Transboundary and Emerging Diseases
ORIGINAL ARTICLE
A Molecular Survey of Anaplasma spp., Rickettsia spp., Ehrlichia canis and Babesia microti in Foxes and Fleas from Sicily A. Torina1,2, V. Blanda1, F. Antoci1, S. Scimeca1, R. D’Agostino1, E. Scariano1, A. Piazza1, P. Galluzzo1, E. Giudice2 and S. Caracappa1 1 2
Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri”, Palermo, Italy Facolt a di Medicina Veterinaria, Universit a degli Studi di Messina, polo universitario dell’Annunziata, Messina, Italy
Keywords: Fox; fleas; Ehrlichia canis; Babesia microti; Rickettsia spp.; Anaplasma spp. Correspondence: V. Blanda. Istituto Zooprofilattico Sperimentale della Sicilia “A.Mirri”, Via Gino Marinuzzi 3, 90129 Palermo, Italy. Tel.: 00390916565360; Fax: 00390916565361; E-mail: [email protected] Received for publication November 15, 2012 doi:10.1111/tbed.12137
Summary Fleas (Insecta: Siphonaptera) are obligate bloodsucking insects, which parasitize birds and mammals, and are distributed throughout the world. Several species have been implicated in pathogen transmission. This study aimed to monitor red foxes and the fleas isolated from them in the Palermo and Ragusa provinces of Sicily, Italy, as these organisms are potential reservoirs and vectors of pathogens. Thirteen foxes (Vulpes vulpes) and 110 fleas were analysed by polymerase chain reaction (PCR) to detect DNA of the pathogens Ehrlichia canis, Babesia microti, Rickettsia spp., Anaplasma phagocytophilum, Anaplasma platys, Anaplasma marginale and Anaplasma ovis. In the foxes, A. ovis was detected in only one animal, whereas the prevalence of the E. canis pathogen was 31%. B. microti and Rickettsia spp. were not detected. Of all of the collected fleas, 75 belonged to the species Xenopsylla cheopis, 32 belonged to Ctenocephalides canis, two belonged to Ctenocephalides felis and one belonged to Cediopsylla inaequalis. In the fleas, the following pathogens were found: A. ovis (prevalence 25%), A. marginale (1%), A. phagocytophilum (1%), Rickettsia felis (2%) and E. canis (3%). X. cheopis was the flea species most frequently infected with Anaplasma, in particular A. ovis (33%), A. marginale (1%) and A. phagocytophilum (1%). Both C. felis exemplars were positive for R. felis. E. canis was found in the lone C. inaequalis and also in 3% of the X. cheopis specimens. No fleas were positive for B. microti or A. platys. As foxes often live in proximity to domestic areas, they may constitute potential reservoirs for human and animal parasites. Further studies should be performed on fleas to determine their vectorial capacity.
Introduction Fleas (Insecta: Siphonaptera) are obligate haematophagous insects, which can parasitize both birds and mammals (including humans) and have a worldwide distribution. Several species have been involved in the transmission of various pathogenic agents, such as Yersinia pestis, Rickettsia typhi, Rickettsia felis and Bartonella sp. Pulicidae includes many species that parasitize humans and domestic animals (Pulex, Ctenocephalides and Spilopsyllus genera). Even wild animals are infested with fleas.
In recent years, climate change and global warming have caused ecological changes in the living conditions of animal reservoirs, and an increase in the incidence of certain diseases transmitted by arthropod vectors has been observed (Blanco and Oteo, 2006). Foxes (Vulpes vulpes Linnaeus, 1758, of the family Canidae), for example, are generally solitary animals that live in burrows and feed on live prey, usually rodents. However, they can also be found in close proximity to urban or agricultural areas and seem to adapt quite well to the presence of humans. This adaptation can lead to increased contact with both humans and
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domestic animals. Consequently, foxes may be infested with many tick and flea species coming from their prey or from other animals living in the same environment. These fox arthropods may be infected with microorganisms, including human and animal pathogens. For example, Rickettsia was found in ectoparasites collected from foxes in Corsica (Marie et al., 2012). These bacteria, although commonly transmitted by ticks, also have fleas, lice and mites as relevant vectors. Fleas, in particular, can be vectors of R. typhi and R. felis. In addition, Anaplasma phagocytophilum has been detected in arthropods collected from foxes in Hungary (Sreter et al., 2004). The aims of this study were to monitor red foxes (Vulpes vulpes) and the fleas collected from them in Sicily to detect the presence of pathogen DNA belonging to Ehrlichia canis, Babesia microti, Rickettsia spp. and Anaplasma spp. and to investigate their potential role as reservoirs and vectors of pathogens. Materials and Methods Sample collection and flea identification The study was conducted in the Sicily region of southern Italy. We analysed 13 foxes killed in the provinces of Palermo and Ragusa during permitted hunting in December 2011 and January 2012. Most of the foxes were killed in a hunting reserve near the coast, located at sea level. The reserve is adjacent to sheep and cattle farms, and farmers have often complained about fox incursions. For each fox, the spleen was removed and processed for DNA extraction. Fleas, if present, were harvested, kept alive for a few days to allow the insects to clean themselves of any ingested blood, rinsed with distilled water and stored in 70% ethyl alcohol. A total of 110 fleas were collected, and the arthropods were identified according to previously described identification keys (Herms, 1923; Chinery, 1998). DNA extraction from fox spleens and fleas DNA was extracted from fox spleens using the PureLink Genomic Mini kit (Life Technologies Corporation, Carlsbad, CA, USA) following the manufacturer’s instructions. After identification, the collected fleas were sectioned longitudinally, and one half of each flea was placed in a pool, according to species and sex, comprised of one to a maximum of six exemplars. DNA was extracted from each pool using the above-mentioned kit. The other half of each flea was maintained in alcohol, awaiting further analysis. In the case of a positive result in a pool, DNA was extracted from the other half of every single flea present in the positive pool and individually subjected to amplification by polymerase chain reaction (PCR) to evaluate the number of positive fleas in each pool. 126
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Molecular analysis The extracted nucleic acids were analysed by PCR to detect the presence of DNA from Anaplasma spp. (Stuen et al., 2003), Rickettsia spp. (Tzianabos et al., 1989), E. canis (Siarkou et al., 2007) and B. microti (Persing et al., 1992). PCRs were performed using GoTaq Polymerase (Promega, Madison, WI, USA). For each reaction, a positive control, consisting of pathogen DNA, and a negative control, in which DNA was replaced by water, were used. PCR products were visualized after electrophoretic migration on an agarose gel containing 10 lg/ml ethidium bromide. Fleas positive for Anaplasma spp. were analysed by PCR for A. phagocytophilum (de la Fuente et al., 2005), A. platys (Inokuma et al., 2002), A. marginale and A. ovis (de la Fuente et al., 2003; Torina et al., 2012). Fleas positive for Rickettsia spp. were subjected to PCR amplification of the ompA (Oteo et al., 2006), ompB (Choi et al., 2005) and gltA genes (Roux et al., 1997) to identify the Rickettsia species using a multigene assay. PCR products obtained by amplifying the Rickettsia ompA, ompB and gltA genes were purified by the Wizard SV Gel and PCR Clean-up System (Promega), quantified and sent to Macrogen Inc., (Seoul, South Korea) for sequencing. The obtained sequences were aligned using Bioedit software (Tom Hall, Ibis Biosciences, Carlsbad, CA, USA) and ClustalW 2.0.10 (Larkin et al., 2007) and analysed for nucleotide sequence identity by comparing them with reference strains. Results Flea identification A total of 110 fleas were collected from 13 foxes (Vulpes vulpes) in Sicily. Of these fleas, 75 (68%) belonged to the species Xenopsylla cheopis, 32 (29%) to Ctenocephalides canis, two (2%) belonged to Ctenocephalides felis and one (1%) belonged to Cediopsylla inaequalis, as reported in Fig. 1. Fleas were grouped into 28 pools according to species and sex, thereby obtaining 18 pools of X. cheopis, eight
Fleas species (%) 1 2 29
68
Xenopsylla cheopis
Ctenocephalides canis
Ctenocephalides felis
Cediopsylla inaequalis
Fig. 1. Percentage of different flea species found on foxes in Sicily.
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pools of C. canis, one pool of C. felis and one pool containing the sole C. inaequalis exemplar. Molecular analysis of foxes Among the thirteen analysed fox samples, one was positive for Anaplasma spp., which was further characterized as A. ovis, and four were positive for E. canis (31% prevalence). No foxes were positive for B. microti or Rickettsia spp. Molecular analysis of fleas Of the 28 pools of fleas, 15 were positive for Anaplasma spp., three were positive for E. canis and one was positive for Rickettsia spp. No pools were positive for B. microti. With regard to the pool positive for Rickettsia spp., it was composed of the two C. felis exemplars. Sequence analysis of the ompA, ompB and gltA genes showed that both were infected with R. felis. Table 1 reports the prevalence values of A. phagocytophilum, A. marginale, A. ovis, E. canis and R. felis for each flea species. Table 2 shows the distribution of fleas on foxes and
indicates the PCR results for comparison between analysed foxes and the fleas collected from them. One X. cheopis showed coinfection with A. ovis and A. phagocytophilum, whereas another one, belonging to the same species, was simultaneously infected with A. ovis and A. marginale. No positivity for A. platys was detected. Discussion In this study, we performed a molecular analysis by PCR of DNA extracted from 13 foxes found in Sicily and 110 fleas collected from them. Anaplasma spp. was detected in one fox and in 30% of the fleas. Most of the fleas positive for Anaplasma were collected from the same heavily infested fox, even though the animal tested negative for the pathogen. This observation indicates that positivity in fleas is not necessarily due to the presence of just-eaten, infected blood. The red fox is a mammalian predator that shares an optimal habitat with its prey (e.g. rodents), which could be reservoirs of different pathogens. The prey may also be infested with a number of ectoparasites. Thus, X. cheopis, which has micromammals as its preferred hosts, may be
Table 1. Prevalence values (%) of the analysed pathogens for flea species, calculated as N. fleas of a species positive for a pathogen/N. fleas of the same species. The overall value refers to the total number of fleas positive for a pathogen/total number of fleas
Xenopsylla cheopis Ctenocephalides canis Cediopsylla inaequalis Overall
Rickettsia felis
Ehrlichia canis
Anaplasma ovis
Anaplasma marginale
Anaplasma phagocytophilum
0 0 0 2
3 0 100 3
33 6 0 25
1 0 0 1
1 0 0 1
Table 2. Flea distribution on foxes, with the indication of the results of PCRs performed for Ehrlichia canis, Rickettsia felis, Anaplasma phagocytophilum, Anaplasma marginale and Anaplasma ovis in foxes and their fleas
Fox Id.
n. Fleas
Ehrlichia canis Fox n. pos Fleas
1 2 3 4 5 6 7 8 9 10 11 12 13
20 6 68 4 4 4 4 0 0 0 0 0 0
pos pos 0 0 0 pos pos 0 0 0 0 0 0
1 0 1 0 0 1 0 – – – – – –
Rickettsia felis Fox n. pos Fleas
Anaplasma phagocytophilum Fox n. pos Fleas
Anaplasma ovis Fox n. pos Fleas
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 pos 0 0 0 0 0 0
0 0 2 0 0 0 0 – – – – – –
1 0 0 0 0 0 0 – – – – – –
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3 1 23 0 0 0 0 – – – – – –
Anaplasma marginale Fox n. pos Fleas 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 1 0 0 0 0 – – – – – –
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able to parasitize both rodents and foxes. One hypothesis is that fleas from infected rodents are capable of taking up Anaplasma spp. microorganisms and maintaining them. Alternatively, the ingested pathogen may be digested by the flea and excreted in the faeces, thus resulting in negative results for Anaplasma spp. in the arthropod. Only one flea (X. cheopis) was positive for A. phagocytophilum (1%). Many studies based on serological surveying have reported the presence of antibodies against A. phagocytophilum in foxes, and molecular research has detected the presence of this pathogen’s DNA in these mammals and their ectoparasites (Sreter et al., 2004; Gabriel et al., 2009; Karbowiak et al., 2009; Ebani et al., 2011). The limited presence of A. phagocytophilum can be explained by the fact that Ixodes ricinus, its main vector, is scarce in Sicily and is particularly rare in the Ragusa province, from which most of the examined foxes came (Torina et al., 2008). In the Ragusa province, agriculture and zootechnics are extensively practiced, and the territory has mostly low-altitude coastal zones. The discovery of A. ovis in the fleas (25%) and in one fox was a surprising result, as was the detection of A. marginale (1%) in X. cheopis, even though the association between A. marginale and small rodents, which are possible hosts of ectoparasites and potential prey of the fox, has been previously reported (Akinboade et al., 1981). On the other hand, we know that foxes are often found in close proximity to sheep farms, and farmers have often complained about fox incursions. In such an environment, we can assume that fleas may have derived the pathogen from sheep. These fleas can then pass to the fox from the prey that, in this context, would be a lamb. These considerations do not completely demonstrate the role of fleas as a vector of Anaplasma but provide additional information on the ability of the arthropod to maintain this organism. Further studies are needed to understand the role of these arthropods in the life cycles of A. marginale and A. ovis more clearly. These life cycles are particularly complex because of the possible interactions between wild and domestic animals and different types of carriers, such as ticks and fleas. With regards to E. canis, there was a prevalence of 31% in foxes and 3% in the fleas. In particular, two positive fleas were collected from foxes that were positive for the pathogen, and a third came from a fox that was negative for the pathogen. The latter flea is evidence of the maintenance of E. canis within the insect. Previous seroepidemiological studies conducted on free-ranging red foxes in Israel using an indirect fluorescent antibody test showed that 36% of the foxes were seropositive for E. canis (Fishman et al., 2004). This result suggested that canine ehrlichiosis may be endemic in the wild red fox populations of Israel and that foxes may serve as a reservoir for the infection of domestic 128
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dogs and other wild canine species (Fishman et al., 2004). In addition, 7% of red fox sera specimens examined in the Netherlands were seroreactive to the E. canis antigen (Groen et al., 2002), indicating that a sizable portion of the fox population had been exposed to this pathogen. Therefore, although the association between foxes and E. canis has been demonstrated by research data, there is no evidence concerning the role of fleas as vectors of E. canis. Many studies have failed to detect Ehrlichia species in fleas (Loftis et al., 2006; Barrs et al., 2010; Foongladda et al., 2011). This study provides the first report of an association between E. canis and fleas. No foxes tested positive for Rickettsia spp. Meanwhile, the flea pool containing the two exemplars of C. felis tested positive for R. felis, which is an emergent rickettsial zoonotic pathogen with a worldwide distribution (Parola, 2011). Notably, there have been growing reports of the worldwide detection of R. felis in arthropod hosts, and most of these studies assert that the cat flea C. felis is the most prevalent arthropod in places where R. felis has been detected. These fleas primarily come from cats or dogs (Capelli et al., 2009; Silaghi et al., 2012; Troyo et al., 2012), but an association with wild animals has also been reported. For example, R. felis was found in C. felis fleas collected from wild animals in Spain (Lled o et al., 2010); in California, the infection rate of R. felis was 13% in pooled samples collected from opossums (Didelphis virginiana) trapped in suburban residential and industrial zones (Abramowicz et al., 2012). If it is associated with an infectious agent, the worldwide distribution of C. felis would represent a threat to the human population because the cat flea has a lack of host specificity and it has the ability to bite people. Our findings emphasize the potential risk of the transmission of rickettsias (namely, R. felis) to humans in Sicily. Finally, no pools of flea specimens and no foxes were positive for B. microti, even though a previous study (Torina et al., 2007) in Sicily reported an 11% prevalence of B. microti-positive foxes, suggesting that these mammals seem to have a role in the biological cycle of Babesia species. Many positive findings for B. microti have been reported in wild animals, especially rodents. For example, a molecular survey performed in Croatia on 120 small wild mammals found that 16% of them were infected with B. microti. Sequence analysis demonstrated that some of them were identical to that of the human Jena/Germany strain, showing that wild species may constitute a potential risk of human health (Beck et al., 2011). Moreover, B. microti-like piroplasms infect dogs, causing diseases (Solano-Gallego and Baneth, 2011). The phylogenetic proximity between foxes and dogs, along with their overlapping habitats, supports the hypothesis that foxes may be susceptible to infection with B. microti.
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ORIGINAL ARTICLE
A Molecular Survey of Anaplasma spp., Rickettsia spp., Ehrlichia canis and Babesia microti in Foxes and Fleas from Sicily A. Torina1,2, V. Blanda1, F. Antoci1, S. Scimeca1, R. D’Agostino1, E. Scariano1, A. Piazza1, P. Galluzzo1, E. Giudice2 and S. Caracappa1 1 2
Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri”, Palermo, Italy Facolt a di Medicina Veterinaria, Universit a degli Studi di Messina, polo universitario dell’Annunziata, Messina, Italy
Keywords: Fox; fleas; Ehrlichia canis; Babesia microti; Rickettsia spp.; Anaplasma spp. Correspondence: V. Blanda. Istituto Zooprofilattico Sperimentale della Sicilia “A.Mirri”, Via Gino Marinuzzi 3, 90129 Palermo, Italy. Tel.: 00390916565360; Fax: 00390916565361; E-mail: [email protected] Received for publication November 15, 2012 doi:10.1111/tbed.12137
Summary Fleas (Insecta: Siphonaptera) are obligate bloodsucking insects, which parasitize birds and mammals, and are distributed throughout the world. Several species have been implicated in pathogen transmission. This study aimed to monitor red foxes and the fleas isolated from them in the Palermo and Ragusa provinces of Sicily, Italy, as these organisms are potential reservoirs and vectors of pathogens. Thirteen foxes (Vulpes vulpes) and 110 fleas were analysed by polymerase chain reaction (PCR) to detect DNA of the pathogens Ehrlichia canis, Babesia microti, Rickettsia spp., Anaplasma phagocytophilum, Anaplasma platys, Anaplasma marginale and Anaplasma ovis. In the foxes, A. ovis was detected in only one animal, whereas the prevalence of the E. canis pathogen was 31%. B. microti and Rickettsia spp. were not detected. Of all of the collected fleas, 75 belonged to the species Xenopsylla cheopis, 32 belonged to Ctenocephalides canis, two belonged to Ctenocephalides felis and one belonged to Cediopsylla inaequalis. In the fleas, the following pathogens were found: A. ovis (prevalence 25%), A. marginale (1%), A. phagocytophilum (1%), Rickettsia felis (2%) and E. canis (3%). X. cheopis was the flea species most frequently infected with Anaplasma, in particular A. ovis (33%), A. marginale (1%) and A. phagocytophilum (1%). Both C. felis exemplars were positive for R. felis. E. canis was found in the lone C. inaequalis and also in 3% of the X. cheopis specimens. No fleas were positive for B. microti or A. platys. As foxes often live in proximity to domestic areas, they may constitute potential reservoirs for human and animal parasites. Further studies should be performed on fleas to determine their vectorial capacity.
Introduction Fleas (Insecta: Siphonaptera) are obligate haematophagous insects, which can parasitize both birds and mammals (including humans) and have a worldwide distribution. Several species have been involved in the transmission of various pathogenic agents, such as Yersinia pestis, Rickettsia typhi, Rickettsia felis and Bartonella sp. Pulicidae includes many species that parasitize humans and domestic animals (Pulex, Ctenocephalides and Spilopsyllus genera). Even wild animals are infested with fleas.
In recent years, climate change and global warming have caused ecological changes in the living conditions of animal reservoirs, and an increase in the incidence of certain diseases transmitted by arthropod vectors has been observed (Blanco and Oteo, 2006). Foxes (Vulpes vulpes Linnaeus, 1758, of the family Canidae), for example, are generally solitary animals that live in burrows and feed on live prey, usually rodents. However, they can also be found in close proximity to urban or agricultural areas and seem to adapt quite well to the presence of humans. This adaptation can lead to increased contact with both humans and
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domestic animals. Consequently, foxes may be infested with many tick and flea species coming from their prey or from other animals living in the same environment. These fox arthropods may be infected with microorganisms, including human and animal pathogens. For example, Rickettsia was found in ectoparasites collected from foxes in Corsica (Marie et al., 2012). These bacteria, although commonly transmitted by ticks, also have fleas, lice and mites as relevant vectors. Fleas, in particular, can be vectors of R. typhi and R. felis. In addition, Anaplasma phagocytophilum has been detected in arthropods collected from foxes in Hungary (Sreter et al., 2004). The aims of this study were to monitor red foxes (Vulpes vulpes) and the fleas collected from them in Sicily to detect the presence of pathogen DNA belonging to Ehrlichia canis, Babesia microti, Rickettsia spp. and Anaplasma spp. and to investigate their potential role as reservoirs and vectors of pathogens. Materials and Methods Sample collection and flea identification The study was conducted in the Sicily region of southern Italy. We analysed 13 foxes killed in the provinces of Palermo and Ragusa during permitted hunting in December 2011 and January 2012. Most of the foxes were killed in a hunting reserve near the coast, located at sea level. The reserve is adjacent to sheep and cattle farms, and farmers have often complained about fox incursions. For each fox, the spleen was removed and processed for DNA extraction. Fleas, if present, were harvested, kept alive for a few days to allow the insects to clean themselves of any ingested blood, rinsed with distilled water and stored in 70% ethyl alcohol. A total of 110 fleas were collected, and the arthropods were identified according to previously described identification keys (Herms, 1923; Chinery, 1998). DNA extraction from fox spleens and fleas DNA was extracted from fox spleens using the PureLink Genomic Mini kit (Life Technologies Corporation, Carlsbad, CA, USA) following the manufacturer’s instructions. After identification, the collected fleas were sectioned longitudinally, and one half of each flea was placed in a pool, according to species and sex, comprised of one to a maximum of six exemplars. DNA was extracted from each pool using the above-mentioned kit. The other half of each flea was maintained in alcohol, awaiting further analysis. In the case of a positive result in a pool, DNA was extracted from the other half of every single flea present in the positive pool and individually subjected to amplification by polymerase chain reaction (PCR) to evaluate the number of positive fleas in each pool. 126
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Molecular analysis The extracted nucleic acids were analysed by PCR to detect the presence of DNA from Anaplasma spp. (Stuen et al., 2003), Rickettsia spp. (Tzianabos et al., 1989), E. canis (Siarkou et al., 2007) and B. microti (Persing et al., 1992). PCRs were performed using GoTaq Polymerase (Promega, Madison, WI, USA). For each reaction, a positive control, consisting of pathogen DNA, and a negative control, in which DNA was replaced by water, were used. PCR products were visualized after electrophoretic migration on an agarose gel containing 10 lg/ml ethidium bromide. Fleas positive for Anaplasma spp. were analysed by PCR for A. phagocytophilum (de la Fuente et al., 2005), A. platys (Inokuma et al., 2002), A. marginale and A. ovis (de la Fuente et al., 2003; Torina et al., 2012). Fleas positive for Rickettsia spp. were subjected to PCR amplification of the ompA (Oteo et al., 2006), ompB (Choi et al., 2005) and gltA genes (Roux et al., 1997) to identify the Rickettsia species using a multigene assay. PCR products obtained by amplifying the Rickettsia ompA, ompB and gltA genes were purified by the Wizard SV Gel and PCR Clean-up System (Promega), quantified and sent to Macrogen Inc., (Seoul, South Korea) for sequencing. The obtained sequences were aligned using Bioedit software (Tom Hall, Ibis Biosciences, Carlsbad, CA, USA) and ClustalW 2.0.10 (Larkin et al., 2007) and analysed for nucleotide sequence identity by comparing them with reference strains. Results Flea identification A total of 110 fleas were collected from 13 foxes (Vulpes vulpes) in Sicily. Of these fleas, 75 (68%) belonged to the species Xenopsylla cheopis, 32 (29%) to Ctenocephalides canis, two (2%) belonged to Ctenocephalides felis and one (1%) belonged to Cediopsylla inaequalis, as reported in Fig. 1. Fleas were grouped into 28 pools according to species and sex, thereby obtaining 18 pools of X. cheopis, eight
Fleas species (%) 1 2 29
68
Xenopsylla cheopis
Ctenocephalides canis
Ctenocephalides felis
Cediopsylla inaequalis
Fig. 1. Percentage of different flea species found on foxes in Sicily.
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pools of C. canis, one pool of C. felis and one pool containing the sole C. inaequalis exemplar. Molecular analysis of foxes Among the thirteen analysed fox samples, one was positive for Anaplasma spp., which was further characterized as A. ovis, and four were positive for E. canis (31% prevalence). No foxes were positive for B. microti or Rickettsia spp. Molecular analysis of fleas Of the 28 pools of fleas, 15 were positive for Anaplasma spp., three were positive for E. canis and one was positive for Rickettsia spp. No pools were positive for B. microti. With regard to the pool positive for Rickettsia spp., it was composed of the two C. felis exemplars. Sequence analysis of the ompA, ompB and gltA genes showed that both were infected with R. felis. Table 1 reports the prevalence values of A. phagocytophilum, A. marginale, A. ovis, E. canis and R. felis for each flea species. Table 2 shows the distribution of fleas on foxes and
indicates the PCR results for comparison between analysed foxes and the fleas collected from them. One X. cheopis showed coinfection with A. ovis and A. phagocytophilum, whereas another one, belonging to the same species, was simultaneously infected with A. ovis and A. marginale. No positivity for A. platys was detected. Discussion In this study, we performed a molecular analysis by PCR of DNA extracted from 13 foxes found in Sicily and 110 fleas collected from them. Anaplasma spp. was detected in one fox and in 30% of the fleas. Most of the fleas positive for Anaplasma were collected from the same heavily infested fox, even though the animal tested negative for the pathogen. This observation indicates that positivity in fleas is not necessarily due to the presence of just-eaten, infected blood. The red fox is a mammalian predator that shares an optimal habitat with its prey (e.g. rodents), which could be reservoirs of different pathogens. The prey may also be infested with a number of ectoparasites. Thus, X. cheopis, which has micromammals as its preferred hosts, may be
Table 1. Prevalence values (%) of the analysed pathogens for flea species, calculated as N. fleas of a species positive for a pathogen/N. fleas of the same species. The overall value refers to the total number of fleas positive for a pathogen/total number of fleas
Xenopsylla cheopis Ctenocephalides canis Cediopsylla inaequalis Overall
Rickettsia felis
Ehrlichia canis
Anaplasma ovis
Anaplasma marginale
Anaplasma phagocytophilum
0 0 0 2
3 0 100 3
33 6 0 25
1 0 0 1
1 0 0 1
Table 2. Flea distribution on foxes, with the indication of the results of PCRs performed for Ehrlichia canis, Rickettsia felis, Anaplasma phagocytophilum, Anaplasma marginale and Anaplasma ovis in foxes and their fleas
Fox Id.
n. Fleas
Ehrlichia canis Fox n. pos Fleas
1 2 3 4 5 6 7 8 9 10 11 12 13
20 6 68 4 4 4 4 0 0 0 0 0 0
pos pos 0 0 0 pos pos 0 0 0 0 0 0
1 0 1 0 0 1 0 – – – – – –
Rickettsia felis Fox n. pos Fleas
Anaplasma phagocytophilum Fox n. pos Fleas
Anaplasma ovis Fox n. pos Fleas
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 pos 0 0 0 0 0 0
0 0 2 0 0 0 0 – – – – – –
1 0 0 0 0 0 0 – – – – – –
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3 1 23 0 0 0 0 – – – – – –
Anaplasma marginale Fox n. pos Fleas 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 1 0 0 0 0 – – – – – –
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able to parasitize both rodents and foxes. One hypothesis is that fleas from infected rodents are capable of taking up Anaplasma spp. microorganisms and maintaining them. Alternatively, the ingested pathogen may be digested by the flea and excreted in the faeces, thus resulting in negative results for Anaplasma spp. in the arthropod. Only one flea (X. cheopis) was positive for A. phagocytophilum (1%). Many studies based on serological surveying have reported the presence of antibodies against A. phagocytophilum in foxes, and molecular research has detected the presence of this pathogen’s DNA in these mammals and their ectoparasites (Sreter et al., 2004; Gabriel et al., 2009; Karbowiak et al., 2009; Ebani et al., 2011). The limited presence of A. phagocytophilum can be explained by the fact that Ixodes ricinus, its main vector, is scarce in Sicily and is particularly rare in the Ragusa province, from which most of the examined foxes came (Torina et al., 2008). In the Ragusa province, agriculture and zootechnics are extensively practiced, and the territory has mostly low-altitude coastal zones. The discovery of A. ovis in the fleas (25%) and in one fox was a surprising result, as was the detection of A. marginale (1%) in X. cheopis, even though the association between A. marginale and small rodents, which are possible hosts of ectoparasites and potential prey of the fox, has been previously reported (Akinboade et al., 1981). On the other hand, we know that foxes are often found in close proximity to sheep farms, and farmers have often complained about fox incursions. In such an environment, we can assume that fleas may have derived the pathogen from sheep. These fleas can then pass to the fox from the prey that, in this context, would be a lamb. These considerations do not completely demonstrate the role of fleas as a vector of Anaplasma but provide additional information on the ability of the arthropod to maintain this organism. Further studies are needed to understand the role of these arthropods in the life cycles of A. marginale and A. ovis more clearly. These life cycles are particularly complex because of the possible interactions between wild and domestic animals and different types of carriers, such as ticks and fleas. With regards to E. canis, there was a prevalence of 31% in foxes and 3% in the fleas. In particular, two positive fleas were collected from foxes that were positive for the pathogen, and a third came from a fox that was negative for the pathogen. The latter flea is evidence of the maintenance of E. canis within the insect. Previous seroepidemiological studies conducted on free-ranging red foxes in Israel using an indirect fluorescent antibody test showed that 36% of the foxes were seropositive for E. canis (Fishman et al., 2004). This result suggested that canine ehrlichiosis may be endemic in the wild red fox populations of Israel and that foxes may serve as a reservoir for the infection of domestic 128
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dogs and other wild canine species (Fishman et al., 2004). In addition, 7% of red fox sera specimens examined in the Netherlands were seroreactive to the E. canis antigen (Groen et al., 2002), indicating that a sizable portion of the fox population had been exposed to this pathogen. Therefore, although the association between foxes and E. canis has been demonstrated by research data, there is no evidence concerning the role of fleas as vectors of E. canis. Many studies have failed to detect Ehrlichia species in fleas (Loftis et al., 2006; Barrs et al., 2010; Foongladda et al., 2011). This study provides the first report of an association between E. canis and fleas. No foxes tested positive for Rickettsia spp. Meanwhile, the flea pool containing the two exemplars of C. felis tested positive for R. felis, which is an emergent rickettsial zoonotic pathogen with a worldwide distribution (Parola, 2011). Notably, there have been growing reports of the worldwide detection of R. felis in arthropod hosts, and most of these studies assert that the cat flea C. felis is the most prevalent arthropod in places where R. felis has been detected. These fleas primarily come from cats or dogs (Capelli et al., 2009; Silaghi et al., 2012; Troyo et al., 2012), but an association with wild animals has also been reported. For example, R. felis was found in C. felis fleas collected from wild animals in Spain (Lled o et al., 2010); in California, the infection rate of R. felis was 13% in pooled samples collected from opossums (Didelphis virginiana) trapped in suburban residential and industrial zones (Abramowicz et al., 2012). If it is associated with an infectious agent, the worldwide distribution of C. felis would represent a threat to the human population because the cat flea has a lack of host specificity and it has the ability to bite people. Our findings emphasize the potential risk of the transmission of rickettsias (namely, R. felis) to humans in Sicily. Finally, no pools of flea specimens and no foxes were positive for B. microti, even though a previous study (Torina et al., 2007) in Sicily reported an 11% prevalence of B. microti-positive foxes, suggesting that these mammals seem to have a role in the biological cycle of Babesia species. Many positive findings for B. microti have been reported in wild animals, especially rodents. For example, a molecular survey performed in Croatia on 120 small wild mammals found that 16% of them were infected with B. microti. Sequence analysis demonstrated that some of them were identical to that of the human Jena/Germany strain, showing that wild species may constitute a potential risk of human health (Beck et al., 2011). Moreover, B. microti-like piroplasms infect dogs, causing diseases (Solano-Gallego and Baneth, 2011). The phylogenetic proximity between foxes and dogs, along with their overlapping habitats, supports the hypothesis that foxes may be susceptible to infection with B. microti.
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