Detection of Anaplasma marginale DNA in

Larvae of Boophilus microplus Ticks by

Polymerase Chain Reaction
MÁRCIA KIYOE SHIMADA,a MILTON HISSASHI YAMAMURA,a
PAULA MIYUKI KAWASAKI,a KÁTIA TAMEKUNI,a MICHELLE IGARASHI,a
ODILON VIDOTTO,a AND MARILDA CARLOS VIDOTTOb
aDepartamentoMed. Vet. Preventiva, CCA, Universidade Estadual de Londrina,
Campus Universitário, Londrina, PR, Brazil
bDepartamento de Microbiologia, CCB,
Universidade Estadual de Londrina, Londrina, PR, Brazil

ABSTRACT: Boophilus microplus larvae from two different sources were used
for the detection of Anaplasma marginale DNA: larvae A, which were collected
from a pasture of an endemic farm, and larvae B, which originated from en-
gorged female ticks fed on calves with no clinical signs of disease and with low
rickettsemia (approximately 0.01 to 1.0%). Larvae A were collected monthly,
from January to May in 2001. Two hundred engorged female ticks fed on calves
that provided larvae B were divided into groups of 10 and kept in a controlled
environment at either 18°C or 28°C. Fifty larvae were used from each sample
for DNA extraction, and 5 ␮L of DNA were submitted to amplification of the
sequence of msp5 gene of A. marginale by polymerase chain reaction (PCR).
Seven out of 50 samples of larvae A (14%) were positive for the presence of
DNA of A. marginale showing amplified product of 457 bp. Ten out of 91 sam-
ples of larvae B (11%) kept at 18°C were positive, and all larvae B at 28°C were
negative. Thus, this study confirmed the presence of A. marginale DNA in
B. microplus larvae by PCR. The EcoRI restriction enzyme analysis confirmed
the specificity of the amplicon, which resulted in two fragments: 265 bp and
192 bp. The sequencing analysis of the amplicon from larvae demonstrated
98% homology with the msp5 sequence from Florida A. marginale strain .

KEYWORDS: Anaplasma marginale; larvae; Boophilus microplus; polymerase

chain reaction (PCR)

INTRODUCTION

Bovine anaplasmosis is a tick-borne disease of cattle caused by the obligate in-

traerythrocytic bacteria Anaplasma marginale (order Rickettsiales, family Anaplas-
mataceae).1 This disease is endemic in many tropical and subtropical areas, and the

Address for correspondence: Odilon Vidotto, Departamento Med. Vet. Preventiva, CCA,
Universidade Estadual de Londrina, Campus Universitário, C.P. 6001, CEP 86051-990, Lond-
rina, PR, Brazil. Fax: 55-43-3371-4714.
[email protected]

Ann. N.Y. Acad. Sci. 1026: 95–102 (2004). © 2004 New York Academy of Sciences.
doi: 10.1196/annals.1307.012

95
96 ANNALS NEW YORK ACADEMY OF SCIENCES

acute phase causes anemia, abortion, weight loss, and eventually death, resulting in
significant losses to meat and milk production.2 A. marginale is usually transmitted
biologically by feeding ticks, whereas mechanical transmission occurs when infect-
ed blood is transferred to susceptible animals by biting flies or blood-contaminated
fomites. Approximately 20 species of ticks have been implicated as vectors world-
wide.3 In Brazil, Boophilus microplus is considered to be the main vector of A. mar-
ginale,4 but the mechanism of transmission employed by this tick is still
controversial.
B. microplus is a monoxenic tick, but adult stages can migrate between animals
by physical contact.4–6 Intrastadial transmission by male ticks may be important in
maintaining the organism in enzootic areas. The transtadial transmission has been
reported for many ticks including B. microplus.7,8 Transovarial transmission of A.
marginale has also been reported for some ticks, but has not been demonstrated for
B. microplus.4,5,8–11
Ribeiro and Lima12,13 demonstrated the multiplication of A. marginale in B. mi-
croplus, and verified the influence of temperature on the development of colonies of
A. marginale in the midgut epithelial cells of experimentally infected B. microplus
females. The colonies were observed in the midgut epithelial cells in 11.1% of
females that were kept at environmental temperature by 19 days after detachment
from donor calf, suggesting that transovarial transmission occurs in winter (temper-
atures ranged from 10 to 15°C and from 22 to 32°C), when the infection could be
restricted to the last eggs laid.
This work shows the results of a polymerase chain reaction (PCR) study with
larvae of B. microplus collected from pasture and from engorged female ticks infect-
ed with A. marginale incubated at different temperatures.

MATERIALS AND METHODS

Boophilus microplus Ticks

Larvae from two sources were studied: larvae A, from a pasture in a dairy farm
with endemic anaplasmosis, located in Londrina municipality (23° 30′ 17″ S, 51°
07′ 28″ W), Paraná State, Brazil; larvae B, resulting from engorged female ticks fed
on calves infected with A. marginale with rickettsemia varying from 0.01% to 1.0%
(confirmed by PCR), without clinical sign of anaplasmosis.
Larvae A were collected monthly, from January to May 2001, in a pasture of Bra-
chiaria spp and coast-cross grass. The minimum temperatures in this period ranged
from 8.2 to 20.5°C, and the maximum temperatures ranged from 21.2 to 31.5°C.
The humidity ranged from 59.6 to 79.7%. Each month the collected larvae were sep-
arated in 10 samples of 50 larvae each.
The engorged female ticks that provided larvae B were first transferred to a con-
trolled environment under two temperatures. Two hundred female ticks fed on calves
were divided into groups of 10 and kept in Petri plates. Ten plates were incubated at
18 ± 2°C with 80–83% humidity, and the other 10 plates were incubated at 27 ± 2°C
with 86–89% humidity. The pre-oviposition and oviposition periods were monitored
daily, and the eggs were collected approximately every 5 days. All the eggs from
each period were collected and kept in sterile flasks at 28 ± 1°C with 87% relative
SHIMADA et al.: DETECTION OF ANAPLASMA MARGINALE DNA 97

humidity until the hatching of the larvae. Fifty larvae were separated for each sam-
ple. All larvae and tick controls were washed with ethanol 70% and ultra-pure H2O
and kept at –20°C until further use.
B. microplus male ticks collected from a calf naturally infected with A. marginale
(60% of rickettsemia) were used as positive control. The negative control consisted
of larvae originating from female ticks fed on cattle negative for A. marginale, kind-
ly provided by EMBRAPA/CNPGC/Campo Grande/MS.

DNA Extraction
The DNA from ticks and larvae were extracted by the phenol-clorophorm/
isoamilic-alcohol and silica/guanidium thiocyanate14 methods with some modifica-
tions. Fifty larvae from each sample were used. The male ticks or larvae groups were
triturated in 450 µL of TE (Tris-HCl 10 mM; EDTA 1 mM, pH 8.0). After cell lysis
with 10% SDS and proteinase K (10 mg/mL) for 1 hr at 37°C, the same volume of
phenol-clorophorm/isoamilic-alcohol (24:24:1) was added. The suspension was in-
cubated at 56°C during 15 min and centrifuged at 10,000 × g 10 min. The aqueous
phase was then processed in silica/guanidium thiocyanate (120 g guanidium thiocy-
anate; 100 mL Tris-HCl 10 mM pH 6.4; EDTA 20 mM pH 8.0; 2.6 g Triton X-100).
The washes were realized twice with L2 solution (120 g guanidium thiocyanate;
100 mL Tris-HCl 0.1 M, pH 6.4), twice with ethanol 70%, and once with acetone.
The pellet was kept at 56°C for 15 min to dry, and the DNA was eluted in 25 µL of
ultra-pure autoclaved H2O. This procedure was repeated, and 50 µL of DNA was ob-
tained. All extracted DNA was stored at –20°C until further use.

PCR Procedure
PCR was performed with primers obtained from a sequence of the msp5 gene of
Florida A. marginale (GenBank number M93392) according to a procedure
described by de Echaide and colleagues.15 These primers included external forward,
5′-GCATAGCCTCCGCGTCTTTC-3′ (position 254 a 273), and external reverse,
5′-TCCTCGCCTTGGCCCTCAGA-3′ (position 710 a 692). The analysis of these
primers did not show genomic similarity with microorganisms from the Ehrlichia
and Wolbachia genera. Amplification reactions were performed in a final volume of
25 µL containing 5 µL of DNA, 20 pmol of each primer, 0.2 mM of deoxynucleoside
triphosphate, 20 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 1.25 U of
Taq DNA polymerase (GIBCO-Life Technology, Rockville, MD) with thermocycler
(MJ Research, Watertown, MA) for 5 min at 95°C, 35 cycles at 95° C for 1 min,
65°C for 2 min, and 72°C for 1 min with a final extension at 72° C for 10 min fol-
lowed by cooling to 4°C. Ultra-pure H2O was used as a reaction control in the same
conditions. After the amplification reaction, 10 µL of the amplicon was electro-
phoresed on polyacrylamide gel (10%) and stained by silver. A 123-bp ladder
(GIBCO-Life Technology) was used as molecular size standard.

Specificity of PCR Product

To determine the specificity of the amplicon, the bands with 457 bp were digested
with EcoRI restriction endonuclease (Bethesda Research Laboratories, Rockville,
MD), according to the manufacturer’s instructions. The digests were subjected to
98 ANNALS NEW YORK ACADEMY OF SCIENCES

eletrophoresis through polyacrylamide gel (10%) and stained by silver. 123-bp lad-
der was used as a molecular mass marker.
The sequence analysis of the amplicon from larvae was carried out with the nested
PCR product obtained with external forward 5′-CACCATGAGAATTTTCAAGAT-
TGTG-3′, external reverse 5′-TCCTCGCCTTGCCCCTCAGA-3′, and internal for-
ward 5′-AGAATTAAGCATGTGACCGCTG-3′. The amplicon was submitted at
sequencing (3100, Applied Biosystems, Foster City, CA), and the sequence analysis
was done with BLAST (<http://www.ncbi.nih.gov/BLAST/>) program.

RESULTS AND DISCUSSION

Seven out of the 50 samples (14%) of larvae A collected from pasture in different
months and 10 of the 91 samples (11%) of larvae B from engorged female ticks fed
on calves were positive to presence of msp5 gene of A. marginale by PCR, amplify-
ing the fragment of 457 bp (TABLE 1). The EcoRI restriction enzyme analysis of this
amplicon showed its specificity, showing two fragments, one of 265 bp and one of
192 bp (FIG. 1). The nucleotide sequence of this amplicon was determined and is dis-
played in FIGURE 2. The sequencing analysis of the amplicon from larvae demon-
strated 98% homology with the msp5 sequence from Florida A. marginale strain.

FIGURE 1. Polyacrylamide (10%) gel stained by silver of PCR product from msp5
gene of A. marginale. Lane 1: molecular size markers (123-bp ladder). Lane 2: PCR product
from infected blood of calf not cleaved (457 bp). Lane 3: PCR product from infected blood
of calf cleaved with EcoRI. Lanes 4 & 5: B. microplus male ticks not cleaved and cleaved.
Lanes 6 & 7: engorged female ticks not cleaved and cleaved. Lanes 8 & 9: larvae from pas-
ture not cleaved and cleaved. Lanes 10–13: larvae from engorged female ticks not cleaved
and cleaved.
SHIMADA et al.: DETECTION OF ANAPLASMA MARGINALE DNA 99

The occurrence of positive larvae from pasture was higher in April (four sam-
ples), and in January all samples were negative for the presence of A. marginale
DNA. The reported influence of environmental temperature on the multiplication of
the A marginale in the B. microplus females12 might explain the higher number of
positive larvae samples in April (autumn) than in January (summer).
The period of incubation of engorged female ticks that provided larvae B at 18°C
was longer than at 28°C. The pre-oviposition period of engorged female ticks that
provide larvae B was 8–9 and 2–3 days at 18°C and 28°C, respectively. The ovipo-
sition period in larvae B at 18°C was 13–44 days, whereas the period at 28°C was

FIGURE 2. Comparison of sequence obtained GenBank (number M93392) with se-

quence of msp5 of A. marginale from positive larvae of B. microplus ticks, showing 98%
homology.
100 ANNALS NEW YORK ACADEMY OF SCIENCES

TABLE 1. Positive samples of Boophilus microplus larvae collected from pasture and
engorged females by collection period using polymerase chain reaction (PCR)
Larvae from pasture
(ambient T °C) Larvae from females incubated in chambers
(Larvae A) (Larvae B)
28°C 18°C
Total Total
Larvae Egg number Number of number Number of
collection Samples/ Positive collection of positive of positive
month collectiona samples days samples samples samples samples
January 10 0 7–8 20 0 0 0
February 10 1 12–14 20b 0 20 2
March 10 1 18–19 20 2
April 10 4 23–24 20 0
May 10 1 28–29 20 3
33–34 10 3
43–44 01b 0
Total 50 7 (14%) 40 0 91
aFifty larvae each sample.
bEnd of oviposition period.

7–13 days. All larvae B at 28°C were negative for the presence of A. marginale
DNA. Ten samples of larvae B were positive between 13 and 34 days of egg
collection (TABLE 1).
These results show that longer pre-oviposition and oviposition periods, which oc-
cur at lower temperatures, contributed to an increase in the number of positive lar-
vae, a result that is in agreement with Ribeiro et al.11 and Ribeiro and Lima,12 who
observed colonies of A. marginale in the midgut of B. microplus engorged females
only at low environmental temperatures, when the pre-oviposition was 6–11 days
and the oviposition period was 28–41 days. Also, these authors observed colonies
only in 11% of female ticks by the 19th day after detachment from the donor calf,
which increased to 33% of infected vectors in the following weeks, suggesting that
winter is the most probable period for the occurrence of vertical transmission of this
agent in nature, and that it is limited to the last eggs laid.
The increased number of positive larvae obtained in this work, when the tick fe-
males were incubated at 18°C, can be explained because oviposition periods longer
than 14 days can enable the migration of A. marginale from midgut to ovaries,
whereas in optimal conditions the oviposition in B. microplus is bigger (99.9%) from
10th to the 13th days after detachment of engorged female ticks,16,17 limiting the
passage of Anaplasma to ovaries. Most of the unsuccessful attempts of transovarial
transfer of A. marginale infection were performed with larvae originated from
B. microplus females incubated under high temperatures (28 to 30°C).8–10 Such
conditions of engorged tick female incubations could explain the failure of these at-
tempts of transovarial transfer of A. marginale infection.
SHIMADA et al.: DETECTION OF ANAPLASMA MARGINALE DNA 101

The rickettsemia level of persistently infected animals influences A. marginale

transmission by Dermacentor andersoni and B. microplus.18,19 Rickettsemia levels
during persistence are cyclical and vary from 102 to 107 mL−1. Although the tick in-
fection rate correlates with the level of rickettsemia during acquisition feeding, a
substantial percentage of ticks (27%) become infected even while feeding at the low
levels of persistent rickettsemia that occur between the cyclic peaks.20 Our results
show an increase in the number of positive sample in larvae from engorged female
ticks fed on calves with low parasitemia.
This study determined the presence of A. marginale DNA in B. microplus larvae
by PCR. These larvae originated from a long oviposition period of engorged female
ticks. Even though these data are not conclusive, they indicate that A. marginale can
migrate from the midgut of B. microplus engorged female ticks to ovaries and con-
sequently infect larvae.

ACKNOWLEDGMENTS

This research was supported by Conselho Nacional de Desenvolvimento

Cientifico e Tecnológico, CAPES, and CPG-UEL. We thank the Instituto de
Biologia Molecular do Parana/Fiocruz for the DNA sequencing analysis.

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