Simultaneous Exposure to Angiostrongylus vasorum and Vector-Borne Pathogens in Dogs from Italy

Several drivers have recently fostered the expansion of Angiostrongylus vasorum throughout Europe, where Vector-Borne Pathogens (VBPs) are also spreading. However, the level of simultaneous risk of infection is still unknown in canine populations. This study evaluated the simultaneous exposure to A. vasorum and major canine VBPs in dogs of Italy. Sera of 294 dogs were subjected to two ELISAs, detecting A. vasorum circulating antigens and antibodies against the parasite, and to the following assays: (i) SNAP® 4DX (IDEXX Laboratories Inc.) detecting Dirofilaria immitis antigens, and antibodies vs. Borrelia burgdorferi, Anaplasma spp. and Ehrlichia spp. and (ii) IFAT for the detection of antibodies vs. Leishmania infantum, Babesia canis and Rickettsia conorii. Twenty-two (7.5%, CI: 4.8–11.1%) and six (2%, CI: 0.7–4.4%) dogs scored positive for circulating A. vasorum antibodies and antigens, respectively. Seventeen dogs (5.8%, CI: 3.4–9.1%) were positive for A. vasorum antibodies + at least one VBP, three (1%, CI: 0.2–3%) for A. vasorum antigen + at least one VBP, while one dog (0.3%, CI: 0.01–1.88%) was positive for A. vasorum antigen + A. vasorum antibodies + B. canis antibodies. These results show that dogs living in different regions of Italy are at risk of simultaneous infections with both A. vasorum and VBPs. Despite the same scenario being likely in other countries of Europe, the current knowledge is scant. Therefore, further studies are warranted to amplify current epizootiological information and to understand whether control programs should be improved.


Introduction
Over the last decade, nematodes of the genus Angiostrongylus have changed their distribution in Europe, with the spread of Angiostrongylus vasorum in canids [1,2], and new or unexpected findings of other species like Angiostrongylus cantonensis, Angiostrongylus chabaudi and Angiostrongylus daskalovi in different animal hosts [3][4][5]. Among them, A. vasorum is now regarded as a primary parasite of dogs throughout European regions, e.g., Iberian Peninsula, Mediterranean Basin, and northern, central, and eastern Countries [6][7][8][9].
Detailed information on the different combinations of seropositivity to the tested pathogens are reported in Tables 1 and 2, respectively.

Discussion
The present results show that dogs living in Italy can be simultaneously exposed to A. vasorum and several VBPs, with different positivity rates according to the investigated geographic areas.
Exposure to A. vasorum was herein found in all the studied macro-areas. Thus far, this metastrongyloid has been rarely recorded outside Central and Southern Italy, in particular seldom in Northern-Western territories [41]. Nevertheless, a relatively high proportion of dogs of the present study tested seropositive in Site A. This and other recent findings [42] indicate that A. vasorum has currently expanded its distribution range and confirm that also dogs living in North-Eastern regions of Italy are at risk of canine angiostrongylosis. A past study investigated the occurrence of A. vasorum in dogs from a small region (Liguria) of North-Western Italy, showing~1 and~3% positivity to Ag and AB, respectively [47]. The present data indicate that A. vasorum can occur in Northern Italy more frequently than previously recorded, although the nematode remains more distributed in Central Italy rather than in the North of the country [41]. This difference is likely due to the chemoprevention for D. immitis using macrocyclic lactones (i.e., milbemycin oxime and moxidectin) that are active also against A. vasorum, routinely performed in Northern Italy [44]. No positive dogs were detected in Giglio Island of Site B, most probably attributable to the absence of foxes, the natural reservoirs of A. vasorum, that are not present in minor Italian islands [48]. However, a future introduction of the parasite in such spots, e.g., via dogs travelling with their owners in touristic areas, is not unlikely, as metastrongyloids can perpetuate their lifecycle in absence of their natural wild reservoirs [49].
As in previous studies, the number of dogs with detectable A. vasorum Ab was here higher if compared to the number of dogs showing circulating Ag [8,47,50]. Many reasons can account for this discrepancy, e.g., the inclusion of dogs (i) sampled during prepatency when Ag is not detectable yet, (ii) after a natural clearance of the infection (iii) after antiparasitic treatment, based on longer persistence of Ab [51,52]. Moreover, individual variations in the production of Ab against A. vasorum (i.e., the production of antibodies may vary individually and some dogs can even remain antibody negative) may further reduce the number of detected positive dogs [53,54].
Dirofilaria immitis was here found in all study sites and most of the positive dogs lived in central regions, which are now regarded as stably enzootic. Seropositivity to L. infantum was detected in dogs living on the island of Giglio and in Umbria/Abruzzo/Marche, but not in Friuli Venezia Giulia region (i.e., Site B, the easternmost study site). The lack of positive dogs may not reflect a true absence in this latter region, as competent vectors are already established in this area [55]. The presented results and recent analysis [56] ultimately prove that, to date, all Italian regions should be considered potentially enzootic for D. immitis and L. infantum and corroborate the changing epizootiological scenario of dog dirofilariosis in Italy, following an expansion from Northern areas southwards [56,57]. Hence, worthy of note is the establishment of hyperendemic foci of dirofilariosis in the extreme South of Italy, and vice versa for leishmaniosis [58][59][60][61].
The seropositivity of the here studied dog populations to tick borne pathogens fits with past and most recent surveys carried out in Italy and confirm that dogs are exposed to a multitude of pathogens throughout the country [17,39,62,63] (Table 3). The level of exposure to given pathogens in each geographic area is in accordance with the biology of their vectors. For instance, R. conorii and Anaplasma spp. are transmitted by ticks (e.g., Rhipicephalus sanguineus and Ixodes ricinus) which are widespread and active throughout the year [64][65][66]. Conversely, the main vector of B. canis (Dermacentor spp.) is little distributed in northern territories and limited to a short season [65,67]. On the other hand, the positivity values in central and southern areas could be due to possible cross-reactions with other Babesia species, e.g., Babesia vogeli or others, transmitted by widely spread ticks like R. sanguineus [63,68,69]. Analogously, the occurrence of B. burgdorferi in northern Italy is explained by the wide distribution of its major vector I. ricinus in northern areas, with forested environments and suitable climate [65].  Worthy of note is the positivity to R. conorii, as dogs are reliable epidemiological sentinels for monitoring potential risk of human exposure to spotted fever group rickettsioses. Regardless of the potential of cross-reactions with other Rickettsia species, R. conorii is the most frequent affecting dogs in the Mediterranean basin, and the present data indicate a high exposure to infected tick arthropods, which act as vectors of these pathogens [63].
Knowledge on the simultaneous risk for angiostrongylosis and VBDs in dog populations of Europe is very poor, as studies have thus far been conducted only in a few countries, e.g., Portugal [37] and Bulgaria [70,71]. In the latter studies, cases of co-exposure /co-infection by A. vasorum and VBPs have not been found [37]. In Italy, a similar study was carried out a few years ago in kenneled dogs of central Italy, though limited to L. infantum, A. vasorum and Dirofilaria spp. (and other endoparasites) [106]. Unfortunately, a comparison with the number of dogs co-infected by VBPs and A. vasorum is not possible, as this information is not shown in ref. [106]. Further differences in the diagnostic methods and/or protocols, e.g., the use of Baermann test for A. vasorum vs. antigen detection by ELISA or rapid assay [52], of higher screening dilution for L. infantum and of a different commercial kit for D. immitis antigen detection, impair any comparison with the present results.
This study has confirmed once again the applicability of serological methods in large-numbers epizootiological studies and investigations on pet metastrongyloid angiostrongylids [47,50,51, [132][133][134][135]. The usefulness of serology should be considered in planning future studies relying on standardized methods towards comparable results from different regions and settings.
Many European countries are enzootic for A. vasorum and VBPs (Table 3). Hence, the simultaneous exposure to these pathogens is a realistic threat even where this has not been verified by purposed studies, especially if one considers that these pathogens are spreading in both enzootic and previously free regions [35,36,136,137].
As the scenario of the distribution of canine parasites and VBDs is in continuous evolution, further large-scale studies are warranted for more comprehensive knowledge on the epizootiological risks in European territories, and to understand if and how the routine use of broad-spectrum endo-ectoparasiticides needs a refinement based on local settings. The use of ectoparasiticides and/or repellents is the most effective strategy to minimize the risk of diseases transmitted by arthropod vectors, while dog angiostrongylosis and dirofilariosis can be prevented by the monthly administration of macrocyclic lactones like milbemycin oxime and moxidectin [138][139][140][141]. Although cases of LOE/ML-resistant strain of heartworm are known to occur in North America, this is not the case in Europe [142,143]. Several broad-spectrum formulations are available on the market to protect dogs and those containing an isoxazoline and either or milbemycin oxime or moxidectin may be used for the prevention of ticks, D. immitis and A. vasorum infections. Although isoxazolines do not have a repellent activity, their fast onset of action has the potential to reduce the risk of disease transmission of tick-borne pathogens that are not immediately transmitted to the host, such as Babesia or Borrelia [144]. Repellents (e.g., synthetic pyrethroids) have a fast onset of action on many insects and ticks, and they are also irritant or repulsive for many ectoparasites [144,145]. Their use is thus particularly important for the prevention of L. infantum [146,147] and of pathogens that are passed within a few hours or immediately after the tick's bite [148]. In fact, in recent years, the use of systemic isoxazolines to reduce the transmission of leishmaniosis or human VBDs has been evaluated [149][150][151]. Further studies in this field are still necessary and, to date, the combination of repellency and parasiticidal activity is still the best approach to prevent pathogen transmission by arthropods. Therefore, an adequate preventative measure should be put in place on a case-by-case basis considering the lifestyle of the dog, the geographical distribution of vectors and of the related transmitted diseases.
In conclusion, canine populations of Italy and in parts of other European countries are at factual risk of angiostrongylosis and VBDs at the same time. A standardization of the diagnostic techniques used in future epizootiological surveys is here advocated, along with the establishment of international monitoring tools with real-time mapping of positive animals in determined areas, e.g., as available in the USA via the CAPC (capcvet.org).

Materials and Methods
Overall, 294 privately owned dogs from North-Eastern (Friuli Venezia-Giulia and Veneto = site A), Central-Western (Giglio Island of Tuscany, Latium = Site B) and Central-Eastern (Umbria, Abruzzo, Marche = Site C) Italy were enrolled in the study, i.e., 67, 82 and 145 in each site, respectively. All the dogs were apparently healthy at the clinical examination. Overall, 150 dogs were female, 144 were male and the median age was 48 months. Of the 294 dogs included, 187 lived permanently outdoors, while 107 lived mostly indoors.
Dogs were enrolled during routine medical checks by local veterinarians and all dog owners signed a written consent form before sampling. All sera samples were subjected to two ELISAs detecting A. vasorum circulating antigen (Ag) (Sensitivity 95.7%, Specificity 94.0%) and specific antibodies (Ab) (Sensitivity 81.0%, Specificity 98.8%) against the parasite [54,152]. The optical density (OD) threshold was determined for both Ag (A 405 nm = 0.153) and Ab test (A 405 nm = 0.266) using the mean OD value plus three standard deviations of 291 (Ag) and 244 (Ab) samples. All dogs were tested also for VBPs using Institutional Review Board Statement: Ethical review and approval were waived for this study, as dogs were sampled in the framework of their routine medical checks coordinated by local veterinarians. In addition, according to local laws and regulations, a consent form signed by each single dog owner or those legally responsible.