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Detection of Leishmania infantum, Babesia canis, and rickettsiae in ticks removed from dogs living in Italy
Ticks and Tick-borne Diseases 3 (2012) 293–296
Contents lists available at SciVerse ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Detection of Leishmania infantum, Babesia canis, and rickettsiae in ticks removed from dogs living in Italy Michele Trotta a , Martina Nicetto a , Alessandro Fogliazza b , Fabrizio Montarsi c , Marco Caldin d , Tommaso Furlanello a , Laia Solano-Gallego a,e,∗ a
Laboratorio d’Analisi Veterinarie “San Marco” Padova, Italy Merial Italia Spa, Milano, Italy c Dipartimento di Ecopatologia, Isitituto Zooprofilattico Sperimentale delle Venezie, Italy d Clinica Veterinaria “San Marco” Padova, Italy e Department of Pathology and Infectious Diseases, Royal Veterinary College, University of London, UK b
a r t i c l e Keywords: Ticks PCR Leishmania infantum Rickettsia conorii Rickettsia massiliae Babesia canis
i n f o
a b s t r a c t The aims of this study were to determine natural infections by Anaplasma phagocytophilum/Anaplasma platys, Bartonella henselae, Ehrlichia canis, Leishmania infantum, Rickettsia spp., Babesia spp., and Hepatozoon spp. by molecular methods in ticks (n = 91) removed from dogs with clinical signs and laboratory abnormalities compatible with tick-borne diseases (n = 22) living in Italy and to assess the distribution and species of ticks encountered. Ticks from dogs living in southern Italy were all identified as Rhipicephalus sanguineus (n = 25), ticks from central Italy included Rh. sanguineus (n = 8) and Ixodes ricinus (n = 9), ticks from northern Italy included Rh. sanguineus (n = 45), Dermacentor marginatus (n = 3), and one I. ricinus. Leishmania infantum, Rickettsia spp., and Babesia canis were the only pathogens detected in 7 (8%), 4 (4%), and 2 (2%) out of 91 ticks, respectively. L. infantum was detected in I. ricinus from central Italy and in Rh. sanguineus from northern and central Italy. Rickettsia conorii and Ri. massiliae were detected in Rh. sanguineus ticks from central and southern Italy (Sicily), respectively. Bab. canis was detected in D. marginatus ticks from northern Italy. © 2012 Elsevier GmbH. All rights reserved.
Introduction Ticks are important vectors of human and animal pathogens including bacteria, viruses, and protozoa (Parola and Raoult, 2001). There has been an increasing spread of parasitic arthropods and the pathogens they transmit across the world. Dogs are infested with blood-feeding arthropods and might act as reservoir hosts of several zoonotic pathogens. The risk of exposure of dogs to numerous vector-borne pathogens transmitted by ticks has increased notably in many parts of the world including Europe, and the increasingly close relationships with humans in urban areas pose new concerns for human public health (Beugnet and Marié, 2009). In addition, the advancement in molecular techniques in the past decades has resulted in increased detection of vector-borne pathogens DNA in ticks (Sparagano et al., 1999). However, limited information is available about the rickettsial and protozoan pathogens that are detected in several species of ticks removed from dogs in Europe. The aims of this study were to evaluate natural infections with
∗ Corresponding author at: Departament de Medicina i Cirurgia Animal Facultat de Veterinaria Universitat Autonoma de Barcelona, Spain. E-mail address: [email protected] (L. Solano-Gallego). 1877-959X/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2012.10.031
vector-borne pathogens in ticks removed from dogs with clinicopathological abnormalities compatible with tick-borne diseases living in Italy and to assess the distribution and species of ticks encountered. Materials and methods A total of 91 ticks were removed from 22 dogs with clinical signs and laboratory findings compatible with tick-borne diseases from April to November 2007 from many veterinarian clinics throughout Italy (Trotta et al., 2009). The ticks were placed in tubes with 75% ethanol and stored at −20 ◦ C. Later, the ticks were morphologically identified (Iori et al., 2005). The DNA was isolated from individual ticks by using the High Pure PCR template preparation kit according to the manufacturer’s instructions with some modifications (Roche, Mannheim, Germany). The ticks were mechanically crushed with a sterile micropestle, suspended in 200 l of tissue lysis buffer and 40 l of proteinase K (100 g/ml) and incubated overnight at 65 ◦ C. The final elution volume was 100 l for each sample. The efficiency of tick DNA extraction was evaluated by amplification of the tick mitochondrial 16S rRNA gene by using tick-specific primers in a conventional PCR (Halos et al., 2004). Tick speciation based on morphological characteristics was not possible
294
M. Trotta et al. / Ticks and Tick-borne Diseases 3 (2012) 293–296
Table 1 Species of ticks removed from Italian dogs and their geographical distribution. Tick species (number of ticks)
Rhipicephalus sanguineus (78) Dermacentor marginatus (3) Ixodes ricinus (10) Total (91)
Stage of ticks
Geographical region in Italy
Adult
Male
Female
Nymph
South
Central
North
70 3 9 82
38 0 2 40
32 3 7 42
8 0 1 9
25 0 0 25
8 0 9 17
45 3 1 49
in 6 ticks (5 Rhipicephalus spp. and one Ixodes spp.) and therefore, direct sequencing from amplified 16S rRNA gene and comparison with GenBank database was performed for speciation. Real-time PCRs for Anaplasma phagocytophilum/Anaplasma platys, Ehrlichia canis, Leishmania infantum, and Rickettsia spp. were performed by using the LightCycler Instrument 1.3.1 (Roche) (Solano-Gallego et al., 2006a, 2007, 2008a). Real-time PCR for Bartonella henselae was performed by using the QuantiTect® SYBR® Green PCR Kit (Qiagen, Hilden, Germany) as previously reported (Anderson et al., 1994). Babesia spp. (Solano-Gallego et al., 2008b) and Hepatozoon spp. (Inokuma et al., 2002) conventional PCRs were also carried out. Babesia-positive samples were submitted for direct sequencing. The positive samples for Rickettsia spp. by real-time PCR were further characterized by using a conventional PCR and direct sequencing for a fragment (170 bp) of the ompA gene (SolanoGallego et al., 2006b). The positive samples for L. infantum in I. ricinus ticks and the dog from which ticks were collected were further characterized by using a nested PCR and direct sequencing for a fragment (182 bp) of the internal transcribed spacer (ITS) locus with outer primers as previously described (Schönian et al., 2003) and inner primers, ITS76: 5 -CCTTTCCCACATACACAGCA-3 and ITS258: 5 -ATCGCGACACGTTATGTGAG-3 , designed by using Primer3Plus (Untergasser et al., 2007). The sequencing reactions were performed with the Applied Biosystem 3730XI DNA Analyzer on both strands. Consensus sequences were aligned with sequences in GenBank® using the basic local alignment search tool (nBLAST). Clinical data and vector-borne pathogen serological and molecular testing from dogs with detection of vector-borne pathogens in ticks removed from them were evaluated. Serological testings were carried out as previously described (Trotta et al., 2009; Riera et al., 1999). Results The mean number of ticks collected per dog and standard deviation were 4.5 ± 4 (range of ticks per dog 1–15). The species of ticks removed from dogs and their geographical distributions are listed in Table 1. A 320-bp fragment of the tick mitochondrial 16S rRNA
gene was amplified in all DNA tick samples. The species identification performed by direct sequencing allowed the identification of 5 Rhipicephalus sanguineus (Rh. sanguineus) and one I. ricinus. The vector-borne pathogens detected in ticks and their geographical distributions are displayed in Table 2. L. infantum, Rickettsia spp., and Bab. canis were the only pathogens detected in 7 (8%), 4 (4%), and 2 (2%) out of 91 ticks, respectively. One out of 73 (1%) and 3 out of 73 (4%) Rh. sanguineus ticks were positive for Rickettsia conorii (Ri. conorii) and Ri. massiliae, respectively. Five out of 9 I. ricinus (55%) and 2 out of 73 Rh. sanguineus (3%) ticks were positive for L. infantum, and 2 out of 3 Dermacentor marginatus (67%) ticks were positive for Bab. canis. The clinical data and the serological and molecular findings in dogs and on vector-borne pathogens in ticks removed from them are described in Table 3. Discussion In the present study L. infantum DNA was detected in I. ricinus and in Rh. sanguineus ticks. To our knowledge, this is the first report of L. infantum DNA in I. ricinus ticks. Moreover, these ticks were collected from a dog with clinical leishmaniosis based on the clinical findings, with a high antileishmanial antibody level, and a positive PCR of the blood. In agreement with the present study, L. infantum DNA was detected in Rh. sanguineus ticks from Brazil and Italy (Dantas-Torres et al., 2010) and in ticks from Brazil (Colombo et al., 2011) collected from L. infantum-seropositive dogs, although in the present study, some L. infantum DNA was detected for the first time in Rh. sanguineus ticks from dogs that were seronegative and PCRnegative. The proportion of L. infantum-positive Rh. sanguineus ticks were lower, but similar to those described in Brazil (Dantas-Torres et al., 2010). Transstadial and transovarial transmission of L. infantum in Rh. sanguineus ticks have been demonstrated (Dantas-Torres, 2011; Dantas-Torres et al., 2011), and Leishmania DNA has been detected in the salivary glands of ticks (Dantas-Torres et al., 2010). However, evidence of transmission of Leishmania infection by tick bite was reported only in 2 dogs (McKenzie, 1984; Dantas-Torres, 2011). Therefore, the significance of the present findings and the role of transmission of Leishmania infection to dogs by ticks remain
Table 2 Vector-borne pathogens detected in ticks in this study. Dog ID
Tick species
Gender of ticks
Pathogens
Geographical region in Italy
105A1 10H 86D 86D 86D 86D 86D 24A 25A 105A2 107A1 107A4 108A3
Rhipicephalus sanguineus Rh. sanguineus Ixodes ricinus I. ricinus I. ricinus I. ricinus I. ricinus Dermacentor marginatus D. marginatus Rh. sanguineus Rh. sanguineus Rh. sanguineus Rh. sanguineus
Male Male Engorged female Female Female Female Female Female Female Male Engorged female Engorged female Female
Leishmania infantum L. infantum L. infantum L. infantum L. infantum L. infantum a L. infantum Babesia canisa Bab. canisa Rickettsia conoriia Ri. massiliaea Ri. massiliaea Ri. massiliaea
Central (Rome) North Central Central Central Central Central North North Central (Rome) South (Sicily) South (Sicily) South (Sicily)
a Species identification by direct sequencing. 100% identity with GenBank® sequences of Ri. massiliae (HM014444), Ri. conorii (HM050291), L. infantum (FJ555210), and Bab. canis (DQ181653).
M. Trotta et al. / Ticks and Tick-borne Diseases 3 (2012) 293–296
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Table 3 Clinical data and vector-borne pathogen testing in dogs and detection of vector-borne pathogens in the ticks collected from dogs. Dog ID and signalment
Species of tick/pathogen detected
Vector-borne pathogen testing
Clinical signs and laboratory abnormalities 1st visit
Serology
Positive PCR
Antigen
1st visit
2nd visit
#107A mixed breed, male, 2 years
Rh. sanguineus/Ri. massiliae
A. phagocytophilum E. canis L. infantum Ri. conorii
NEG 1:320 NEG 1:320
Not available
None
Pyoderma
#105A mixed breed, female, 1 year
Rh. sanguineus/Ri. conorii Rh. sanguineus/L. infantum
A. phagocytophilum
NEG
Not available
A. platys
Anorexia, lethargy, hyperthermia
E. canis
NEG
L. infantum Ri. conorii
NEG 1:5120
#108A mixed breed, female, 4 years
Rh. sanguineus/Ri. massiliae
A. phagocytophilum E. canis L. infantum Ri. conorii
1:320 NEG NEG 1:1280
Not available
None
Anorexia, lethargy, hyperthermia, dyspnoea
#25A female, Labrador retriever, 5 years
D. marginatus/Bab. canis
A. phagocytophilum E. canis L. infantum Ri. conorii
NEG NEG NEG NEG
NEG NEG NEG NEG
Bab. canis
Anorexia, lethargy, pigmenturia, hyperthermia, thrombocytopenia, leukopenia, large-form Babesia in erythrocytes (babesiosis), improved after therapy with imidocarb dipropionate and doxycycline
#24A mixed breed, male 4 years
D. marginatus/Bab. Canis
A. phagocytophilum E. canis L. infantum Ri. conorii
1:80 1:160 NEG 1:320
Not available
Bab. canis
Hyperthermia, lethargy, tonsillitis, thrombocytopenia, neutrophilia, large-form Babesia in erythrocytes (babesiosis), improved after therapy with imidocarb dipropionate and doxycycline
#10H mixed breed
Rh. sanguineus/L. infantum
A. phagocytophilum E. canis L. infantum Ri. conorii
1:80 NEG NEG NEG
NEG 1:80 NEG 1:80
None
Not available
#86D mixed breed, female, 6 years
I. ricinus/L. infantuma
A. phagocytophilum E. canis L. infantum Ri. conorii
1:1280 1:320 HIGH 1:160
Not available
L. infantuma
Pale mucous membranes, conjunctivitis
a
L. infantum sequences were identical in the tick and in canine blood.
unclear (Coutinho et al., 2005; Dantas-Torres, 2011). Further studies need to elucidate the potential of Leishmania transmission to dogs via ticks. Ri. conorii is the classical agent of Mediterranean spotted fever (Raoult and Roux, 1997), and Ri. massiliae is an emerging pathogen described for the first time in a human patient from Sicily (Vitale et al., 2006) and first isolated from a Rh. sanguineus tick in Marseille (Beati and Raoult, 1993). Thereafter, it was detected in ticks in other Mediterranean regions including Sardinia (Márquez et al., 2008; Mura et al., 2008; Harrus et al., 2010). The present study demonstrated the detection of Ri. conorii (1%) and Ri. massiliae (4%) in Rh. sanguineus ticks collected from dogs living in central and southern Italy, respectively. The low rates of Ri. conorii infection in Rh. sanguineus ticks in the present study are similar to those reported from several countries in the Mediterranean basin (Parola et al., 2005; Psaroulaki et al., 2006), whilst the Ri. massiliae rate of infection in ticks was slightly lower than in previous studies performed in Algeria, Spain, and Israel (Bitam et al., 2006; Márquez et al., 2008; Harrus et al., 2010) and similar to previous studies from Greece and Morocco (Psaroulaki et al. 2006; Sarih et al., 2008). In addition, to the author’s best knowledge, the detection of Ri. massiliae in ticks has not been previously described from Sicily. Interestingly, despite the high seroprevalence described in Italian dogs with rickettsial disease including Rickettsia, Ehrlichia, and Anaplasma in another study (Trotta et al., 2009), only a low number of ticks were positive for Rickettsia spp. in the present study.
In the present study, Bab. canis DNA was detected in 2 D. marginatus ticks from northern Italy collected from 2 dogs with babesiosis infected with the same species. This tick species is considered the main vector for Bab. canis in Italy (Cassini et al., 2009). In conclusion, the most prevalent ticks found on dogs were Rh. sanguineus, and they were present throughout the country. D. marginatus and I. ricinus ticks were only found in central and northern Italy. The pathogens detected in the ticks were L. infantum, Ri. massiliae, Ri. conorii, and Bab. canis, and their geographical distributions were correlated with those of human and canine vector-borne diseases in Italy. Our results demonstrated the first detection of Ri. massiliae, an emerging human pathogen, in Rh. sanguineus ticks from Sicily and the first detection of L. infantum in I. ricinus ticks removed from Italian dogs. References Anderson, B., Sims, K., Regnery, R., Robinson, L., Schmidt, M.J., Goral, S., Hager, C., Edwards, K., 1994. Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR. J. Clin. Microbiol. 32, 942–948. Beati, L., Raoult, L., 1993. Rickettsia massiliae sp. nov., a new spotted fever group rickettsia. Int. J. Syst. Bacteriol. 43, 839–840. Beugnet, F., Marié, J.L., 2009. Emerging arthropod-borne diseases of companion animals in Europe. Vet. Parasitol. 163, 298–305. Bitam, I., Parola, P., Matsumoto, K., Rolain, J.M., Baziz, B., Boubidi, S.C., Harrat, Z., Belkaid, M., Raoult, D., 2006. First molecular detection of R. conorii, R. aeschlimannii, and R. massiliae in ticks from Algeria. Ann. N. Y. Acad. Sci. 1078, 368–372. Cassini, R., Zanutto, S., Frangipane di Regalbono, A., Gabrielli, S., Calderini, P., Moretti, A., Tampieri, M.P., Pietrobelli, M., 2009. Canine piroplasmosis in Italy: epidemiological aspects in vertebrate and invertebrate hosts. Vet. Parasitol. 165, 30–35.
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Contents lists available at SciVerse ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Detection of Leishmania infantum, Babesia canis, and rickettsiae in ticks removed from dogs living in Italy Michele Trotta a , Martina Nicetto a , Alessandro Fogliazza b , Fabrizio Montarsi c , Marco Caldin d , Tommaso Furlanello a , Laia Solano-Gallego a,e,∗ a
Laboratorio d’Analisi Veterinarie “San Marco” Padova, Italy Merial Italia Spa, Milano, Italy c Dipartimento di Ecopatologia, Isitituto Zooprofilattico Sperimentale delle Venezie, Italy d Clinica Veterinaria “San Marco” Padova, Italy e Department of Pathology and Infectious Diseases, Royal Veterinary College, University of London, UK b
a r t i c l e Keywords: Ticks PCR Leishmania infantum Rickettsia conorii Rickettsia massiliae Babesia canis
i n f o
a b s t r a c t The aims of this study were to determine natural infections by Anaplasma phagocytophilum/Anaplasma platys, Bartonella henselae, Ehrlichia canis, Leishmania infantum, Rickettsia spp., Babesia spp., and Hepatozoon spp. by molecular methods in ticks (n = 91) removed from dogs with clinical signs and laboratory abnormalities compatible with tick-borne diseases (n = 22) living in Italy and to assess the distribution and species of ticks encountered. Ticks from dogs living in southern Italy were all identified as Rhipicephalus sanguineus (n = 25), ticks from central Italy included Rh. sanguineus (n = 8) and Ixodes ricinus (n = 9), ticks from northern Italy included Rh. sanguineus (n = 45), Dermacentor marginatus (n = 3), and one I. ricinus. Leishmania infantum, Rickettsia spp., and Babesia canis were the only pathogens detected in 7 (8%), 4 (4%), and 2 (2%) out of 91 ticks, respectively. L. infantum was detected in I. ricinus from central Italy and in Rh. sanguineus from northern and central Italy. Rickettsia conorii and Ri. massiliae were detected in Rh. sanguineus ticks from central and southern Italy (Sicily), respectively. Bab. canis was detected in D. marginatus ticks from northern Italy. © 2012 Elsevier GmbH. All rights reserved.
Introduction Ticks are important vectors of human and animal pathogens including bacteria, viruses, and protozoa (Parola and Raoult, 2001). There has been an increasing spread of parasitic arthropods and the pathogens they transmit across the world. Dogs are infested with blood-feeding arthropods and might act as reservoir hosts of several zoonotic pathogens. The risk of exposure of dogs to numerous vector-borne pathogens transmitted by ticks has increased notably in many parts of the world including Europe, and the increasingly close relationships with humans in urban areas pose new concerns for human public health (Beugnet and Marié, 2009). In addition, the advancement in molecular techniques in the past decades has resulted in increased detection of vector-borne pathogens DNA in ticks (Sparagano et al., 1999). However, limited information is available about the rickettsial and protozoan pathogens that are detected in several species of ticks removed from dogs in Europe. The aims of this study were to evaluate natural infections with
∗ Corresponding author at: Departament de Medicina i Cirurgia Animal Facultat de Veterinaria Universitat Autonoma de Barcelona, Spain. E-mail address: [email protected] (L. Solano-Gallego). 1877-959X/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2012.10.031
vector-borne pathogens in ticks removed from dogs with clinicopathological abnormalities compatible with tick-borne diseases living in Italy and to assess the distribution and species of ticks encountered. Materials and methods A total of 91 ticks were removed from 22 dogs with clinical signs and laboratory findings compatible with tick-borne diseases from April to November 2007 from many veterinarian clinics throughout Italy (Trotta et al., 2009). The ticks were placed in tubes with 75% ethanol and stored at −20 ◦ C. Later, the ticks were morphologically identified (Iori et al., 2005). The DNA was isolated from individual ticks by using the High Pure PCR template preparation kit according to the manufacturer’s instructions with some modifications (Roche, Mannheim, Germany). The ticks were mechanically crushed with a sterile micropestle, suspended in 200 l of tissue lysis buffer and 40 l of proteinase K (100 g/ml) and incubated overnight at 65 ◦ C. The final elution volume was 100 l for each sample. The efficiency of tick DNA extraction was evaluated by amplification of the tick mitochondrial 16S rRNA gene by using tick-specific primers in a conventional PCR (Halos et al., 2004). Tick speciation based on morphological characteristics was not possible
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M. Trotta et al. / Ticks and Tick-borne Diseases 3 (2012) 293–296
Table 1 Species of ticks removed from Italian dogs and their geographical distribution. Tick species (number of ticks)
Rhipicephalus sanguineus (78) Dermacentor marginatus (3) Ixodes ricinus (10) Total (91)
Stage of ticks
Geographical region in Italy
Adult
Male
Female
Nymph
South
Central
North
70 3 9 82
38 0 2 40
32 3 7 42
8 0 1 9
25 0 0 25
8 0 9 17
45 3 1 49
in 6 ticks (5 Rhipicephalus spp. and one Ixodes spp.) and therefore, direct sequencing from amplified 16S rRNA gene and comparison with GenBank database was performed for speciation. Real-time PCRs for Anaplasma phagocytophilum/Anaplasma platys, Ehrlichia canis, Leishmania infantum, and Rickettsia spp. were performed by using the LightCycler Instrument 1.3.1 (Roche) (Solano-Gallego et al., 2006a, 2007, 2008a). Real-time PCR for Bartonella henselae was performed by using the QuantiTect® SYBR® Green PCR Kit (Qiagen, Hilden, Germany) as previously reported (Anderson et al., 1994). Babesia spp. (Solano-Gallego et al., 2008b) and Hepatozoon spp. (Inokuma et al., 2002) conventional PCRs were also carried out. Babesia-positive samples were submitted for direct sequencing. The positive samples for Rickettsia spp. by real-time PCR were further characterized by using a conventional PCR and direct sequencing for a fragment (170 bp) of the ompA gene (SolanoGallego et al., 2006b). The positive samples for L. infantum in I. ricinus ticks and the dog from which ticks were collected were further characterized by using a nested PCR and direct sequencing for a fragment (182 bp) of the internal transcribed spacer (ITS) locus with outer primers as previously described (Schönian et al., 2003) and inner primers, ITS76: 5 -CCTTTCCCACATACACAGCA-3 and ITS258: 5 -ATCGCGACACGTTATGTGAG-3 , designed by using Primer3Plus (Untergasser et al., 2007). The sequencing reactions were performed with the Applied Biosystem 3730XI DNA Analyzer on both strands. Consensus sequences were aligned with sequences in GenBank® using the basic local alignment search tool (nBLAST). Clinical data and vector-borne pathogen serological and molecular testing from dogs with detection of vector-borne pathogens in ticks removed from them were evaluated. Serological testings were carried out as previously described (Trotta et al., 2009; Riera et al., 1999). Results The mean number of ticks collected per dog and standard deviation were 4.5 ± 4 (range of ticks per dog 1–15). The species of ticks removed from dogs and their geographical distributions are listed in Table 1. A 320-bp fragment of the tick mitochondrial 16S rRNA
gene was amplified in all DNA tick samples. The species identification performed by direct sequencing allowed the identification of 5 Rhipicephalus sanguineus (Rh. sanguineus) and one I. ricinus. The vector-borne pathogens detected in ticks and their geographical distributions are displayed in Table 2. L. infantum, Rickettsia spp., and Bab. canis were the only pathogens detected in 7 (8%), 4 (4%), and 2 (2%) out of 91 ticks, respectively. One out of 73 (1%) and 3 out of 73 (4%) Rh. sanguineus ticks were positive for Rickettsia conorii (Ri. conorii) and Ri. massiliae, respectively. Five out of 9 I. ricinus (55%) and 2 out of 73 Rh. sanguineus (3%) ticks were positive for L. infantum, and 2 out of 3 Dermacentor marginatus (67%) ticks were positive for Bab. canis. The clinical data and the serological and molecular findings in dogs and on vector-borne pathogens in ticks removed from them are described in Table 3. Discussion In the present study L. infantum DNA was detected in I. ricinus and in Rh. sanguineus ticks. To our knowledge, this is the first report of L. infantum DNA in I. ricinus ticks. Moreover, these ticks were collected from a dog with clinical leishmaniosis based on the clinical findings, with a high antileishmanial antibody level, and a positive PCR of the blood. In agreement with the present study, L. infantum DNA was detected in Rh. sanguineus ticks from Brazil and Italy (Dantas-Torres et al., 2010) and in ticks from Brazil (Colombo et al., 2011) collected from L. infantum-seropositive dogs, although in the present study, some L. infantum DNA was detected for the first time in Rh. sanguineus ticks from dogs that were seronegative and PCRnegative. The proportion of L. infantum-positive Rh. sanguineus ticks were lower, but similar to those described in Brazil (Dantas-Torres et al., 2010). Transstadial and transovarial transmission of L. infantum in Rh. sanguineus ticks have been demonstrated (Dantas-Torres, 2011; Dantas-Torres et al., 2011), and Leishmania DNA has been detected in the salivary glands of ticks (Dantas-Torres et al., 2010). However, evidence of transmission of Leishmania infection by tick bite was reported only in 2 dogs (McKenzie, 1984; Dantas-Torres, 2011). Therefore, the significance of the present findings and the role of transmission of Leishmania infection to dogs by ticks remain
Table 2 Vector-borne pathogens detected in ticks in this study. Dog ID
Tick species
Gender of ticks
Pathogens
Geographical region in Italy
105A1 10H 86D 86D 86D 86D 86D 24A 25A 105A2 107A1 107A4 108A3
Rhipicephalus sanguineus Rh. sanguineus Ixodes ricinus I. ricinus I. ricinus I. ricinus I. ricinus Dermacentor marginatus D. marginatus Rh. sanguineus Rh. sanguineus Rh. sanguineus Rh. sanguineus
Male Male Engorged female Female Female Female Female Female Female Male Engorged female Engorged female Female
Leishmania infantum L. infantum L. infantum L. infantum L. infantum L. infantum a L. infantum Babesia canisa Bab. canisa Rickettsia conoriia Ri. massiliaea Ri. massiliaea Ri. massiliaea
Central (Rome) North Central Central Central Central Central North North Central (Rome) South (Sicily) South (Sicily) South (Sicily)
a Species identification by direct sequencing. 100% identity with GenBank® sequences of Ri. massiliae (HM014444), Ri. conorii (HM050291), L. infantum (FJ555210), and Bab. canis (DQ181653).
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295
Table 3 Clinical data and vector-borne pathogen testing in dogs and detection of vector-borne pathogens in the ticks collected from dogs. Dog ID and signalment
Species of tick/pathogen detected
Vector-borne pathogen testing
Clinical signs and laboratory abnormalities 1st visit
Serology
Positive PCR
Antigen
1st visit
2nd visit
#107A mixed breed, male, 2 years
Rh. sanguineus/Ri. massiliae
A. phagocytophilum E. canis L. infantum Ri. conorii
NEG 1:320 NEG 1:320
Not available
None
Pyoderma
#105A mixed breed, female, 1 year
Rh. sanguineus/Ri. conorii Rh. sanguineus/L. infantum
A. phagocytophilum
NEG
Not available
A. platys
Anorexia, lethargy, hyperthermia
E. canis
NEG
L. infantum Ri. conorii
NEG 1:5120
#108A mixed breed, female, 4 years
Rh. sanguineus/Ri. massiliae
A. phagocytophilum E. canis L. infantum Ri. conorii
1:320 NEG NEG 1:1280
Not available
None
Anorexia, lethargy, hyperthermia, dyspnoea
#25A female, Labrador retriever, 5 years
D. marginatus/Bab. canis
A. phagocytophilum E. canis L. infantum Ri. conorii
NEG NEG NEG NEG
NEG NEG NEG NEG
Bab. canis
Anorexia, lethargy, pigmenturia, hyperthermia, thrombocytopenia, leukopenia, large-form Babesia in erythrocytes (babesiosis), improved after therapy with imidocarb dipropionate and doxycycline
#24A mixed breed, male 4 years
D. marginatus/Bab. Canis
A. phagocytophilum E. canis L. infantum Ri. conorii
1:80 1:160 NEG 1:320
Not available
Bab. canis
Hyperthermia, lethargy, tonsillitis, thrombocytopenia, neutrophilia, large-form Babesia in erythrocytes (babesiosis), improved after therapy with imidocarb dipropionate and doxycycline
#10H mixed breed
Rh. sanguineus/L. infantum
A. phagocytophilum E. canis L. infantum Ri. conorii
1:80 NEG NEG NEG
NEG 1:80 NEG 1:80
None
Not available
#86D mixed breed, female, 6 years
I. ricinus/L. infantuma
A. phagocytophilum E. canis L. infantum Ri. conorii
1:1280 1:320 HIGH 1:160
Not available
L. infantuma
Pale mucous membranes, conjunctivitis
a
L. infantum sequences were identical in the tick and in canine blood.
unclear (Coutinho et al., 2005; Dantas-Torres, 2011). Further studies need to elucidate the potential of Leishmania transmission to dogs via ticks. Ri. conorii is the classical agent of Mediterranean spotted fever (Raoult and Roux, 1997), and Ri. massiliae is an emerging pathogen described for the first time in a human patient from Sicily (Vitale et al., 2006) and first isolated from a Rh. sanguineus tick in Marseille (Beati and Raoult, 1993). Thereafter, it was detected in ticks in other Mediterranean regions including Sardinia (Márquez et al., 2008; Mura et al., 2008; Harrus et al., 2010). The present study demonstrated the detection of Ri. conorii (1%) and Ri. massiliae (4%) in Rh. sanguineus ticks collected from dogs living in central and southern Italy, respectively. The low rates of Ri. conorii infection in Rh. sanguineus ticks in the present study are similar to those reported from several countries in the Mediterranean basin (Parola et al., 2005; Psaroulaki et al., 2006), whilst the Ri. massiliae rate of infection in ticks was slightly lower than in previous studies performed in Algeria, Spain, and Israel (Bitam et al., 2006; Márquez et al., 2008; Harrus et al., 2010) and similar to previous studies from Greece and Morocco (Psaroulaki et al. 2006; Sarih et al., 2008). In addition, to the author’s best knowledge, the detection of Ri. massiliae in ticks has not been previously described from Sicily. Interestingly, despite the high seroprevalence described in Italian dogs with rickettsial disease including Rickettsia, Ehrlichia, and Anaplasma in another study (Trotta et al., 2009), only a low number of ticks were positive for Rickettsia spp. in the present study.
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