Exposure of Owned Dogs and Feeding Ticks to Spotted Fever Group Rickettsioses in Central Italy

Abstract

Dogs may be useful sentinels for public health monitoring of spotted fever group rickettsioses (SFGR). The aim of this study was to determine the exposure to SFGR among dogs and feeding ticks in central Italy. A total of 344 dogs and 607 adult ticks (395 Rhipicephalus sanguineus and 212 Ixodes ricinus specimens) collected from the coats of sampled animals were included in the study. Canine serum samples were analyzed by indirect fluorescent antibody technique (IFAT) for IgG antibodies against Rickettsia conorii and Rickettsia rickettsii. All the ticks and buffy coats were processed by a PCR targeting a fragment of gltA followed by sequencing. Overall, 56 dogs (16.3%) tested positive for one or both rickettsial antigens by IFAT with endpoint titers ranging from 1:64 to 1:2048; 38 (11%) serum samples reacted against R. conorii, 46 (13.4%) reacted against R. rickettsii, and 28 (8.1%) reacted simultaneously against both rickettsial agents. All buffy coats were PCR negative. Rickettsial DNA was revealed in 39 (18.4%) I. ricinus and in 10 (2.5%) R. sanguineus specimens. The amplicons sequencing showed three SFGR, that is, R. conorii detected in 10 R. sanguineus specimens and Rickettsia helvetica and Rickettsia monacensis detected in 7 and 32 I. ricinus ticks. Nine out of the 10 R. conorii isolates were obtained from ticks collected from seronegative dogs, and one specimen from a dog tested positive for both R. rickettsii and R. conorii by immunofluorescence assay. Among the seven ticks tested positive for R. helvetica, six were recovered from the coats of seronegative dogs and one from a dog having antibodies against R. conorii; the 32 isolates of R. monacensis were obtained from 28 seronegative and 4 R. conorii/R. rickettsii-positive dogs. The results highlight the non-negligible exposure of the canine population to SFGR in the sampled areas.

Introduction

The spotted fever group rickettsioses (SFGR; Order Rickettsiales, Family Rickettsiaceae) are recognized as important and emerging zoonoses worldwide (Parola et al. 2013). Rickettsia conorii subsp. conorii has long been considered the only tick-borne rickettsial agent belonging to the spotted fever group (SFG) with veterinary and medical importance in southern and eastern Europe (Raoult and Roux 1997, Beninati et al. 2002). However, recent advances in molecular techniques have detected several other SFGR (Parola et al. 2013), for example, Rickettsia helvetica, Rickettsia aeschlimannii, Rickettsia massiliae, Rickettsia monacensis, Rickettsia raoultii, and Rickettsia slovaca, which have been isolated from questing and feeding ixodid ticks, as well as from a wide range of wild and domesticated mammals (Millán et al. 2016). Some of the mentioned rickettsiae are considered a threat to human health because they are isolated from cases of Mediterranean spotted fever (MSF)-like illnesses such as tick-borne lymphadenopathy/Dermacentor-borne necrosis-erythema-lymphadenopathy by R. slovaca (Parola et al. 2013). Knowledge of the impact of SFGR other than Rickettsia rickettsii (the most pathogenic species associated with severe illness in the western hemisphere and that has never been reported in the Old World to date) and R. conorii (which has a lower pathogenic role; Paddock et al. 2002, Solano-Gallego et al. 2006a) in canine illness is still limited. However, acute undifferentiated febrile syndromes, which are responsive to tetracycline treatment, frequently occur in dogs around the world (Faccini-Martínez et al. 2017) and in the Mediterranean basin, including Italy (authors' unpublished observations) and are often empirically attributed to rickettsial infections without providing any diagnostic evidence.

The role of dogs as competent hosts and as reservoir for SFGR is still unclear (Parola et al. 2005a), because little is known about the development and long-term duration of rickettsiemia in this species (Pinter et al. 2008). However, since dogs can act as carriers of infected ticks and may develop a significant antibody response against Rickettsia spp. (Lauzi et al. 2016), they can be used as sensitive sentinels to examine the presence and assess the infective pressure of SFG pathogens in particular regions (Solano-Gallego et al. 2006b, Alexandre et al. 2011).

Serological surveys conducted in dog populations across the Mediterranean basin have shown an SFGR seroprevalence ranging from 38% to 81% (Solano-Gallego et al., 2006b, Torina and Caracappa 2006, Harrus et al. 2007, Alexandre et al. 2011, Pennisi et al. 2012, Traversa et al. 2017). However, due to the cross-reactivity of antibodies within SFG, serological methods do not provide a reliable species discrimination (Pinter et al. 2008). Molecular detection and sequencing are needed to assess the rickettsial species circulating within the canine population of a certain area. The aim of this study was to investigate the occurrence and prevalence of SFGR in owned dogs and their feeding ticks to better understand the level of exposure of canine populations.

Materials and Methods

Sample collection

During 2012–2014, a total of 344 clinically healthy owned dogs living in urban and suburban settings in Umbria (central Italy), presenting to the Veterinary Teaching Hospital of Perugia and local private clinics, were included in the survey. No dog had a history of traveling outside the area or a history of antibiotic administration during the previous 2 months. Oral consensus was obtained from each dog owner. Blood samples were aseptically collected in a serum-separating tube for serologic analyses, and in sterile ethylenediaminetetraacetic acid vacutainers for biomolecular investigation. Sera were obtained by centrifugation of blood at 3000 g for 10 min. Samples were frozen at −20°C until used.

Feeding ticks were collected with sterile tweezers from the sampled dogs and species, and instars were determined based on the morphometric characteristics following conventional keys (Manilla 1998). After collection, ticks were placed individually in vials with 70% ethanol and stored at −20°C until DNA extraction.

Serological tests

Canine serum samples were analyzed by indirect immunofluorescence assay (IFA) using commercial antigens (MegaCor^® Diagnostik, Horbranz, Austria) of two different SFG rickettsial agents (R. conorii and R. rickettsii) to detect IgG antibodies against these agents. All samples were screened at starting dilution of 1:64 in a phosphate-buffered saline solution (pH 7.2), as described in the manufacturer's protocol. Seropositive samples were subsequently diluted to determine the endpoint titer. Positive and negative controls were included in each assay, and slides were examined under a fluorescence microscope (Axioscope 2, Zeiss) at 400× or 1000× magnification.

DNA extraction and molecular analysis

All the buffy coat samples and individual ticks were subjected to genomic DNA extraction using a QIAmp^® Blood and Tissue Extraction Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's guidelines. A negative control was added to each extraction. All of the extracted samples were analyzed using a spectrophotometer (Nanodrop; Thermo Scientific, Middlesex, MA) and electrophoresis on 1% agarose gel to determine the quality of the DNA. A PCR protocol amplifying a fragment of the citrate synthase-encoding gene (gltA) was used to detect Rickettsia spp. DNA (Regnery et al. 1991). PCR products were purified using Wizard SV Gel and PCR Clean-up System (Promega Corporation, Madison, WI) in accordance with the manufacturer's recommended protocol and subjected to direct sequencing (BioFab srl, Italy). All gel-purified PCR products were sequenced using gene-specific primers. The final sequences were subjected to a Basic Local Alignment Search Tool (BLAST) analysis against prokaryotic databases to confirm specific amplification.

Results

Overall 56/344 dogs (16.3%; 95% CI, 12.4–20.2%) tested positive to at least one of the SFGR tested by indirect fluorescent antibody technique (IFAT). An overall seroreactivity against R. conorii and R. rickettsii was detected in 38 (11%; 95% CI, 7.8–14.4%) and 46 samples (13.4%; 95% CI, 9.8–16.9%), respectively. Specifically, 10 dogs (2.9%) tested positive exclusively for R. conorii, 18 (5.2%) were positive exclusively for R. rickettsii, and 28 (8.1%) reacted against both rickettsial species. Serum endpoint titers ranged from 1:64 to 1:2048 for R. conorii, and from 1:64 to 1:256 for R. rickettsii. The distribution of seroreactivities according to the antibody endpoint titers is given in Table 1. There were no PCR-positive samples from canine buffy coats.

Table 1.

Seroreactivity to Rickettsia rickettsii and Rickettsia conorii Antigens and Distribution of Serum Endpoint Antibody Titers by IFAT

		Endpoint titers Rickettsia rickettsii
		Negative	1:64	1:128	1:256	1:512	1:1024	1:2048	No. of dogs
Endpoint titers R. conorii	Negative	288	6	9	3	0	0	0	306
	1:64	2	2	3	1	0	0	0	8
	1:128	4	3	1	0	0	0	0	8
	1:256	4	0	0	1	0	0	0	5
	1:512	0	4	4	5	0	0	0	13
	1:1024	0	2	0	0	0	0	0	2
	1:2048	0	2	0	0	0	0	0	2
No. of dogs		298	19	17	10	0	0	0	344

IFAT, indirect fluorescent antibody technique.

A total of 607 adult ticks were removed from the coats of 298 out of 344 sampled dogs; 395 specimens (304 females, 80 males, and 11 nymphs) were morphologically identified as Rhipicephalus sanguineus and 212 (148 females, 50 males, and 14 nymphs) as Ixodes ricinus. Female ticks presented a different grading of engorgement.

PCR products of the expected length were obtained in 39 (18.4%; 95% CI, 13.2–23.6%) I. ricinus and in 10 (2.5%; 95% CI 1–4.1%) R. sanguineus specimens. Sequencing of the rickettsial gltA products and BLAST analysis revealed identity with R. conorii in all the 10 specimens (2.5%) isolated from R. sanguineus. Nine out of the 10 R. conorii isolates were obtained from tick specimens collected from seronegative dogs, and one specimen from a dog tested positive for both R. rickettsii and R. conorii by IFA (Table 2).

Table 2.

Correspondence Between PCR Performed on Ticks and Antibody Detection in Dogs

		Seronegative (N = 288)	R. conorii-seropositive (N = 10)	R. rickettsii-seropositive (N = 18)	R. rickettsia + R. conorii–seropositive (N = 28)	Overall no. of ticks
Rhipicephalus sanguineus (N = 395)	R. conorii—positive PCR	9	0	0	1	10
Rhipicephalus sanguineus (N = 395)	Negative PCR	342	6	22	15	385
Ixodes ricinus (N = 212)	Rickettsia helvetica—positive PCR	6	1	0	0	7
	Rickettsia monacensis—positive PCR	28	0	0	4	32
	Negative PCR	147	8	8	10	173

Seven isolates (3.3%) from I. ricinus had identical sequences with R. helvetica (GenBank accession nos. KY231194–KY231200) and the remaining 32 (15.1%) were confirmed as R. monacensis (GenBank accession nos. KY231201, KY213882, KY213883, KY203361–KY203389), as already stated in one of our previous articles (Morganti et al. 2017). Among the seven ticks testing positive for R. helvetica, six were recovered from the coats of seronegative dogs and one from a dog that tested positive for R. conorii antibodies; the 32 isolates of R. monacensis were obtained from 28 seronegative and 4 R. conorii/R. rickettsii-positive dogs, respectively (Table 2).

Discussion

The seroprevalence detected against Rickettsia spp. in the sampled dog population (16.3%) is within the range observed in previous surveys conducted in Italy (15–77.9%; Torina and Caracappa 2006, Pennisi et al. 2012, Solano-Gallego et al. 2015, Vascellari et al. 2016, Traversa et al. 2017) and shows a non-negligible circulation of SFGR in the investigated geographic areas. The IFAT is considered the gold standard for antibody detection against rickettsiae both in humans and in animals, but it has limitations linked to the cross-seroreactivity existing within the SFGRs. However, the seroreactivity stimulated by an SFGR, which is homologous to the antigenic source, is often higher than that developed by an heterologous species (Pinter et al. 2008). In a multicenter study conducted in central northern Italy, antibodies against rickettsiae were detected in 113 out of 150 (77.9%) sampled dogs, of which 27 and 1 dogs were seropositive to R. conorii and Rickettsia typhi alone, respectively, and all the other dogs reacted to two or more rickettsial antigens (Traversa et al. 2017). Although seropositivity due to a real contemporary exposure to different rickettsiae should not be ruled out, the authors suggest the occurrence of cross-reactions within rickettsiae antigens.

In this study, the IFAs were carried out using two commercial kits having R. conorii and R. rickettsii-infected cells as source of antigens, respectively; in fact, a very limited selection of antigens for SFGR (e.g., R. rickettsii in the United States or R. conorii and R. rickettsii in Europe) is commercially available.

A total of 42 out of 56 seroreactivities detected here were probably not due to a specific SFGR; in fact 18 reacted exclusively against R. rickettsii at medium–low titers, and 24 samples tested positive for both R. conorii and R. rickettsii with medium–low titers. Since the agent of the Rocky Mountain Spotted Fever has never been reported in the Old World to date, the evidence of a degree of cross-reactivity among these two rickettsiae suggests that these seroreactivities could be stimulated by other SFGR, antigenically correlated to R. conorii and R. rickettsii, as speculated elsewhere and stated in the instructions accompanying the serological kit used (Vascellari et al. 2016). In contrast, 14 reacting sera were likely stimulated specifically by R. conorii because 10 reacted exclusively against this species and a further 4 showed titers against R. conorii, which were at least four-fold higher than R. rickettsii.

Notwithstanding the seroprevalence observed against SFGR, no DNA was detected from canine blood by molecular assay, which could have helped to assess the identity of the infection source stimulating the antibody response. The lack of concordance between serological and molecular tools may be attributed to the fact that the dogs were likely to have been exposed to rickettsiae; however, rickettsiemia lasts 2–10 days and the bacteria probably would get cleared rapidly from the blood (Parola et al. 2005b). In fact, it is frequently difficult to find a proven etiology in dogs with suspected rickettsiosis, which is why a negative PCR result in canine whole blood should not be used to guide treatment decisions (Nicholson et al. 2010).

The present findings differ from those obtained by Traversa et al. (2017) who detected 44 (30.3%) Rickettsia spp. DNA from 113 dogs tested seropositive for Rickettsia spp. This represents a very high rate of positive molecular detection; in fact, molecular surveys carried out, also on sick dogs, from several regions in Italy showed a very low positivity rate, ranging from 0.4% to 3.3% (Solano-Gallego et al. 2008, 2015).

The canine population sampled in this study did not show clinical signs referable to SFG diseases. However, Solano-Gallego et al. (2006a) reported R. conorii infection in three acutely ill febrile Yorkshire terrier dogs from Sicily, supported by PCR and seroconversion.

Molecular surveys carried out on sick dogs from various regions in Italy showed a very low positivity rate, ranging from 0.4% to 3.3% (Solano-Gallego et al. 2008), and, in most cases, Rickettsia spp. infections were diagnosed based on seroconversion in IFAT rather than molecular tools (Solano-Gallego et al. 2015).

A molecular study reported the detection of only three SFGR, that is, R. conorii, R. helvetica, and R. monacensis in feeding ticks, whereas the entire variability of rickettsiae in ixodid populations across Italy is known to be higher (Otranto et al. 2014). The rate of infection of R. conorii detectable in R. sanguineus was moderate (2.5%) although in accordance with the biomolecular findings obtained in molecular surveys conducted in Italy and in Europe on feeding ticks collected from dogs (Psaroulaki et al. 2003, Scarpulla et al. 2016).

These data have a notable epidemiological value given the efficient transovarial transmission of R. conorii (Parola et al. 2013), and the fact that all life stages of R. sanguineus, which are mainly associated with dogs, may be transported in domestic and peridomestic environments. There have been reports of a concurrent outbreak of R. conorii infection in both dogs and humans living in proximity with each other (Paddock et al. 2002, Nanayakkara et al. 2013). R. helvetica and R. monacensis have been detected in questing and feeding ticks in various parts of Europe (Parola et al. 2013). Although it has been generally accepted that I. ricinus is the main vector of these pathogens, with a rate of detection ranging from 1.5% to 27% for R. helvetica (Stańczak et al. 2018) and from 1% to 42.8% for R. monacensis (Rizzoli et al. 2014), isolates have also been detected in other tick species, that is, Dermacentor reticulatus and Ixodes hexagonus (Dobec et al. 2009, Sprong et al. 2009). In our study, the prevalence of R. helvetica in I. ricinus was 3.0%, which is in the range of infection observed in feeding I. ricinus (2.6–13.37%) collected in wild habitats from other areas of Italy (Corrain et al. 2012, Pistone et al. 2017). R. monacensis has also been observed in ticks from central and northern Italy with a prevalence rate of infection up to 13% (Beninati et al. 2002, Capelli et al. 2012), but never before in specimens collected from domestic animals from periurban or urban habitats. Surprisingly, we did not detect any R. massiliae in R. sanguineus ticks in contrast to other colleagues in central Italy (Mancini et al. 2015, Scarpulla et al. 2016) and in Sardinia (Chisu et al. 2017).

Dogs may act as a competent reservoir for R. conorii (Parola et al. 2009); however, the role of dogs in the ecoepidemiology of R. monacensis and R. helvetica infections still needs to be defined through future field and experimental studies. R. conorii may cause clinical illness both in humans (Solano-Gallego et al. 2015) and dogs (Solano-Gallego et al. 2008). In contrast, the clinical significance of R. monacensis and R. helvetica has to date only been demonstrated in human patients with clinical manifestations of MSF-like illness (Madeddu et al. 2012). The pathogenic role of SFGR other than in domestic animals is still poorly understood.

In our study, the PCR-positive ticks were removed from dogs whose buffy coats tested negative. However, five PCR-positive tick specimens were collected from dogs tested seropositive for SFGR. No correlation between the positive recovery of SFGR from ticks feeding on dogs and the serological status was shown. In fact, because ticks spend more days feeding on dogs than the time needed for seroconversion, the pathogens inoculated from the collected infected ticks cannot be the same as those stimulated detectable level of antibodies in dogs tested serologically positives. At the same time, the lack of bacteremia in the canine blood samples does not suggest that the PCR-positive ticks acquired SFGR infections by feeding on the sampled dogs, but most probably through previous blood meals conducted on other hosts and transstadially transmitted.

Ticks removed from dogs can act as vectors or only as carriers of infection acquired in previous other hosts (Leulmi et al. 2016), as in the present case, and dogs might facilitate the transport of infected ticks in peridomestic environments, representing a public health concern.

In conclusion, our study highlights the non-negligible circulation of SFGR in dog populations and their ticks in central Italy. Further studies on rickettsioses should be performed to assess their epidemiological and clinical relevance in canine populations in Italy. This will enable their prevalence to be estimated and promote the use of antivectorial measures (e.g., antifeeding products) to protect both animal and public health.

Footnotes

Acknowledgment

The authors thank Carlo Sanesi for his technical assistance.

Author Disclosure Statement

No competing financial interests exist.

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