Babesia canis canis and Babesia canis vogeli infections in dogs

from northern Portugal

Luıs Cardoso a,b,*, Alvaro Costa c, Joana Tuna c, Lisete Vieira c,Osnat Eyal d, Yael Yisaschar-Mekuzas d, Gad Baneth d

a Department of Veterinary Sciences and CECAV, University of Tras-os-Montes e Alto Douro, P.O. Box 1013, 5001-801 Vila Real, Portugalb Parasite Disease Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Portugal

c Os Bichos–Veterinary Clinic, 5400-266 Chaves, Portugald School of Veterinary Medicine, Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel

Received 8 February 2008; received in revised form 7 May 2008; accepted 15 May 2008

Abstract

Canine babesiosis represents an important veterinary medical problem. This study describes the molecular characterization of

babesial parasites detected in eight clinically suspected dogs from northern Portugal, affected by lethargy, muscle tremors, weight

loss, pale mucous membranes, hyperthermia or red-coloured urine. Microscopic examination of peripheral blood smears showed

large intraerythrocytic piroplasms morphologically compatible with Babesia canis in all eight animals. DNA was extracted from

blood on filter paper, and a Babesia spp. infection confirmed by polymerase chain reaction (PCR) amplification of a 408 bp

fragment of the 18S rRNA gene. Analysis of PCR-derived sequences revealed that seven dogs were infected with B. canis canis and

one with B. canis vogeli. This is the first molecular identification report of both the species B. canis and the subspecies B. canis canis

and B. canis vogeli in dogs from Portugal.

# 2008 Elsevier B.V. All rights reserved.

Keywords: Babesia canis canis; Babesia canis vogeli; Babesiosis; Dog; Portugal; 18S rRNA gene

www.elsevier.com/locate/vetpar

Available online at www.sciencedirect.com

Veterinary Parasitology 156 (2008) 199–204

1. Introduction

Canine babesiosis (or piroplasmosis), caused by tick-

borne protozoa, represents an important veterinary

medical problem worldwide (Lobetti, 1998). Two

species have traditionally been identified as the

aetiological agents of the disease in dogs: Babesia

canis and Babesia gibsoni, which correspond to large

(3–5 mm) and small (0.5–2.5 mm) intraerythrocytic

* Corresponding author at: Department of Veterinary Sciences,

University of Tras-os-Montes e Alto Douro, P.O. Box 1013, 5001-

801 Vila Real, Portugal. Tel.: +351 259 350 458;

fax: +351 259 350 629.

E-mail address: lcardoso@utad.pt (L. Cardoso).

0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2008.05.027

parasitic forms (or piroplasms), respectively (Boozer

and Macintire, 2003). The clinical features of canine

babesiosis often include hyperthermia, anaemia, hae-

moglobinuria, lethargy and anorexia (Bourdoiseau,

2006), but clinical signs in dogs may vary depending

on the pathogen (Schetters et al., 1997) and host

immunity (Brandao et al., 2003).

The species B. canis has been subdivided into three

subspecies – B. canis canis, B. canis vogeli and B. canis

rossi – on the basis of differences in vector specificity,

geographical distribution, pathogenicity and antigenic

properties (Uilenberg et al., 1989). Characterization

with molecular methods confirmed the existence of

three distinct genotypes of B. canis, and it has

additionally been suggested that each of the subspecies

L. Cardoso et al. / Veterinary Parasitology 156 (2008) 199–204200

that infect dogs might, in fact, correspond to a

genetically distinct species (Zahler et al., 1998; Carret

et al., 1999). A large Babesia sp. that shares a high

genetic identity with Babesia bigemina from cattle was

recently reported in a dog from North Carolina

(Birkenheuer et al., 2004), and a new subspecies, B.

canis presentii, has been characterized in cats from

Israel (Baneth et al., 2004).

Babesia canis canis, transmitted by Dermacentor

reticulatus ticks, is mostly found in temperate regions of

Europe (Duh et al., 2004; Foldvari et al., 2005) and causes

haemolytic anemia and coagulation abnormalities with

variable degrees of severity (Schetters et al., 1997).

Babesia canis vogeli, transmitted by Rhipicephalus

sanguineus, has mainly been detected in tropical or

subtropical areas of northern, eastern and southern Africa

(Lobetti, 1998; Matjila et al., 2004; Oyamada et al.,

2005), Europe (Caccio et al., 2002; Criado-Fornelio et al.,

2003b), Asia (Baneth et al., 2004; Inokuma et al., 2004),

northern and central Australia (Jefferies et al., 2003), and

North and South America (Birkenheuer et al., 2003;

Passos et al., 2005). It is considered a mildly virulent

subspecies, commonly inducing moderate clinical signs

in dogs. Finally, B. canis rossi, transmitted by

Haemaphysalis leachi, is prevalent in eastern and

southern Africa (Lobetti, 1998; Oyamada et al., 2005)

and causes a severe and often fatal haemolytic syndrome,

notoriously being the most virulent of these three

subspecies (Boozer and Macintire, 2003).

In addition to B. gibsoni, a small Babesia sp. reported

from five continents (Lobetti, 1998; Muhlnickel et al.,

2002; Criado-Fornelio et al., 2003a; Inokuma et al.,

2004; Birkenheuer et al., 2005), other genetically

distinct small piroplasms capable of infecting dogs have

been described. These include Theileria annae, closely

related to Babesia microti and endemic in northwestern

Spain (Zahler et al., 2000a; Camacho et al., 2001) and

Babesia conradae, from southern California (Kjemtrup

et al., 2000; Zahler et al., 2000b).

As variations in pathogenesis and clinical manifes-

tations are known to exist between species and

subspecies of the canine piroplasms, knowledge of

their distribution is important for the therapy against

canine babesiosis and also has potential implications

for vaccine development (Uilenberg et al., 1989;

Schetters, 2005). The diagnosis of infections with

Babesia spp. is usually based on the size and

morphology of the intraerythrocytic piroplasms

observed in peripheral blood smears, but the similarity

between species and subspecies has been a limiting

factor (Kjemtrup et al., 2000; Jefferies et al., 2007).

Serology does not definitively discriminate species and

subspecies, as antibodies to Babesia spp. are often

cross-reactive (Birkenheuer et al., 2003). Molecular

techniques, including polymerase chain reaction (PCR)

and sequence analysis, represent an objective and

precise method of species identification and have been

used for the individual diagnosis and epidemiological

studies of canine Babesia infections (Zahler et al.,

2000a,b; Caccio et al., 2002; Inokuma et al., 2003).

Canine babesiosis caused by large piroplasms is

known to occur in Portugal (Diz-Lopes et al., 2005), but

no data on the genetic characterization of Babesia

species or subspecies has been reported from dogs. In

the present study, we describe the molecular analysis of

parasites from several cases of canine babesiosis

detected in northern Portugal.

2. Materials and methods

2.1. Animals and samples

Between October and December 2006, eight domes-

tic dogs living in or close to the city of Chaves

(4184403500N, 782801700W), northern Portugal, were

tested for the presence of babesial parasites. Routine

physical examination had previously revealed clinical

signs compatible with babesiosis, including lethargy,

muscle tremors, weight loss, pale mucous membranes,

hyperthermia, or red-coloured urine. These suspected

animals were of different breeds, both genders, aged

from 12 to 36 months (Table 1), and each of them had

access to outdoors as reported by their owners. No

history of travelling outside Portugal was recorded for

any of the eight dogs. Samples of peripheral blood from

the ear tip were collected from each animal to prepare

thin smears and to assess haematocrit. The slide smears

were air-dried, fixed with methanol, Giemsa-stained

and then examined under light microscopy (magnifica-

tion 1000�) for detection of possible intraerythrocytic

piroplasms. Peripheral blood was also spotted onto

individual filter papers allowed to air-dry and stored at

�20 8C until use.

2.2. DNA extraction

Blood samples spotted onto the filter paper,

corresponding to approximately 20 ml of fluid, were

cut out by use of individual sterile scalpel blades and put

into sterile tubes for DNA extraction (Strauss-Ayali

et al., 2004). DNA was extracted by adding 300 ml

of lysis buffer [50 mM NaCl, 50 mM Tris, 10 mM

EDTA (pH 8.0)], proteinase K to a final concentration

of 250 mg/ml and Triton X-100 (20%) to a final

L. Cardoso et al. / Veterinary Parasitology 156 (2008) 199–204 201

Table 1

Physical examination, haematocrit and level of parasitemia in eight dogs from northern Portugal infected with Babesia spp. by Giemsa-stained smear

microscopy and PCR with further characterization of infective subspecies by sequence analysis

Dog Breed Gender Age

(months)

Clinical signs HCT (%) Observed

parasitemia

PCR for

Babesia

Subspecies (% relatedness; closest

GenBank accession number)

1 Mongrel M 36 Lethargy, hyperthermia, RU 33 ++ + B. canis canis (99.0; AY321119)

4 Pointer M 24 Muscle tremors, weight loss, RU 20 +++ + B. canis canis (99.0; AY321119)

5 Mongrel M 17 PMM, hyperthermia, RU 20 + + B. canis canis (100; AY648872)

7 Rottweiler M 20 PMM, RU 20 + + B. canis canis (100; DQ181653)

8 Mongrel F 12 Yellow mucous membranes, RU 15 + + B. canis canis (99.0; DQ181653)

9 Podengo F 12 Lethargy, PMM, hyperthermia 35 + + B. canis vogeli (100; AY371197)

11 ND M 30 Hyperthermia, RU 38 + + B. canis canis (99.0; DQ181653)

12 Podengo ND 24 PMM, hyperthermia, RU 28 ++ + B. canis canis (100; DQ181653)

HCT: haematocrit (normal range: 37–55%); M: male; F: female; ND: not determined; RU: red urine; PMM: pale mucous membranes.

concentration of 1%. Following a 2 h incubation at

56 8C and an inactivation of proteinase K at 90 8C for

10 min, 300 ml of phenol (75%), chloroform (24%) and

isoamylalcohol (1%) mixture were added, vortexed and

centrifuged (12,000 � g) for 4 min. The supernatant

was collected and 300 ml of a mixture of phenol (50%),

chloroform (48%) and isoamylalcohol (2%) were

added, vortexed and centrifuged (12,000 � g) for

4 min. The supernatant was collected and 300 ml of a

mixture of chloroform (96%) and isoamylalcohol (4%)

were added, vortexed and centrifuged (12,000 � g) for

4 min. The supernatant was collected, and 1:10 volume

of Na-acetate (3 M) and 1 volume of ice cold 100%

isopropanol (�20 8C) were added and incubated over

night at �20 8C. Following centrifugation (14,000 � g)

at 4 8C for 30 min, the supernatant was discarded and

the pellet was washed with 150 ml of ethanol (75%,

�20 8C) and centrifuged (13,000 � g, 4 8C) for 15 min.

The supernatant was discarded and the pellet was air-

dried. The DNAwas then hydrated with 30 ml of ddH2O

for 1 h at 50 8C.

2.3. PCR

Primers PIRO-A (50-AAT ACC CAA TCC TGA

CAC AGG G-30) and Piro-B (50-TTA AAT ACG AAT

GCC CCC AAC-30) were used to amplify a 408 bp

fragment of the 18S rRNA gene of Babesia spp. by

PCR (Olmeda et al., 1997). Amplification was done

under the following conditions: 94 8C for 1 min

followed by 39 cycles of 94 8C for 45 s, 62 8C for

45 s, and 72 8C for 45 s.

2.4. Sequence analysis

The PCR DNA products were sequenced using

BigDye1 Terminator v3.1 Cycle Sequencing Kit

(PerkinElmer, Applied Biosystems Divisions, Foster

City, CA) and ABI PRISM 3100 Genetic Analyzer

(Applied Biosystems Divisions, Foster City, CA) at the

Hylabs laboratories (Rehovot, Israel) according to the

recommendations of the manufacturer. Obtained

sequences were evaluated with ChromasPro software

version 1.33 and compared to sequence data available

from GenBank using the BLAST 2.2.9 program (http://

www.ncbi.nlm.nih.gov/BLAST/).

3. Results

Intraerythrocytic pear-shaped parasitic forms 3–

5 mm long were detected in Giemsa-stained blood

smears from all eight clinically suspected dogs (Fig. 1).

These large piroplasms mainly occurred in pairs inside

red blood cells and were morphologically compatible

with B. canis.

Results concerning physical examination, haemato-

crit, level of parasitemia, PCR for Babesia spp. and

subspecies relatedness of the infected dogs are

presented in Table 1. Red-coloured urine, hyperthermia

and pale mucous membranes were frequent clinical

signs, found in seven, five and four animals, respec-

tively. Seven out of the eight dogs with babesiosis were

anemic with a haematocrit value below the normal

range (37–55%). Dogs number 5, 7, 8 and 9 were

reported by the owners to have had ticks in the past, but

no external parasites could be identified at the time of

consultation.

The eight animals found to be infected by blood

smear microscopy were confirmed as positive by PCR

specific for Babesia spp. Further analysis of the partial

18S rRNA gene sequences determined seven dogs

infected with B. canis canis (99–100% relatedness to

the GenBank closest sequence) and one with B. canis

vogeli (100% relatedness).

L. Cardoso et al. / Veterinary Parasitology 156 (2008) 199–204202

Fig. 1. Peripheral blood smear showing pairs of intraerythrocytic large piroplasms compatible with Babesia canis (filled arrows) and phagocytosis of

infected erythrocytes by a mononuclear cell (open arrows). These piroplasms were further characterized as B. canis canis by DNA sequence analysis.

Giemsa; bar = 5 mm.

4. Discussion

The diagnosis of Babesia spp. infections is often based

on intraerythrocytic piroplasm observation in peripheral

blood smears. However, this method does not allow

differentiation between morphologically similar strains,

subspecies or species (Jefferies et al., 2003). In addition,

with antigenic cross-reactivity between species,

serology-based diagnosis lacks specificity (Yamane

et al., 1993; Birkenheuer et al., 2003). PCR represents

a great advantage over microscopical detection due to its

high sensitivity and specificity (Inokuma et al., 2004).

Furthermore, the phylogenetic analysis of DNA

sequences derived from the amplification of the 18S

rRNA gene provides strain characterization, including

that of subspecies B. canis canis, B. canis vogeli and B.

canis rossi (Zahler et al., 1998; Carret et al., 1999). The

collection of blood on filter paper facilitates convenient

sample storage and transport to the diagnostic laboratory

(Jefferies et al., 2007).

The present study was carried out to characterize, by

means of molecular methods, the aetiological agents of

babesiosis in dogs from northern Portugal. The PCR

primers used were not species-specific (Olmeda et al.,

1997; Foldvari et al., 2005); however, subsequent

sequence analysis of PCR products showed that seven

dogs were infected with B. canis canis and one with B.

canis vogeli.

This is the first report of the molecular identification

of both the species (B. canis) and subspecies (B. canis

canis and B. canis vogeli) of babesial parasites in

naturally infected dogs from Portugal. On the basis of

size and morphology of intraerythrocytic piroplasms, it

has been demonstrated that the agents of canine

babesiosis in northern Portugal comprised large

babesial parasites (Diz-Lopes et al., 2005). Based on

presumed vector specificity and clinical aspects, it was

previously assumed that B. canis could be the species

causing disease in dogs. However, identification and

molecular characterization at the subspecies level were

not available.

The presence of B. canis canis and B. canis vogeli in

northern Portugal is supported by the geographical

distribution of their vector ticks. In fact, D. reticulatus

and R. sanguineus, the vectors of B. canis canis and B.

canis vogeli, respectively, have been found to be

abundant in the Montesinho Natural Park (Santos-Silva

et al., 2006), which is contiguous to the municipality of

Chaves where the dogs were sampled. These facts

strongly suggest the autochthonous nature of B. canis

canis and B. canis vogeli infection among dogs in

northern Portugal, with D. reticulatus and R. sanguineus

as their probable vectors. However, additional studies

are essential to confirm the vector candidates and

evaluate endemicity levels.

Infections with Babesia spp. can range from an

asymptomatic or mild clinical condition to severe

disease depending on the virulence of the infecting

protozoan and the susceptibility of the individual host.

The fact that B. canis canis was more frequently

L. Cardoso et al. / Veterinary Parasitology 156 (2008) 199–204 203

detected may be due to its more virulent nature or to a

higher prevalence of infected tick vectors. Dogs

infected with B. canis canis would potentially be

brought in more often for veterinary consultation,

because of the severity of clinical presentation as

compared with the relatively mild signs of B. canis

vogeli infection.

The different subspecies of B. canis have been

described to cause somewhat diverse clinical manifes-

tations, and this finding may have implications for

treatment and vaccination (Uilenberg et al., 1989;

Matjila et al., 2004; Schetters, 2005).

In the present study, most of the dogs infected either

with B. canis canis or B. canis vogeli showed clinical

abnormalities such as low haematocrit, hyperthermia

and red urine. Although data from a complete

assessment are not available, a clear correlation could

not be drawn between B. canis subspecies and clinical

signs in those animals diagnosed with each of the two

subspecies.

In Europe, canine babesiosis has been found to be

caused mainly by B. canis canis and B. canis vogeli

(Caccio et al., 2002; Criado-Fornelio et al., 2003b; Duh

et al., 2004; Foldvari et al., 2005; Gulanber et al., 2006),

and cases due to small species are more rarely reported.

However, T. annae causes severe illness in dogs and is

endemic in Galicia, northwestern Spain (Camacho

et al., 2001), which borders the area of Portugal where

the present study was carried out. This small piroplasm

has Ixodes hexagonus as its main candidate vector

(Camacho et al., 2003), a tick species which has

also been found in Portugal (Caeiro, 1999). To our

knowledge there are no reports of T. annae infection in

dogs from Portugal, but this might change in the future.

Indeed, the increasing mobility of dogs and the

existence of competent vectors may allow piroplasms

to spread into previously non-endemic areas (Caccio

et al., 2002).

In conclusion, studies on the prevalence of B. canis

canis and B. canis vogeli in larger dog populations in

Portugal, consisting of both symptomatic and asympto-

matic animals, are necessary as well as studies in ticks.

These are important in order to define endemic areas

and to promote effective control measures against

canine babesiosis.

References

Baneth, G., Kenny, M.J., Tasker, S., Anug, Y., Shkap, V., Levy, A.,

Shaw, S.E., 2004. Infection with a proposed new subspecies of

Babesia canis, Babesia canis subsp. presentii, in domestic cats. J.

Clin. Microbiol. 42, 99–105.

Birkenheuer, A.J., Correa, M.T., Levy, M.G., Breitschwerdt, E.B.,

2005. Geographic distribution of babesiosis among dogs in the

United States and association with dog bites: 150 cases (2000–

2003). J. Am. Vet. Med. Assoc. 227, 942–947.

Birkenheuer, A.J., Levy, M.G., Stebbins, M., Poore, M., Breitsch-

werdt, E., 2003. Serosurvey of antiBabesia antibodies in stray

dogs and American pit bull terriers from North Carolina. J. Am.

Anim. Hosp. Assoc. 39, 551–557.

Birkenheuer, A.J., Neel, J., Ruslander, D., Levy, M.G., Breitschwerdt,

E.B., 2004. Detection and molecular characterization of a novel

large Babesia species in a dog. Vet. Parasitol. 124, 151–160.

Boozer, A.L., Macintire, D.K., 2003. Canine babesiosis. Vet. Clin.

Small Anim. 33, 885–904.

Bourdoiseau, G., 2006. Canine babesiosis in France. Vet. Parasitol.

138, 118–125.

Brandao, L.P., Hagiwara, M.K., Myiashiro, S.I., 2003. Humoral

immunity and reinfection resistance in dogs experimentally inocu-

lated with Babesia canis and either treated or untreated with

imidocarb dipropionate. Vet. Parasitol. 114, 253–265.

Caccio, S.M., Antunovic, B., Moretti, A., Mangili, V., Marinculic, A.,

Baric, R.R., Slemenda, S.B., Pieniazek, N.J., 2002. Molecular

characterisation of Babesia canis canis and Babesia canis vogeli

from naturally infected European dogs. Vet. Parasitol. 106, 285–

292.

Caeiro, V., 1999. General review of tick species present in Portugal.

Parassitologia 41 (Suppl. 1), 11–15.

Camacho, A.T., Pallas, E., Gestal, J.J., Guitian, F.J., Olmeda, A.S.,

Goethert, H.K., Telford, S.R., 2001. Infection of dogs in north-

west Spain with a Babesia microti-like agent. Vet. Rec. 149, 552–

555.

Camacho, A.T., Pallas, E., Gestal, J.J., Guitian, F.J., Olmeda, A.S.,

Telford III, S.R., Spielman, A., 2003. Ixodes hexagonus is the main

candidate as vector of Theileria annae in northwest Spain. Vet.

Parasitol. 112, 157–163.

Carret, C., Walas, F., Carcy, B., Grande, N., Precigout, E., Moubri, K.,

Schetters, T.P., Gorenflot, A., 1999. Babesia canis canis, Babesia

canis vogeli, Babesia canis rossi: differentiation of the three

subspecies by a restriction fragment length polymorphism analysis

on amplified small subunit ribosomal RNA genes. J. Eukaryot.

Microbiol. 46, 298–303.

Criado-Fornelio, A., Gonzalez-del-Rıo, M.A., Buling-Sarana, A.,

Barba-Carretero, J.C., 2003a. Molecular characterization of a

Babesia gibsoni isolate from a Spanish dog. Vet. Parasitol. 117,

123–129.

Criado-Fornelio, A., Martinez-Marcos, A., Buling-Sarana, A., Barba-

Carretero, J.C., 2003b. Molecular studies on Babesia, Theileria

and Hepatozoon in southern Europe. Part I. Epizootiological

aspects. Vet. Parasitol. 113, 189–201.

Diz-Lopes, D., Rodrigues, F.T., Alves, S., Santos-Silva, M., 2005.

Babesiose em Cao de Gado Transmontano. Vet. Tec. 11 (Set/Out),

36.

Duh, D., Tozon, N., Petrovec, M., Strasek, K., Avsic-Zupanc, T., 2004.

Canine babesiosis in Slovenia: molecular evidence of Babesia

canis canis and Babesia canis vogeli. Vet. Res. 35, 363–368.

Foldvari, G., Hell, E., Farkas, R., 2005. Babesia canis canis in dogs

from Hungary: detection by PCR and sequencing. Vet. Parasitol.

127, 221–226.

Gulanber, A., Gorenflot, A., Schetters, T.P.M., Carcy, B., 2006. First

molecular diagnosis of Babesia vogeli in domestic dogs from

Turkey. Vet. Parasitol. 139, 224–230.

Inokuma, H., Yoshizaki, Y., Matsumoto, K., Okuda, M., Onishi, T.,

Nakagome, K., Kosugi, R., Hirakawa, M., 2004. Molecular survey

L. Cardoso et al. / Veterinary Parasitology 156 (2008) 199–204204

of Babesia infection in dogs in Okinawa, Japan. Vet. Parasitol.

121, 341–346.

Inokuma, H., Yoshizaki, Y., Shimada, Y., Sakata, Y., Okuda, M.,

Onishi, T., 2003. Epidemiological survey of Babesia species in

Japan performed with specimens from ticks collected from dogs

and detection of new Babesia DNA closely related to Babesia

odocoilei and Babesia divergens DNA. J. Clin. Microbiol. 41,

3494–3498.

Jefferies, R., Ryan, U.M., Irwin, P.J., 2007. PCR-RFLP for the

detection and differentiation of the canine piroplasm species

and its use with filter paper-based technologies. Vet. Parasitol.

144, 20–27.

Jefferies, R., Ryan, U.M., Muhlnickel, C.J., Irwin, P.J., 2003.

Two species of canine Babesia in Australia: detection and

characterization by PCR. J. Parasitol. 89, 409–412.

Kjemtrup, A.M., Kocan, A.A., Whitworth, L., Meinkoth, J., Birken-

heuer, A.J., Cummings, J., Boudreaux, M.K., Stockham, S.L.,

Irizarry-Rovira, A., Conrad, P.A., 2000. There are at least three

genetically distinct small piroplasms from dogs. Int. J. Parasitol.

30, 1501–1505.

Lobetti, R.G., 1998. Canine babesiosis. Compend. Cont. Educ. Pract.

Vet. 20, 418–431.

Matjila, T.P., Penzhorn, B.L., Bekker, C.P.J., Nijhof, A.M., Jongejan,

F., 2004. Confirmation of occurrence of Babesia canis vogeli in

domestic dogs in South Africa. Vet. Parasitol. 122, 119–125.

Muhlnickel, C.J., Jefferies, R., Morgan-Ryan, U.M., Irwin, P.J., 2002.

Babesia gibsoni infection in 3 dogs in Victoria. Aust. Vet. J. 80,

606–610.

Olmeda, A.S., Armstrong, P.M., Rosenthal, B.M., Valladares, B., del

Castillo, A., de Armas, F., Miguelez, M., Gonzalez, A., Rodriguez,

J.A., Spielman, A., Telford, S.R., 1997. A subtropical case of

human babesiosis. Acta Trop. 67, 229–234.

Oyamada, M., Davoust, B., Boni, M., Dereure, J., Bucheton, B.,

Hammad, A., Itamoto, K., Okuda, M., Inokuma, H., 2005. Detec-

tion of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis

in dogs in a village of eastern Sudan by using a screening PCR and

sequencing methodologies. Clin. Diagn. Lab. Immunol. 12, 1343–

1346.

Passos, L.M.F., Geiger, S.M., Ribeiro, M.F.B., Pfister, K., Zahler-

Rinder, M., 2005. First molecular detection of Babesia vogeli in

dogs from Brazil. Vet. Parasitol. 127, 81–85.

Santos-Silva, M., Sousa, R., Santos, A.S., Lopes, D., Queijo, E.,

Doreta, B., Vitorino, L., Bacellar, F., 2006. Ticks and tick-borne

rickettsiae surveillance in Montesinho Natural Park, Portugal.

Ann. N. Y. Acad. Sci. 1078, 137–142.

Schetters, T., 2005. Vaccination against canine babesiosis. Trends

Parasitol. 21, 179–184.

Schetters, T.M.P., Moubri, K., Precigout, E., Kleuskens, J., Scholtes,

N.C., Gorenflot, A., 1997. Different Babesia canis isolates, dif-

ferent diseases. Parasitology 115, 485–493.

Strauss-Ayali, D., Jaffe, C.L., Burshtain, O., Gonen, L., Baneth, G.,

2004. Polymerase chain reaction using noninvasively obtained

samples, for the detection of Leishmania infantum DNA in dogs. J.

Infect. Dis. 189, 1729–1733.

Uilenberg, G., Franssen, F.F.J., Perie, M., Spanjer, A.M.M., 1989.

Three groups of Babesia canis distinguished and a proposal for

nomenclature. Vet. Q. 11, 33–40.

Yamane, I., Thomford, J.W., Gardner, I.A., Dubey, J.P., Levy, M.,

Conrad, P.A., 1993. Evaluation of the indirect fluorescent antibody

test for diagnosis of Babesia gibsoni infections in dogs. Am. J. Vet.

Res. 54, 1579–1584.

Zahler, M., Rinder, H., Schein, E., Gothe, R., 2000a. Detection of a

new pathogenic Babesia microti-like species in dogs. Vet. Para-

sitol. 89, 241–248.

Zahler, M., Rinder, H., Zweygarth, E., Fukata, T., Maede, Y., Schein,

H., Gothe, R., 2000b. ‘Babesia gibsoni’ of dogs from North

America and Asia belong to different species. Parasitology 120,

365–369.

Zahler, M., Schein, E., Rinder, H., Gothe, R., 1998. Characteristic

genotypes discriminate between Babesia canis isolates of differ-

ing vector specificity and pathogenicity to dogs. Parasitol. Res. 84,

544–548.