The document describes the development of a reverse line blot (RLB) hybridization kit that can simultaneously detect four genera of common tick-borne pathogens - Anaplasma, Ehrlichia, Babesia and Theileria. This provides a sensitive and cost-effective diagnostic tool that facilitates the study of tick-borne diseases. The RLB technique involves PCR amplification of the pathogens, followed by hybridization of the products on a membrane containing genus-specific probes. This allows for simultaneous identification of any of the pathogens present in one sample. The kit will support improved diagnosis and epidemiological research on these important diseases affecting both animals and humans.

1. as published in BTi September 2004RLB HYBRIDISATION
Ticks transmit a greater variety of pathogenic micro-organisms
than any other arthropod vector group. These include tick-
borne protozoa and tick-borne bacteria of both medical and vet-
erinary importance. Two important tick-borne protozoan dis-
eases are theileriosis and babesiosis [Figure 1]. The former is one
of the most important diseases of cattle in southern, eastern and
central Africa. Commonly called East Coast Fever, the more
pathogenic strains of Theileria can decimate herds. Babesiosis,
commonly called Red Water Fever, is a disease with a high mor-
tality rate in cattle that can also affect other domestic animals. It
was only in the late 1960s that a human infection with Babesia
was identified, but since then the reported incidence of human
infections has risen, probably because of increased contact
between humans and the tick vectors.
Examples of tick-borne bacteria include Anaplasma species and
Ehrlichia species.Anaplasmosis is a global problem affecting cat-
tle, sheep, goats and other ruminants, with a high mortality rate,
particularly in older animals. Human anaplasmosis was only
recognised a decade ago, but has since been reported in both
North America and Europe. Ehrlichiosis is another disease of
domestic animals which, whilst it was only recently identified as
a potentially life-threatening condition in humans, is increasing
in incidence and geographic range.
INTEGRATED MOLECULAR DIAGNOSIS OF TICK-BORNE
PATHOGENS
Although many useful species-specific PCR assays have been
developed to detect a particular tick-borne pathogen, these
organisms frequently occur together with other species trans-
mitted by ticks within the same host. Thus a universal test is
needed where it is possible to simultaneously detect and differ-
entiate all protozoan and ehrlichial parasites that could possibly
be present in the blood of an infected host or in vector ticks.
Reverse line blot (RLB) hybridisation, where multiple samples
can be analysed against multiple probes to enable simultaneous
detection, fulfils these criteria. RLB was originally developed for
the identification of Streptococci serotypes [1]. The first applica-
tion of RLB for the detection and differentiation of pathogens in
ticks was developed for Borrelia spirochetes [2] and was subse-
quently combined with Ehrlichia spp [3]. RLB was then success-
fully applied for the detection and differentiation of all known
Theileria and Babesia species [4].The subsequent development
of an RLB suitable for detection and differentiation of Ehrlichia
and Anaplasma species provided a further basis for the present
RLB hybridisation kit, which includes 36 probes for Anaplasma,
Ehrlichia, Babesia and Theileria species.
RLB is rapidly becoming a standard molecular tool for diagnos-
tic and epidemiological studies in an increasing number of lab-
oratories all over the world. There are many examples. For
instance, RLB was used for the characterisation of Babesia diver-
gens in a human case [5], and novel Theileria and Babesia species
were discovered through the application of RLB [6].
Furthermore, RLB was used for detection and differentiation of
many Babesia and Theileria spp occurring in small ruminants
[7]. RLB was also successfully applied to the study of protozoan
Reverse line blot hybridisation in
the detection of tick-borne diseases
Many diseases of both medical and veterinary
importance are transmitted by ticks, which can
carry several pathogens at the same time.
Mammalian hosts also frequently harbour more
than one species of tick-borne pathogen simultane-
ously. The development of a novel reverse line blot
(RLB) hybridisation kit that can simultaneously
detect four different genera of common tick-borne
pathogens will facilitate diagnostic and epidemio-
logical studies. This article describes the RLB tech-
nology, which is much more sensitive than PCR
alone, but also cost-effective and robust enough for
use in the field.
Figure 1. Babesia (left) and Theileria, protozoans transmitted by
ticks.

2. haemoparasites in
Uganda and in
Portugal [8, 9,10].
THE REVERSE LINE
BLOT HYBRIDISA-
TION KIT
The new RLB test
kitcombines naplas-
ma/Ehrlichia detec-
t i o n w i t h
Theileria/Babesia
detection. The
availability of stan-
dardised mem-
branes for a fixed
number of tick-
borne pathogens
makes it possible to
conduct meaningful
comparative epi-
demiological stud-
ies between different laboratories. A further advantage of the
current test kit is the incorporation of cloned plasmid controls
that can be used as standardised positive controls. The assay is a
versatile diagnostic tool, which sensitively and simultaneously
detects and differentiates haemoparasites in blood, tissue or
ticks. RLB is based on simultaneous PCR amplification of relat-
ed species, the Erhlichia/Anaplasma cluster of species and the
cluster of Theileria/Babesia species, making specific PCR reac-
tions for each individual species unnecessary. Each species can
be identified by a species-specific oligonucleotide probe using a
line-blotter apparatus. RLB thus combines PCR amplification
followed by a hybridisation step resulting in sensitivity up to
1000 fold or higher than PCR only. Moreover, detection is based
on chemiluminescence instead of radioactivity, making the kit
more user-friendly. Because the blot containing the oligonu-
cleotides can be reused from 10 to 20 times, and only a limited
number of PCR amplifications are required, RLB makes eco-
nomic use of resources which are frequently limited in the coun-
tries where tick-borne diseases present the greatest challenge.
The first step is PCR amplification of a variable region in the 16S
ribosomal RNA gene (Erhlichia and Anaplasma) or 18S riboso-
mal RNA gene (Theileria and Babesia) using PCR-primers locat-
ed within conserved parts of the rRNA gene. The primers are
designed for the specific amplification of the rRNA gene of the
target organisms and they are not complementary to the rRNA
genes of either the hosts or the ticks, resulting in a high speci-
ficity of the PCR reaction. Two sets of PCR-primers are required
for the amplification of either the Ehrlichia /Anaplasma or
Theileria/Babesia rRNA gene. However, both PCR primer sets
have matching melting temperatures and thus the same PCR-
program can be used for both reactions. In the second step the
PCR products are hybridised on a blot on which a specific
oligonucleotide for each (known) Ehrlichia, Anaplasma,
Theileria and Babesia species has been covalently linked.
The species-specific oligonucleotides are deduced in the hyper-
variable region that is amplified in the first step by PCR. The
species-specific oligonucleotides are applied in lines using a
miniblotter and are covalently linked to the membrane by a 5|
terminal aminolinker [Figure 2]. The PCR-products are applied
to the membrane, also using the miniblotter, so that the direc-
tion of the PCR-products is perpendicular to the direction of the
species-specific oligonucleotides [Figure 3]. In this way the dif-
ferent pathogen species simultaneously amplified by PCR can
each hybridise specifically at the cross-sections of the line con-
taining the specific oligonucleotide and the line containing the
PCR product. A control oligonucleotide for either the Ehrlichia/
Anaplasma or the Theileria/Babesia species deduced from a
region conserved in the amplified PCR product ensures detec-
tion of a species for which no specific oligonucleotide is incor-
porated. After stringent washing to remove unbound PCR prod-
ucts, the hybridised PCR products are visualised using chemilu-
minescence.Visualisation makes use of a biotin label attached to
the PCR primer. The biotin label, presented at a site where the
PCR-product is hybridised to the probe, is subsequently detect-
ed by incubation with its streptavidin ligand conjugated to an
enzymatic label, HRP. Incubation of the blot with the peroxidase
substrate, ECL, results in a reaction producing light which can
be detected on a suitable film. After development of the film,
spots occur at the sites where species-specific oligonucleotide
and PCR-product hybridised and the identity of the micro-
organism(s) in the sample can be identified.
The development of the RLB kit for the simultaneous detection
of four different genera of tick-borne pathogens will greatly
facilitate epidemiological and diagnostic studies on tick-borne
diseases in a cost-effective way, hopefully leading to better con-
trol of these diseases, healthier life-stock and a reduction in inci-
dence of human infections.
REFERENCES
1. Kaufhold A, Podbieldski A, Baumgarten G, Blokpoel M, Top J and
Schouls L. Rapid typing of group A streptococci by the use of DNA
amplification and non-radioactive allele-specific oligonucleotide
probes. FEMS Microbiol letters 1994; 119: 19 - 26.
2. Rijpkema SG, Molkenboer MJ, Schouls LM, Jongejan F and
Schellekens JF. Simultaneous detection and genotyping of three genom-
ic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks
by characterization of the amplified intergenic spacer region between
5S and 23S rRNA genes. J Clin Microbiol 1995; 33: 3091 - 3095.
3. Schouls LM, van de Pol I, Rijpkema SG and Schot CS. Deletion and
as published in BTi September 2004RLB HYBRIDISATION
Figure 2. Schematic representation of the
hybridisation principle.

3. identification of Ehrlichia, Borrelia burgdorferi
sensu lato and Bartonella species in Dutch
Ixodes ricinus ticks. J Clin Microbiol 1999; 37:
2215 - 2222.
4. Gubbels MJ, de Vos S, van der Weide M,
Viseras J, Schouls LM, de Vries E and Jongejan
F. Simultaneous detection of bovine Theileria
and Babesia species using reverse line blot
hybridization. J Clin Microbiol 1999; 37: 1782
- 1789.
5. Centeno-Lima S, do Rosario V, Parreira R,
Maia AJ, Freudenthal AM, Nijhof AM and
Jongejan F. A fatal case of human babesiosis in
Portugal: molecular and phylogenetic analysis.
Trop Med Int Health 2003; 8: 760 - 764.
6. Nijhof AM, Penzhorn BL, Lynen G, Mollel
JO, Morkel P, Bekker CP and Jongejan F.
Babesia bicornis sp. nov. and Theileria bicornis
sp. nov.: tick-borne parasites associated with
mortality in the black rhinoceros (Diceros
bicornis). J Clin Microbiol 2003; 41: 2249 -
2254.
7. Schnittger L,Yin H, Qi B, Gubbels MJ, Beyer
D, Niemann S, Jongejan F and Ahmed JS.
Simultaneous detection and differentiation of
Theileria and Babesia parasites infecting small
ruminants by reverse line blotting. Parasitol
Res 2004; 92: 189 - 196.
8. Oura CA, Bishop RP, Wampande EM,
Lubega GW and Tait A. Application of a
reverse line blot assay to the study of
haemoparasites in cattle in Uganda. Int J
Parasitol 2004; 34: 603 - 613.
9. Oura CA, Bishop RP, Wampande EM,
Lubega GW and Tait A. The persistence of
component Theileria parva stocks in cattle
immunized with the “Muguga cocktail” live
vaccine against East Coast Fever in Uganda.
Parasitol 2004; 129: 27 - 42.
10. Brigido C, Pereira da Fonseca I, Parreira R,
Fazendeiro I, do Rosario VE and Centeno-
Lima S. Molecular and phylogenetic character-
ization of Theileria spp. parasites in autochto-
nous bovines in Portugal. Vet Parasitol 2004;
123: 17 - 23.
Isogen Life Science
P.O. Box 1779
NL-3600 BT Maarssen
The Netherlands
Tel +31 346 550 556
Fax +31 346 554 619
www.isogen-lifescience.com
Based on the work of Amar Taoufik, Ard
Nijhof, Radi Hamidjaja and Frans
Jongejan of the Division of Parasitology and
Tropical Veterinary Medicine, Utrecht
University, The Netherlands; Visva Pillay
of the Department of Veterinary Tropical
Diseases, University of Pretoria, South
Africa; and Marieke Sonnevelt and Marco
de Boer of Isogen Life Science.
as published in BTi September 2004RLB HYBRIDISATION
Figure 3. Schematic representation of the RLB assay.