, 2,5 μl of dNTPs nucleotide mixture (2 mM), 0,25 μl
of thermostable polymerase Hypernova, and 1,0 μl of DNA.
Fig. 1. Percentage of ticks females infected by Borrelia
burgdorferi s. l. [A], Anaplasma phagocytophilum [B] i Babesia spp. [C].
The DNA amplification reaction was conducted in the Personal Cycler (BIOMETRA, Germany) – 40 cycles. The first
initial denaturation was carried out at 94 ºC for 2 min. Each
cycle involved: 45-s DNA denaturation at 94 ºC, 45-s addition of starters at 48 ºC, 45-s elongation of DNA chain at
72 ºC, and 300-s final elongation at 72 ºC.
Anaplasma phagocytophilum was detected with PCR
method. A fragment of 16s rDNA gene was amplified. The
isolated DNA was used in the PCR reaction (diagnostic kit
PCR-Anaplasma, DNA Gdańsk). The PCR solution contained: 21,85 μl of master mix, 2,5 μl of dNTPs nucleotide
mixture (2 mM), 0,15 μl of thermostable polymerase Delta3,
and 0,5 μl of DNA. The DNA amplification reaction was conducted in the Personal Cycler (BIOMETRA, Germany) – 35
cycles. The first initial denaturation was carried out at 94 ºC
for 5 min. Each cycle involved: 30-s DNA denaturation at
94 ºC, 60-s addition of starters at 56 ºC, 30-s elongation of
DNA chain at 72 ºC, and 300-s final elongation at 72 ºC.
The reaction products were separated in 1,5% agarose
gel with ethidium bromide (Sigma Aldrich, Germany) during 1 h at 120 V. The marker of DNA size, DNA M1 and
M2 (DNA Gdańsk) was used as a standard. The amplification products were analyzed in the UV transilluminator (BIOMETRA, Germany) and archived using the UVI - DOC
software (Eppendorf, Germany). The expected size of the
amplified gene fla fragment (Borrelia) was 442 base pairs,
gene 18s rDNA fragment (Babesia) was 560 b.p. and gene
16s rDNA fragment (Anaplasma) 227 b.p.
Results
PCR analysis
Among the analysed material the largest number of infected females of I. ricinus are those with the detected presence of Borrelia burgdorferi s. 1. (73%). A significantly
less numerous group are females infected with Anaplasma
phagocytophilum (25%) and only few (1%) are vectors
for Babesia spp. [Fig. 1]. It is worth a mention that in case
&ARM0RZEGL.AUK
Fig. 2. PCR amplification product of Anaplasma phagocytophilum [2a] and Borrelia burgdorferi
s. l. [2b] in Ixodes ricinus on agarose gel after ethidium bromide staining. M; molecular size marker, +; positive control, -;negative control, lane E♀, 1♀, E embryo and 1 embryo with a positive
product of amplification (arrows).
tinctly visible. Many
of them occur individually within the basic ooplasm, whereas
some of them form
clusters within the area
of vacuoles or infiltrate
mitochondria between
their external and internal membranes [Fig.
4].
The electron microscopic analysis of
embryos and larvae
allows to observe the
locations of microorganisms in the forming tissues and organs.
Some microorganisms
remain extracellular
within the midgut [not
visible data], but a considerable part thereof
is located intracellularly within the midgut,
salivary glands or Malpighian tubules [5].
Discussion
As the PCR tests
revealed, among the
Fig. 3. Ovary structure of I. ricinus. Diagrammatic [3a] and semithin section – fragment of the representatives
of
ovary wall [3b]. Fc; funiculus, OL; lumen of ovary, Oo; oocyte, Ov; oviducts.
I. ricinus subjected to
the analysis the frequency
of
infections
with
Borrelia
burgdorferi
s. 1., Anaof some females co-infection with other microorganisms is
plasma
phagocytophilum
and
Babesia
spp.
is
different.
The
observed, e.g. both Borrelia burgdorferi s. 1., and Anaplasresults
obtained
here
are
not
significant
from
the
statistical
ma phagocytophilum.
The PCR method examination of females, embryos and point of view. Their task was solely to reveal which of the
larvae after hatching, and therefore the offspring derived females examined are vectors for specific microorganisms.
from particular females, allows to check the occurrence of At the same time, they allow to state that the group examthe same pathogens in these forms [Fig. 2]. The occurrence ined is representative. Although the frequencies of infecting
of microorganisms in case of embryos and larvae is clear, ticks with specific pathogens in different regions of Poland
however the proportion of infections requires further stud- and the world are different [4-6], the proportions of those
values in the group examined are similar.
ies.
Apart from B. burgdorferi s. 1, the most commonly
mentioned
in literature, ticks are vectors for numerous othMorphological examinations
er
bacteria,
among which intracellular endosymbionts and
An ovary of I. ricinus is a tubular U-shaped structure, located in the posterior section of the opisthosoma. Both ends parasites are obligatorily encountered. This group includes
of the ovary are connected to a pair of oviducts, directed representatives of the genera Rickettsia, Ehrlichia and Anatowards the front. The ovarian wall consists of one layer of plasma [7]. The list of microorganisms living in tissues and
somatic cells, between which there are single female repro- organs of ticks is constantly increasing. These intracellular
ductive cells in various oogenetic stages. During vitellogen- bacteria are closely related; they belong to Rickettsiales [8]
esis oocytes reach quite big dimensions, bulging towards the and often are difficult to differentiate. They may occur in
outside of the ovary. From the side of the body cavity they the cytoplasm, vacuoles or mitochondria. In the latter case,
are covered solely with the basement membrane, whereas microorganisms named by Sasser et al [9] Candidatus Midfrom the side of the lumen of the ovary they are supported ichloria mitochondrii, live and reproduce at the expense of
mitochondria. In spite of this the physiological balance of
by somatic cells, constituting a funiculus.
Within the cytoplasm of growing oocytes there occur the invaded cells is maintained, and therefore these bacteria
numerous inclusions, among which bacteria are quite dis- are treated as endosymbionts. They occur in female repro-
COPYRIGHT‚'RUPADR!2+WIECIÊSKIEGO)33.†
subjected to the analysis it is difficult to explicitly specify the way
of expansion of these
Protista between generations. However, as
it was observed in case
of Rhipicephalus microplus, 12% to 48% of
larvae inherit Babesia
bovis from the mother
females transovarially
[16]. Babesia equi, on
the other hand, can be
transovarially transmitted in case of species
from the genera of DerFig. 4. Oocyte structure. Ultrathin sections – fragments of oocyte with rickettsialike bacteria in macentor, Hyalomma,
cytoplasm (arrows), in a vacuole V(Ric) and in mitochondria (arrowheads). L; lipid droplets, Mt; Rhipicephalus
and
mitochondria.
Boophilus [17]. Therefore, this transmission
route of Babesia spp. also seems possible in case of I. ricinus.
Assuming that the microorganisms subjected to the analysis are transmitted to the next generations directly from females or males via their reproductive cells, the arrangement
of these pathogens within a developing embryo is extremely
important. As revealed in our research, they can remain in the
reproductive cells, the cells of developing salivary glands, in
spaces filled with hemolymph, in the cells of Malpighian
Fig. 5. Transmission electron micrograph of the transverse tubules. Similarly, this type of positioning of Rickettsia spp.
section of a Malpighian tubule. Arrows; rickettsialike bac- and Rickettsiella-like bacteria was observed by Kurtti et
al. [18] in case if I. woodi, Barldridge et al. [19] in case of
teria, LTM; light of Malpighian tubule, Mt; mitochondria.
I. scapularis and by many other authors. Nevertheless, quite
ductive cells of I. ricinus, in 100% causing no changes in a peculiar case are bacteria occurring in deutoplasm, because
the reproduction process [10]. An interesting fact which is of which in a fully formed larva they are initially closed in
worth further research is a link between these bacteria, as the lumen of the midgut [research in progress]. The last of
well as all those transovarially transmitted with the line of the aforementioned situations most probably refers to Borreproductive cells, especially as this line emerges very early relia burgdorferi s. 1., although this thesis requires further
research which would confirm it.
during the embryogenesis of I. ricinus [11].
The problem of the vertical transfer of microorganisms
between generations of ticks, and therefore via reproduc- Conclusions
tive cells, constitutes a subject matter of numerous studies,
The research results presented in this study confirm the
sometimes postulating contradictory hypotheses. For inpossibility
of spreading via the transovarial transmission of
stance, the transovarial transmission of B. burgdorferi s. 1.
both
Borrelia
burgdorferi s. 1. and Anaplasma phagocywas described as dominating in case of I. pacificus [12]. In
tophilum
in
populations
of Ixodes ricinus. Also Babesia
other cases this type of dissemination of spirochaetes from
spp.
can
be
transmitted
directly
from females to embryos.
the genus of Borrelia is treated as marginal, whereas the
Among
these
three
microorganisms
the most frequent obmain expansion mechanism within young forms of ticks is
servation
was
the
transmission
of
Borrelia burgdorferi
based on their close co-feeding [13, 14]. It is also believed
s.
1
by
oocytes
onto
the
next
generations
of the ticks exthat A. phagocytophilum in I. pacificus is not spread transoamined,
however
these
observations
require
further analvarially [15]. Our research suggests that in I. ricinus both
yses.
B. burgdorferi and A. phagocytophilum are transferred between generations in a transovarial fashion. The proportion
of B. burgdorferi inherited from females is clearly higher Acknowledgements
than A. phagocytophilum [unpublished data], nevertheless,
This investigation was financially supported by the Polish
this issue requires further studies.
State
Committee for Scientific Research (KNW-1-050/09).
There are still numerous ambiguities in the subject of
Praca
jest wykonana dzięki finansowaniu badań statutothe vertical transmission of Babesia spp. Most of all, conwych
nr
KNW-1-050/09.
sidering the relatively small number of infected females
&ARM0RZEGL.AUK
References
1. Sonenshine DE. Biology of ticks. Oxford University
Press. New York 1993; vol. 2.
2. Bowman AS, Sauer JR. Tick salivary glands: function,
physiology and future. Parasitology 2004; 129: 67-81.
3. Rymaszewska A. Symbiotic bacteria in oocyte and ovarian
cell mitochondria of the tick Ixodes ricinus: biology and
phylogenetic position. Parasitol Res 2007; 100: 917-920.
4. Wodecka B. Rozpowszechnienie genogatunków z
kompleksu Borrelia burgdorferi s. l. w populacjach
kleszczy I. ricinus w krajach europejskich. W: Biologia
molekularna patogenów przenoszonych przez kleszcze.
Red. Skotarczak B. Wydawnictwo Lekarskie PZWL.
Warszawa 2006, 105-110.
5. Zwoliński J et al. Human granulocytic anaplasmosis as an
emerging problem of public health. Zdr Publ 2007; 213-219.
6. Gray J et al. Transmission Studies of Babesia microti in
Ixodes ricinus Ticks and Gerbils. J Clin Microbiol 2002;
40: 1259–1263.
7. Sukumaran B et al. An Ixodes scapularis protein required for survival of Anaplasma phagocytophilum in
tick salivary glands. J Exp Med 2006; 203: 1507-1517.
8. Dumler JS et al. Reorganization of genera in the families
Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with
Anaplasma, Cowdria with Ehrlichia and Ehrlichia with
Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and “HGE agent”
as subjective synonyms of Ehrlichia phagocytophila. Int
J Syst Evol Microbiol 2001; 51: 2145–2165.
9. Sassera D et al. “Candidatus Midichloria mitochondrii”, an endosymbiont of the tick Ixodes ricinus with
a unique intramitochondrial lifestyle. Int J Syst Evol
Microbiol 2006; 56: 2535–2540
10. Sassera D et al. ”Candidatus Midichloria” Endosymbionts Bloom after the Blood Meal of the Host, the Hard
Tick Ixodes ricinus. Appl Environ Microbiol 2008; 74:
6138–6140.
11. Jasik K. Embryonic development of Ixodes ricinus (L.).
Zool Pol 2007; 52: 5-60.
12. Lane RS, Burgdorfer W. Transovarial and Transstadial
Passage of Borrelia burgdorferi in the Western BlackLegged Tick, Ixodes pacificus (Acari: Ixodidae). Am J
Trop Med Hyg 1987; 37: 188-192.
13. Gatewood AG et al. Climate and Tick Seasonality
Are Predictors of Borrelia burgdorferi Genotype
Distribution. Appl Environ Microbiol 2009; 75:
2476-2483.
14. Piesman J et al. Transovarially acquired Lyme disease
spirochetes (Borrelia burgdorferi) in field-collected larval Ixodes dammini (Acari: Ixodidae). J Med Entomol
1986; 23: 219.
15. Foley JE et al. Anaplasma phagocytophilum Infection
in mall Mammal Host of Ixodes Ticks, Western United
States. Emerg Infect Dis 2008; 14: 1147-1150.
16. Howell JM et al. Transovarial transmission efficiency
of Babesia bovis tick stages acquired by Rhipicephalus (Boophilus) microplus during acute infection. J Clin
Microbiol 2007; 45: 426-431.
17. Ikadai H et al. Molecular evidence of Babesia equi
transmission in Haemaphysalis longicornis. Am J Trop
Med Hyg 2007; 76: 694–697.
18. Kurtti TJ et al. Rickettsiella-like Bacteria in Ixodes woodi (Acari: Ixodidae). J Med Entomol 2002;
39:534-540.
19. Baldridge GD et al. Infection of Ixodes scapularis ticks
with Rickettsia monacensis expressing green fluorescent protein: A model system. J Invertebr Pathol 2007;
94: 163–174.
data otrzymania pracy: 09.09.2010 r.
data akceptacji do druku: 28.10.2010 r.
Adres do korespondencji:
dr hab. Krzysztof Jasik
Katedra i Zakład Mikrobiologii
Wydział Farmaceutyczny z Oddziałem Medycyny Laboratoryjnej
Śląski Uniwersytet Medyczny w Katowicach
ul. Jagiellońska 4
41-200 Sosnowiec
e-mail: [email protected]