Serologic and molecular characterization of

Anaplasma species infection in farm

animals and ticks from Sicily§

Jose de la Fuente a,b,*, Alessandra Torina c, Santo Caracappa c, Giovanni Tumino c,Roberto Furla d, Consuelo Almazan a, Katherine M. Kocan a

a Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University,

250 McElory Hall, Stillwater, OK 74078, USAb Instituto de Investigacion en Recursos Cinegeticos IREC (CSIC-UCLM-JCCM),

Ronda de Toledo s/n, 13005 Ciudad Real, Spainc Istituto Zooprofilattico Sperimentale della Sicilia, Via G. Marinuzzi no. 3, 90129 Palermo, Italy

d Azienda Unita Sanitaria, Locale no. 7, Ragusa, Italy

Received 25 August 2004; received in revised form 20 March 2005; accepted 28 May 2005

Abstract

Although Anaplasma marginale was known to be endemic in Italy, the diversity of Anaplasma spp. from this area have not

been characterized. In this study, the prevalence of Anaplasma spp. antibodies in randomly selected farm animals collected on

the island of Sicily was determined by use of a MSP5 cELISA for Anaplasma spp. and an immunofluorescence test specific for

Anaplasma phagocytophilum. Genetic variation among strains of Anaplasma spp. from animals and ticks was characterized

using the A. marginale msp1a and the Anaplasma spp. msp4 genes. Eight species of ticks were collected and tested by PCR.

Seropositivity for Anaplasma spp. and A. phagocytophilum was detected in bovine and ovine samples. All the donkeys were

seropositive for A. phagocytophilum but not for Anaplasma spp. Four A. marginale genotypes were identified by msp4 sequences

from bovine and tick samples. Two new genotypes of Anaplasma ovis were characterized in sheep. The sequences of A.

phagocytophilum from three donkeys proved to be identical to the sequence of the MRK equine isolate from California. Six A.

marginale genotypes were found in cattle and one tick using the A. marginale msp1a sequences. All genotypes had four repeated

sequences in the N-terminal portion of the MSP1a, except for one that had five repeats. The Italian strains of A. marginale

contained three repeat sequences that were not reported previously. Definition of the diversity of Anaplasma spp. in Sicily

reported, herein is fundamental to development of control strategies for A. marginale, A. ovis and A. phagocytophilum in Sicily.

# 2005 Published by Elsevier B.V.

Keywords: Anaplasmosis; A. marginale; A. phagocytophilum; A. ovis

www.elsevier.com/locate/vetpar

Veterinary Parasitology 133 (2005) 357–362

§ The GenBank accession numbers for msp4 sequences of A. marginale, A. ovis and A. phagocytophilum strains are AY702917–AY702925

and for A. marginale msp1a are AY702926–AY702932.

* Corresponding author. Tel.: +1 405 744 0372; fax: +1 405 744 5275.

E-mail address: jose_delafuente@yahoo.com (J. de la Fuente).

0304-4017/$ – see front matter # 2005 Published by Elsevier B.V.

doi:10.1016/j.vetpar.2005.05.063

J. de la Fuente et al. / Veterinary Parasitology 133 (2005) 357–362358

1. Introduction

The genus Anaplasma (Rickettsiales: Anaplasma-

taceae) includes three species that infect ruminants:

Anaplasma marginale (the type species), Anaplasma

centrale and Anaplasma ovis (reviewed by Kocan

et al., 2003). Bovine anaplasmosis is caused primarily

by A. marginale while A. ovis is a pathogen of sheep

and is not infectious for cattle. A. centrale, a less

pathogenic organism, is used as a live vaccine for

cattle in Israel, South Africa, South America and

Australia. As the result of a recent reclassification of

the Anaplasmataceae, the genus Anaplasma now also

includes Anaplasma phagocytophilum (formerly Ehr-

lichia equi, Ehrlichia phagocytophila and the HGE

agent of human granulocytic ehrlichiosis, now

recognized as synonymous), which causes a febrile

disease in ruminants, and human, equine and canine

granulocytic anaplasmosis (Dumler et al., 2001).

Ticks are biological vectors of Anaplasma spp., and

mammalian, bird or tick hosts with persistent

Anaplasma spp. infection may serve as natural

reservoirs of infection (reviewed by Dumler et al.,

2001; Kocan et al., 2003; Rikihisa, 2003).

Many geographic strains of A. marginale and A.

phagocytophilum have been identified which differ in

biology, genetic characteristics and/or pathogenicity

(Dumler et al., 2001; Massung et al., 2000, 2002,

2003; Stuen et al., 2003; Kocan et al., 2003; Polin

et al., 2004; de la Fuente et al., 2005a,b). Genetic

diversity has not been widely characterized for A. ovis.

Recent research on Anaplasma spp. has focused on

major surface proteins (MSPs) that are involved in

interactions with vertebrate and invertebrate host cells

(Kocan et al., 2003; Rikihisa, 2003; Lin et al., 2004; de

la Fuente et al., 2005b). Selected MSPs, such as

MSP1a, MSP2 and MSP4, have been used to

characterize the genetic diversity of Anaplasma spp.

(reviewed by de la Fuente et al., 2005b). These MSPs,

involved in host–pathogen interactions, may have

evolved more rapidly than other genes because of

selective pressures exerted by the host immune

system.

A. marginale is endemic in Sicily and in other

regions of the world (reviewed by Kocan et al., 2003)

and has been described previously in Italy (Cringoli

et al., 2002; Tassi et al., 2002). However, the Italian

strains of Anaplasma spp. have not been characterized

at the molecular level. In this study, we examine the

genetic variation among strains of Anaplasma spp.

obtained from infected cattle, sheep, donkeys and

ticks in the island of Sicily in southern Italy using the

A. marginale msp1a and the Anaplasma spp. msp4

genes.

2. Materials and methods

2.1. Samples

Blood from 50 cattle, 8 sheep and 3 donkeys, plus

88 ticks, were collected in Sicily, Italy for these

studies. Blood was collected from randomly selected

farm animals mainly in the province of Palermo but

also in Trapani, Ancona and Ragusa. Ticks were

collected from cattle in the province of Palermo,

district of Carleone, and stored in 70% ethanol at room

temperature. Sixty-eight adult ticks and 20 nymphs

that were pooled from each host were collected. Ticks

were identified using morphological keys for Italian

Ixodidae (Manilla, 1998). Blood was collected into

sterile tubes with and without anticoagulant (lithium

heparin) and maintained at 4 8C until arrival at the

laboratory. Plasma and serum were then separated

after centrifugation and stored at �20 8C.

2.2. Serologic tests for detection of Anaplasma

spp

The anaplasmosis cELISAwas performed using the

Anaplasma Antibody Test Kit, cELISA from VMRD

Inc. (Pullman, WA, USA) following the manufac-

turer’s instructions. This assay detects serum anti-

bodies against the MSP5 protein of Anaplasma spp.

(Knowles et al., 1996).

The immunofluorescence test for A. phagocyto-

philum was performed using the IFA Antibody Test

Kit from Fuller Laboratories (Fullerton, CA, USA)

following the manufacturer’s instructions.

2.3. DNA extraction, PCR and sequence analysis

DNA was extracted from blood and tick samples

using the GenElute Mammalian Genomic DNA

Miniprep Kit (Sigma, St. Louis, MO, USA). The A.

marginale msp1a and the Anaplasma spp. msp4 genes

J. de la Fuente et al. / Veterinary Parasitology 133 (2005) 357–362 359

Table 1

Prevalence of antibodies to Anaplasma spp. in farm animals examined in Sicily

Serologic testa Serum sample

Bovine Ovine Equine

Positive Negative ND Positive Negative ND Positive Negative ND

Anaplasma spp. 39 (78%) 1 (2%) 10 6 (75%) 0 (0%) 2 0 (0%) 0 (0%) 0

A. phagocytophilum 13 (26%) 1 (2%) 36 2 (25%) 3 (37%) 3 3 (100%) 0 (0%) 0a The anaplasmosis cELISA was performed using the Anaplasma Antibody Test Kit, cELISA from VMRD Inc. (Pullman, WA, USA) which

detects serum antibodies against MSP5 of Anaplasma spp. An immunofluorescence test was used to detect antibodies specific against A.

phagocytophilium. ND, not determined.

were amplified by PCR and sequenced as reported

previously (de la Fuente et al., 2001, 2003, 2005a).

The msp4 coding region was completely sequenced.

Only the fragment containing the tandem repeats in

the variable region of msp1a was sequenced (de la

Fuente et al., 2001, 2003, 2005a,b).

The A. marginale msp1a variable region and the

msp4 coding region were used for sequence align-

ment. Multiple sequence alignment was performed

using the program AlignX (Vector NTI Suite V 5.5,

InforMax, North Bethesda, MD, USA) with an engine

based on the Clustal W algorithm (Thompson et al.,

1994).

Table 2

Prevalence of Anaplasma spp. infections in farm animals and ticks in

Sicily

Samplea Positive msp4 PCR

A. marginale A. phagocytophilum A. ovis

Bovine blood 25/50 (50%) 0/50 (0%) 0/50 (0%)

Ovine blood 0/8 (0%) 0/8 (0%) 7/8 (87%)

Donkey blood 0/3 (0%) 3/3 (100%) 0/3 (0%)

Ticks 5/88 (6%) 0/88 (0%) 0/88 (0%)a DNA was extracted from blood samples and ticks. A combina-

tion of msp4 PCR and sequence analysis was used to identify

pathogen DNA.

3. Results

3.1. Tick species collected from bovines

Eighty-eight ticks were collected from cattle in the

province of Palermo, Sicily. Forty (45%) were

classified as Rhipicephalus bursa, 15 (17%) as

Rhipicephalus turanicus, 11 (13%) as Haemaphysalis

punctata, 11 (13%) as Hyalomma m. marginatum, 5

(6%) as Dermacentor marginatus, 4 (4%) as

Rhipicephalus sanguineus, 1 (1%) as Hyalomma m.

lusitanicum and 1 (1%) as Ixodes ricinus. All the

nymphs collected were identified as R. bursa.

3.2. Prevalence of Anaplasma spp. antibodies in

farm animals

The prevalence of Anaplasma spp. antibodies in

selected farm animals was determined using the

MSP5 cELISA for Anaplasma spp. and an immuno-

fluorescence test specific for A. phagocytophilum

(Table 1). Except for equine samples, the number of

A. phagocytophilum seropositive samples was less than

the number of samples seropositive for Anaplasma spp.

(Table 1). Seropositivity for Anaplasma spp. and A.

phagocytophilum were detected in bovine and ovine

samples(Table1).However, threeofthesheepexamined

were positive for Anaplasma spp., but not for A.

phagocytophilum. All cattle and sheep that tested

positive for A. phagocytophilum by the immunofluor-

escent test were also positive for Anaplasma spp. by the

cELISA. All the donkeys were positive for A.

phagocytophilum but not for Anaplasma spp. (Table 1).

3.3. Prevalence and genetic characterization of

Anaplasma spp. in farm animals and ticks

The prevalence of Anaplasma spp. was analyzed by

PCR and sequence analysis of msp4 amplicons

(Table 2). A. marginale was detected in 50% and

6% of the bovine and tick samples, respectively. R.

turanicus and H. punctata were found to be infected

with A. marginale. A. phagocytophilum was detected

in all three blood samples from donkeys and the

amplicons detected in 87% of sheep samples were

identified as A. ovis (Table 2).

J. de la Fuente et al. / Veterinary Parasitology 133 (2005) 357–362360

Fig. 1. Sequence of MSP1a tandem repeats in Italian strains of A. marginale. (A) The structure of the MSP1a repeats region was represented for

the Italian strains of A. marginale using the repeated forms described in (B). (B) The one letter amino acid code was used to depict the different

sequences found in MSP1a repeats. Repeated sequences were updated after de la Fuente et al. (2004a). Asterisks indicate identical amino acids

with respect to the reference sequence of repeat A. Gaps indicate deletions/insertions.

The sequence analysis of A. marginale msp4 from six

bovine samples and two tick species resulted in

identification of four A. marginale genotypes. Three

of the bovine genotypes had sequences identical to those

found in the tick species. The A. ovis msp4 sequences

were characterized in all seven positive samples and

included two genotypes. Six genotypes had a silent

T � C mutation at position 366 (position one at A in the

translation start codon, ATG) with respect to the Idaho

reference sequence (GenBank accession number

AF393742). A single genotype was found with an

additional C � T mutation at position 470 which

resulted in an A �Vamino acid change. The sequences

of A. phagocytophilum msp4 from three positive donkey

samples were determined and were found to be identical

to the sequence of the MRK isolate obtained originally

from an infected horse in California (AY530196).

Six genotypes were found in the six samples from

cattle and one tick using the A. marginale msp1a

sequences (Fig. 1). Two bovine genotypes had

identical sequences. All genotypes had four MSP1a

repeated sequences in the N-terminal portion of the

protein, except for one bovine strain which had five

repeated sequences (Fig. 1A). The Italian strains of A.

marginale contained three repeated sequences (num-

bered 5–7; Fig. 1B) that have not been reported

previously.

4. Discussion

A. marginale and A. ovis are endemic in Sicily. The

high prevalence of seropositivity of Anaplasma spp. in

serum samples collected from cattle and sheep

correlated with the frequency of A. marginale and

A. ovis infections in these animal species.

Concurrent Anaplasma spp. infections in farm

animals or ticks were not demonstrated by PCR in this

study. However, the results of serological tests suggest

that co-infection with A. phagocytophilum and A.

marginale or A. ovis may occur in cattle and sheep,

respectively. The discrepancies between serology and

PCR results could be explained by the absence of

detectable levels of bacteremia in some samples.

Concurrent infections with these organisms have been

reported to occur both in animals and ticks (Adelson

et al., 2004; Hofmann-Lehmann et al., 2004; Lin et al.,

2004; de la Fuente et al., 2004b) and may increase the

severity of disease (Hofmann-Lehmann et al., 2004).

All tick genera identified on the island can serve as

vectors of A. marginale (reviewed by Kocan et al.,

2004). Identification of R. turanicus and H. punctata

infected with A. marginale suggests that these tick

species may be also vectors of A. marginale in Sicily.

In a previous study, de la Fuente et al. (2001) pre-

sented evidence of the co-evolution of Dermacentor

J. de la Fuente et al. / Veterinary Parasitology 133 (2005) 357–362 361

variabilis and A. marginale in the United States using

MSP1a and MSP4 sequences. The evolutionary

history of vector–pathogen interactions could be

reflected in the sequences of MSP1a, which has been

shown to be an adhesin for bovine erythrocytes and

tick cells (reviewed by Kocan et al., 2003). Therefore,

as suggested before for Latin-American strains of A.

marginale (de la Fuente et al., 2004a), vector–

pathogen interactions could influence the presence

of particular MSP1a repeat sequences in Italian strains

of A. marginale. However, mechanical transmission of

A. marginale strains not transmissible by ticks could

also play an important role in the epidemiology of

bovine anaplasmosis (reviewed by Kocan et al., 2003).

In this study, I. ricinus, the main tick vector of A.

phagocytophilum in northern Europe, was not

abundant in tick samples collected in Sicily, which

suggests that other tick species may transmit this

pathogen in Sicily. Similar findings have been

obtained by our group in central Spain (de la Fuente

et al., 2004b). A. ovis is transmitted by R. bursa, the

most abundant tick species collected in Sicily, and

probably other ticks in the Old World. However, the

identity of the vector ticks for A. ovis is uncertain in

many regions of the world (Friedhoff, 1997).

PCR and sequence analysis in this study provided

evidence of A. marginale, A. phagocytophilum and A.

ovis infection in farm animals in Sicily. msp4

sequences of equine strains of A. phagocytophilum

were genetically homogeneous and identical between

North American and Italian strains. However, Italian

strains of A. marginale, as characterized by MSP1a

and MSP4 sequences, were heterogeneous, as was

demonstrated previously in studies of A. marginale

strains from the United States (Palmer et al., 2001; de

la Fuente et al., 2003), Latin-America (de la Fuente

et al., 2002, 2004a) and Spain (de la Fuente et al.,

2004c). This heterogeneity appears to be common in

endemic areas, independent of the geographic location

and predominant tick vector. Two new A. ovis

genotypes were described in this study, based on

msp4 sequences that were different from a North

American strain reported previously, suggesting that

msp4 genotypes in A. ovis may be geographically

diverse.

Evidence of A. phagocytophilum and A. marginale

infections in cattle and sheep in Sicily is particularly

important because both pathogens could greatly

impact animal production (Stuen et al., 2002; Kocan

et al., 2004). A. phagocytophilum is also infective for

humans, and causes a human granulocytic anaplas-

mosis (Strle, 2004). Although A. ovis is wide spread in

the Old World, outbreaks occur only under particular

conditions (Friedhoff, 1997).

Definition of the diversity of Anaplasma spp. in

Sicily reported herein is fundamental to epidemiolo-

gical studies and development of control strategies for

A. marginale, A. ovis and A. phagocytophilum in

Sicily. These results suggest the need to develop

vaccines for the control of concurrent infections with

Anaplasma spp. with potential impact to animal and

human health.

Acknowledgments

This research was supported by The Ministry of

Health, Italy and the Endowed Chair for Food Animal

Research (K.M. Kocan, College of Veterinary

Medicine, Oklahoma State University, USA). Con-

suelo Almazan is supported by a grant-in-aid from the

CONACYT, Mexico and a grant from Pfizer Animal

Health Inc., Kalamazoo, Michigan, USA. We thank

Mr. S. Scimeca, Mrs. Rosalia D’Agostino and Miss

Angelica Corrente for their skilful technical assistance

during the field work.

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