Parasitol Res DOI 10.1007/s00436-014-3794-z
ORIGINAL PAPER
New insights onto cardiopulmonary nematodes of dogs in Sardinia, Italy A. P. Pipia & A. Varcasia & G. Tosciri & S. Seu & M. L. Manunta & M. C. Mura & G. Sanna & C. Tamponi & E. Brianti & A. Scala
Received: 14 January 2014 / Accepted: 28 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Dog heartworms Angiostrongylus vasorum and Dirofilaria immitis cause severe parasitological diseases; the importance of these parasitosis is growing due to their health impact on animals, the possible zoonotic implications and the recent spreading across several European countries and previously non-endemic areas. The aim of this study is to update the epidemiological scenario of cardiopulmonary nematodes A. vasorum and D. immitis in dogs of Sardinia island and to perform a morphological identification of larvae by the use of the Baermann and Knott techniques respectively and the molecular characterization of the mitochondrial cytochrome c oxidase subunit I (cox1) and the second ribosomal transcribed spacer region (ITS-2) of larvae L1 of A. vasorum. In the present study, 3.4 % (5/146) of dogs resulted positive at Baermann technique for A. vasorum while 8.9 % (61/684) to D. immitis. If on one side A. vasorum can be considered an emerging parasite in Sardinia, the parasitic pressure and the risk of infection for D. immitis in the island seems to be increased compared with the recent past.
A. P. Pipia : A. Varcasia : G. Tosciri : S. Seu : M. L. Manunta : M. C. Mura : G. Sanna : C. Tamponi : A. Scala Dipartimento di Medicina Veterinaria, Università degli Studi di Sassari, Sassari, Italy E. Brianti Dipartimento di Scienze Veterinarie, Università degli Studi di Messina, Messina, Italy A. Varcasia (*) Parassitologia Veterinaria – Dipartimento di Medicina Veterinaria, Via Vienna 2, 07100 Sassari, Italy e-mail: [email protected]
Introduction The nematodes Angiostrongylus vasorum (Nematoda, Metastrongyloidea) and Dirofilaria immitis (Spiruridae, Onchocerchidae) are cardiorespiratory parasites infesting dogs, foxes, and other carnivores, which localise in the pulmonary circulation and right heart of their definitive hosts (Schnyder and Deplazes2012). They have an indirect life cycle requiring an intermediate host for their development represented by gastropods and mosquitos for Angiostrongylus vasorum and D. immitis, respectively (Levine et al. 1980; Traversa et al. 2013). Recently, the attention on cardiopulmonary nematodes has been increased due to their spreading in many European countries and the appearance in areas previously free of infection (Traversa et al. 2010a). This scenario may be due to several factors like global warming; changes in vectorial dynamics (Traversa et al. 2010a); movements of dogs for activities such as tourism, hunting and so on (Pietrobelli 2010). The interaction between host, vectors, and the environment play an important role on the increase of these parasitoses (Traversa et al. 2010a). These nematodes cause clinical forms primarily with different respiratory symptoms and severity in accordance to their localization and burden and hostrelated factors (Di Cesare et al. 2011). Age, immune response and presence of concomitant diseases can also determine fatal consequences in the infected animals (Conboy 2009; Traversa et al. 2010a, b). The metastrongyloid Angiostrongylus vasorum, was described for the first time in the south of France (Serres E. 1854) and has been reported as a common parasite in foxes and less frequently in dogs of several countries of Europe, Africa and North and South America (Guardone et al. 2013).
Parasitol Res
In Sardinia, it was first detected in foxes (Leoni et al. 1986) and recently reported in a dog (Sanna et al. 2012). Recent surveys demonstrate an increasing incidence of angiostrongylosis in different European countries (Morgan et al. 2009; Helm et al. 2010; Traversa et al. 2010a) and its spread in previously non-endemic areas as Holland, Germany and Greece (Papazahariadou et al. 2007; vann Doorn et al. 2009; Barutzki and Shaper 2009, 2011). Some Italian regions can be considered endemic for this parasitoses (Traversa et al. 2013), with the highest prevalences in northern and central ones (Di Cesare et al. 2011; Traversa and Guglielmini 2008), in which ideal climatic conditions for the maintenance and development in intermediate host can be found (Morgan et al. 2009). Dirofilaria immitis is also considered an emerging zoonotic agent in different European countries (Genchi et al. 2011). In Italy, current data on canine dirofilariasis confirms the expanding trend of this parasite as observed in the rest of Europe (Genchi et al. 2009). Today D. immitis is present, not only in the hyperendemic area of the Po River Valley, but also in the northern part of Italy (Friuli-Venezia Giulia), in central Italy (Umbria, Abruzzo and Tuscany), in the south (Campania, Apulia and Calabria) and in the islands (Sicily and Sardinia) (Magi et al. 2011). The first survey on canine heartworm in Sardinia was carried out in the 1960s and showed a prevalence of 1.6 % for D. immitis (Arru et al. 1968). After more than 30 years Scala et al. (2004) with a similar survey concerning the years 1998–2003 found D. immitis in 3.8 % of examined dogs, even if in some areas of the island such as the Oristano and SulcisIglesiente higher prevalences (respectively, 17.1 and 7.4 %) were found. Similarity to other countries dirofilariasis in Sardinia has a spotted distribution, with prevalences varying according to locations and eco-zones suitable for vectors as it was evidenced in one recent survey carried out in the South East coast (Scala et al. 2011). Filaroid species seems to find a good environment in the island, where recently it has been found for the first time after almost one century the adult
Fig. 2 Caudal end of L1 larva of A. vasorum with the typical dorsal spine
specimens of Cercopithifilaria bainae (Otranto et al. 2013; Solinas et al.; 2013). Sardinia Island is known as a holiday destination due to its beautiful beaches. Statistical data on the tourism income in Sardinia estimated more than 1.9 million arrivals in 2012 (1.9 % of Italy tourism), highlighting also the fact that since 2000 to 2012, the number of foreign people coming in Sardinia for their vacation has dramatically increased from 24 to 42 %. Interestingly, movement of tourists and of their pets have been recognised as a potential route for the introduction and spread of non-endemic pathogens (Genchi et al. 2011). Therefore, a constant surveillance of the epidemiology of the most important or emerging parasitoses in Sardinia, like cardiopulmonary filariosis and angiostrongylosis, is of importance considering also the insularity and the suitable climate that could favour their maintenance and spreading. This study aimed to investigate and update the epidemiology of canine heartworm, being that the last extensive survey was carried out in 2004, and to study, for the first time, the epidemiology and genetic make-up of Angiostrongylus vasorum in Sardinia.
Materials and methods From July 2012 to June 2013, a total of 146 faecal samples were obtained from dogs (72 females and 74 males) referred for routine vaccination or clinical visit at the Veterinary Teaching Hospital of the University of Sassari, Italy. For each Table 1 Multiple infections for filarial species in examined dogs
Fig. 1 First-stage larva (L1) of Angiostrongylus vasorum
Kind of infection
Number of positive
Prevalence (%)
D. immitis + D. repens D. immitis + A. reconditum; D. immitis + D. repens + A. reconditum Total
17 12 14
2.5 1.7 2
43
6.3
Parasitol Res Table 2 Prevalences per class of age and statistical analysis of data for D. immitis
Age groups (years)
Examined dogs (no.)
No. of positives
D. immitis (%)
Odds ratio
Statistical analysis
>0≤3 >3≤6 >6 Total
306 187 191 684
31 14 16 61
10.1 7.5 8.4 8.9
1.00 0.72 0. 81 1.00
χ2 for linear trend=0.579;
presence or absence of symptoms of canine filariosis by D. immitis was also performed. The identification of microfilariae was performed according to the morphometric keys as described by Euzeby (1981). L1 larvae (Angiostrongylus vasorum) and mfs (D. immitis) were examined and measured using a digital image processing system (LC micro Image Acquisition Software V.5.2, Olympus). Data collected during sampling were coded and made available for computerised analyses using an electronic spread sheet (Excel®, Microsoft Corp., Redmond, WA, USA). Data were then analysed using the statistical package Epi-Info (version 7.0, CDC/WHO, Atlanta, GA, USA).
sampled animal data on sex, age and presence/absence of respiratory symptoms and clinical signs were also recorded. Faecal samples were individually collected soon after emission and examined with the Baermann technique (Euzéby 1981) for recovering migrating broncho-pulmonary nematodes (BPN) L1 larvae. Briefly, about 5 g of faeces were placed in the centre of a cheesecloth layer forming a pouch put in a funnel filled with water and kept at room temperature. After 24–48 h, a few drops of Lugol’s solution were added to the sediment obtained after centrifugation and thereafter transferred onto a slide and microscopically examined at ×100 magnification for L1 recovery. Larvae were identified using morphometrical keys for Angiostrongylus vasorum (Moeramans et al. 2011; Traversa et al. 2010a). After light microscopy, DNA was extracted from positive sediments with L1 larvae with a commercial kit (High Pure PCR Template Preparation kit, Roche Diagnostics, Mannheim, Germany). Molecular characterization was performed for the mitochondrial cytochrome oxidase subunit 1 (coxI) and for the second ribosomal transcribed spacer region (ITS-2) as described by Jefferies et al. (2008). PCR products were then purified using ChargeSwitch ® PCR Clean-Up Kit (Invitrogen™) and sequenced (MWG Biotech/M-Medical, Milan, Italy). Sequences were then aligned using MEGA V.5.2.1 and compared with those available in the GenBank™ using BLAST analysis. In the same period, a parallel survey was carried out on 684 individual blood samples of dogs that were referred to local practitioners from all Sardinia provinces for routine health care examination. Blood samples were analysed using Knott’s technique (Lindsey 1965) for the detection of circulating microfilariae (mfs). Data on sex, attitude (i.e., hunting, guard, pet), and housing (i.e., outdoor or indoor) were acquired for all dogs. A clinical examination to ascertain the
Table 3 Prevalences (P) and odd ratio for D. immitis and the other filarial species for attitude
Attitude
Hunting Guard dogs Pets Total
Total
420 64 200 684
P=0.446
Results First-stage (L1) larvae of Angiostrongylus vasorum were found in 3.4 % (5/146) of examined dogs. No significative difference between sex was observed (2.7 % of females and 4.1 % of males; Yates corrected χ2 =0.00; P=0.975). L1 larvae of Angiostrongylus vasorum, presents a typical indentation of the cuticle (dorsal notch) and a bulging at the dorsal surface of the tail (dorsal spine) (Moeramans et al. 2011) with a mean length of 340 μm±4.3 (Figs. 1 and 2). Interestingly, all positive cases were found in dogs aged above 24 months except of a 3-months puppy. No significant difference have been observed between prevalences of dogs under or above 24 months aged (χ2 Yates corrected = 0.483; P = 0.487). Among Angiostrongylus vasorum positive animals, a 12year-old dog showed suggestive respiratory symptoms such as chronic cough, while two other dogs of 11 and 10 years old, respectively, presented severe pathological conditions such as cardiac failure and pulmonary hypertension.
D. immitis No.
P (%)
37 13 11 61
8.8 20.3 5.5
O. R.
1.00 2.21 0.60
D. repens No.
P (%)
36 12 13 61
8.7 20.3 6.7
O. R.
1.00 2.46 0.74
A. reconditum No.
P (%)
72 7 7 86
17.1 10.9 3.5
O. R.
1.00 0.59 0.18
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Biomolecular characterization of larvae allows us to confirm the morphological identification of the first-stage larvae of Angiostrongylus vasorum (GenBank accession numbers KF270685.1, KF270683.1). The alignment of our sequences with BLAST showed a homology of 100 % within our samples and with the Cox1 and ITS-2 sequences of An giostrongylus vasorum available in G enBank (EU493165.1, EU627597.1). In the epidemiological survey on filariosis, 20.9 % (143/ 684) of examined animals tested positive for at least one f i l a r i a l s p e c i e s . I n p a r t i c u l a r, m i c r o f i l a r i a e o f Acanthocheilonema reconditum were retrieved in 12.6 % (86/684) of samples, D. immitis in 8.9 % (61/684) and Dirofilaria repens in 8.9 % (61/684). Multiple infections were observed in 43 (6.3 %) dogs (Table 1). Prevalences grouped per sex for D. immitis were higher in males (9.5 %) than females (8.2 %) even if this difference was not statistically significant (χ2 =0.32; P=0.573). Stratification of data per class of age was not statistically significant (χ2 for linear trend=0.579; P=0.446) (Table 2). Regarding aptitude, even if guard dogs were more positive for D. immitis (20.3 %) compared to other evaluated categories, the difference was not statistically significant (χ2 for linear trend=1.005; P= 0.316) (Table 3). Microfilaremia for D. immitis was considerably higher in dogs living outdoor than indoor (10.3 vs 1.8 %; Yates corrected χ2 =7.13; P=0.007). Clinical signs as weakness, cough, wearing, weight loss, exercise intolerance, dyspnoea and tachypnea were reported in 6.6 % of examined dogs, and only 0.6 % of these animals were positive to D. immitis.
Discussion The present survey represents the first epidemiological survey on Angiostrongylus vasorum in Sardinia and allowed also the biomolecular characterization of this nematode in the island. Moreover, the paper updates data on the current distribution of D. immitis and other filarioids in dogs sampled across all the provinces. Particularly, prevalence found for Angiostrongylus vasorum (3.4 %) is consistent with those reported in Abruzzo (4.3 %) (Yates corrected χ2 =0.01; P=0.904) and in Latium (2 %) (Yates corrected χ2 =0.07; P=0.787) by Di Cesare et al. (2012) as well as in other European countries where this parasitoses is considered emergent, like Greece ( 1 . 1 % ) ( Ya t e s c o r r e c t e d χ 2 = 1 . 7 6 ; P = 0 . 1 8 4 ) (Papazahariadou et al. 2007). Sex and age do not seem to represent risk factors for these parasitoses. It seems therefore necessary to increase the attention of the vet practitioners on these parasitoses which should include Angiostrongylus vasorum in the differential diagnoses of dog’s cardiopulmonary diseases even in animals with few
or none respiratory symptoms, as suggested by Traversa et al. (2013). In addition, the research of larvae L1 of Angiostrongylus vasorum in dog’s faeces is not commonly performed by veterinarians in the island. Prevalence herein found for D. immitis shows that the parasitic pressure and the risk of infestation in the island are high, especially in guard dogs (27.9 %) and outdoor housed animals (10.5 %). These findings are in agreement with those reported by Giangaspero et al. (2013) in Apulia region. Age and sex do not seem to represent as risk factors for heartworm disease as previously described by Magi et al. (2011). Microfilaremia for filarial infections (20.9 %) is similar to that detected in Tuscany (23.2 %) by Magi et al. (2011). Particularly Acanthocheilonema reconditum (12.6 %; 86/ 684) seems to have a predominant role in Sardinia island and differs from the two other species for the intermediate hosts (fleas and ticks) (Scala et al. 2004; Genchi et al. 2005; Brianti et al. 2012) and the way of transmission, while D. repens is present with the same prevalences observed for D. immitis (8.9 %; 61/684). This scenario requires an implementation toward an accurate differential diagnosis especially between D. immitis and Acanthocheilonema reconditum that have similar length but differs for some morphological features and for clinical presentation and relevance. The morphometric analysis proved to be a useful, quick and cheap diagnostic tool and it represents the first step in the diagnosis of filarial infections. However, the discrimination between different species can be challenging in cases of mixed infections (Traversa et al. 2010b; Magi et al. 2011) or in cases with low parasitaemia (Magnis et al. 2013). This study shows that due to the high movements of tourists with pets in the island, mainly during spring and summer, the monitoring of cardiopulmonary parasites should be kept at the high level and included in differential diagnosis of dogs’ cardiopulmonary disease (Magnis et al. 2013). This scenario requires greater attention for the implementation of pharmacological prophylaxis, accurate diagnosis and correct treatment by the veterinary, for the protection of both animal and human health, taking into consideration the zoonotic potential of filarial species, particularly for D. repens and D. immitis (Genchi et al. 2011; Magi et al. 2011). In this sense, travelling dogs coming in Sardinia Island should be monitored for circulating mfs, treated with preventive drugs and reexamined for circulating mfs 6–7 months after they stay in the island (Genchi et al. 2011). In conclusion, the present work indicate that cardiopulmonary nematodes are present in Sardinia island where Angiostrongylus vasorum can be considered an emerging parasite while D. immitis confirmed a wide diffusion thus to include these parasites in the differential diagnosis of dogs cardiopulmonary disease (Di Cesare et al. 2011). Pharmacological prophylaxis should always be performed
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for travelling dogs coming in the island from other European and Italian regions, to prevent the risk of infection. Acknowledgements The research was funded by Regional Government of Sardinia, prot. CRP2 134 (L.R. 7, 2007).
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Magi M, Guardone L, Prati MC, Tozzini G, Torracca B, Monni G, Macchioni F (2011) Canine filarial infections in Tuscany, central Italy. J Helminthol 86:113–116 Magnis J, Lorentz S, Guardone L, Grimm F, Magi M, Nauche TJ, Deplazes P (2013) Morphometric analyses of canine blood microfilariae isolated by the Knott’s test enables Dirofilaria immitis and D. repens species-specific and Acanthocheilonema (Syn. Dipetalonema) genus-specific diagnosis. ParsiteVector 6: 48–52 Moeremans I, Binst D, Claerebout E, Van de Maele I, Daminet S (2011) Canine Angiostrongylus vasorum. Vlaams Diergen Tijds 80:319– 326 Morgan ER, Jefferies R, Krajewski M, Ward P, Shaw SE (2009) Canine pulmonary angiostrongylosis: the influence of climate on parasite distribution. Parasitol Int 58(4):406–410 Otranto D, Varcasia A, Solinas C, Scala A, Brianti E, Dantas-Torres F, Annoscia G, Martin C, Mutafchiev Y, Bain O (2013) Redescription of Cercopithifilaria bainae Almeida & Vicente, 1984 (Spirurida, Onchocercidae) from a dog in Sardinia, Italy. Parasit Vector 6(1): 132–142 Papazahariadou M, Founta A, Papadopoulos E, Chliounakis S, Antoniadou-Sotiriadou K, Theodorides Y (2007) Gastrointestinal parasites of shepherd and hunting dogs in the Serres Prefecture, Northern Greece. Vet Parasitol 148:170–173 Pietrobelli M (2010) Dirofilariosi canina: un esempio di federalismo parassitario? La sett Vet 698(5–6):7–18 Sanna G, Pinna Parpaglia ML, Corda A, Manunta L, Petruzzi V, Maciotta L, Varcasia A, Scala A (2012) First report of Angiostrongylus vasorum in a dog of Sardinia: parasitological findings. Proceedings of the XXVII SOIPA National Congress, Alghero, Italy, 26–29 June, p 291. Scala A, Atzori F, Varcasia A, Garippa G, Genchi C (2004) Canine filariosis in Sardinia: epidemiological updating (1998–2003). Atti Soc It Sci Vet 58:120–121 Scala A, Solinas C, Pipia A P, Sanna G, Varcasia A, Tosciri G (2011) Canine filariosis in Sardinia: Epidemiological findings in the Ogliastra Region. Trends in Veterinary Science LXV Annual Meeting of The Italian Society for Veterinary Sciences p. 73. Schnyder M, Deplazes P (2012) Cross-reactions of sera from dogs infected with Angiosrongylus vasorum in commercially available Dirofilaria immitis tests kits. Parasit Vector 5:258–262 Serres E (1854) Entozoaires trouvés dans l’oreille droite, le ventricule correspondant et l’artère pulmonaire d’un chien. J Vet Midi 7:70 Solinas C, Varcasia A, Brianti E, Giannetto S, Pipia AP, Columbano N, Tosciri G, Dantas-Torres F, Garippa G, Otranto D, Scala A (2013) Cercopithifilaria spp. in dogs in Sardinia Island (Italy). Parasitol Res (In press). Traversa D, Guglielmini C (2008) Feline aelurostrongylosis and canine angiostrongylosis: a challenging diagnosis for two emerging verminous pneumonia infections. Vet Parasitol 15:53–60 Traversa D, Di Cesare A, Conboy G (2010a) Canine and feline cardiopulmonary parasitic nematodes in Europe: emerging and underestimated. Parasit Vector 3:62–83 Traversa D, Aste G, Milillo P, Capelli G, Pampurini F, Tunesi C, Santori D, Paoletti B, Boari A (2010b) Autochthonous foci of canine and feline infections by Dirofilaria immitis and Dirofilaria repens in central Italy. Vet Parasitol 169:128–132 Traversa D, Di Cesare A, Meloni S, Frangipane di Regalbono A, Milillo P, Pampurini F, Venco L (2013) Canine angiostrongylosis in Italy: occurrence of Angiostrongylus vasorum in dogs with compatible clinical pictures. Parasitol Res 112(7):2473–2480 van Doorn DC, van de Sande AH, Nijsse ER, Eysker M, Ploeger HW (2009) Autochthonous Angiostrongylus vasorum infection in dogs in The Netherlands. Vet Parasitol 162:163–166
ORIGINAL PAPER
New insights onto cardiopulmonary nematodes of dogs in Sardinia, Italy A. P. Pipia & A. Varcasia & G. Tosciri & S. Seu & M. L. Manunta & M. C. Mura & G. Sanna & C. Tamponi & E. Brianti & A. Scala
Received: 14 January 2014 / Accepted: 28 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Dog heartworms Angiostrongylus vasorum and Dirofilaria immitis cause severe parasitological diseases; the importance of these parasitosis is growing due to their health impact on animals, the possible zoonotic implications and the recent spreading across several European countries and previously non-endemic areas. The aim of this study is to update the epidemiological scenario of cardiopulmonary nematodes A. vasorum and D. immitis in dogs of Sardinia island and to perform a morphological identification of larvae by the use of the Baermann and Knott techniques respectively and the molecular characterization of the mitochondrial cytochrome c oxidase subunit I (cox1) and the second ribosomal transcribed spacer region (ITS-2) of larvae L1 of A. vasorum. In the present study, 3.4 % (5/146) of dogs resulted positive at Baermann technique for A. vasorum while 8.9 % (61/684) to D. immitis. If on one side A. vasorum can be considered an emerging parasite in Sardinia, the parasitic pressure and the risk of infection for D. immitis in the island seems to be increased compared with the recent past.
A. P. Pipia : A. Varcasia : G. Tosciri : S. Seu : M. L. Manunta : M. C. Mura : G. Sanna : C. Tamponi : A. Scala Dipartimento di Medicina Veterinaria, Università degli Studi di Sassari, Sassari, Italy E. Brianti Dipartimento di Scienze Veterinarie, Università degli Studi di Messina, Messina, Italy A. Varcasia (*) Parassitologia Veterinaria – Dipartimento di Medicina Veterinaria, Via Vienna 2, 07100 Sassari, Italy e-mail: [email protected]
Introduction The nematodes Angiostrongylus vasorum (Nematoda, Metastrongyloidea) and Dirofilaria immitis (Spiruridae, Onchocerchidae) are cardiorespiratory parasites infesting dogs, foxes, and other carnivores, which localise in the pulmonary circulation and right heart of their definitive hosts (Schnyder and Deplazes2012). They have an indirect life cycle requiring an intermediate host for their development represented by gastropods and mosquitos for Angiostrongylus vasorum and D. immitis, respectively (Levine et al. 1980; Traversa et al. 2013). Recently, the attention on cardiopulmonary nematodes has been increased due to their spreading in many European countries and the appearance in areas previously free of infection (Traversa et al. 2010a). This scenario may be due to several factors like global warming; changes in vectorial dynamics (Traversa et al. 2010a); movements of dogs for activities such as tourism, hunting and so on (Pietrobelli 2010). The interaction between host, vectors, and the environment play an important role on the increase of these parasitoses (Traversa et al. 2010a). These nematodes cause clinical forms primarily with different respiratory symptoms and severity in accordance to their localization and burden and hostrelated factors (Di Cesare et al. 2011). Age, immune response and presence of concomitant diseases can also determine fatal consequences in the infected animals (Conboy 2009; Traversa et al. 2010a, b). The metastrongyloid Angiostrongylus vasorum, was described for the first time in the south of France (Serres E. 1854) and has been reported as a common parasite in foxes and less frequently in dogs of several countries of Europe, Africa and North and South America (Guardone et al. 2013).
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In Sardinia, it was first detected in foxes (Leoni et al. 1986) and recently reported in a dog (Sanna et al. 2012). Recent surveys demonstrate an increasing incidence of angiostrongylosis in different European countries (Morgan et al. 2009; Helm et al. 2010; Traversa et al. 2010a) and its spread in previously non-endemic areas as Holland, Germany and Greece (Papazahariadou et al. 2007; vann Doorn et al. 2009; Barutzki and Shaper 2009, 2011). Some Italian regions can be considered endemic for this parasitoses (Traversa et al. 2013), with the highest prevalences in northern and central ones (Di Cesare et al. 2011; Traversa and Guglielmini 2008), in which ideal climatic conditions for the maintenance and development in intermediate host can be found (Morgan et al. 2009). Dirofilaria immitis is also considered an emerging zoonotic agent in different European countries (Genchi et al. 2011). In Italy, current data on canine dirofilariasis confirms the expanding trend of this parasite as observed in the rest of Europe (Genchi et al. 2009). Today D. immitis is present, not only in the hyperendemic area of the Po River Valley, but also in the northern part of Italy (Friuli-Venezia Giulia), in central Italy (Umbria, Abruzzo and Tuscany), in the south (Campania, Apulia and Calabria) and in the islands (Sicily and Sardinia) (Magi et al. 2011). The first survey on canine heartworm in Sardinia was carried out in the 1960s and showed a prevalence of 1.6 % for D. immitis (Arru et al. 1968). After more than 30 years Scala et al. (2004) with a similar survey concerning the years 1998–2003 found D. immitis in 3.8 % of examined dogs, even if in some areas of the island such as the Oristano and SulcisIglesiente higher prevalences (respectively, 17.1 and 7.4 %) were found. Similarity to other countries dirofilariasis in Sardinia has a spotted distribution, with prevalences varying according to locations and eco-zones suitable for vectors as it was evidenced in one recent survey carried out in the South East coast (Scala et al. 2011). Filaroid species seems to find a good environment in the island, where recently it has been found for the first time after almost one century the adult
Fig. 2 Caudal end of L1 larva of A. vasorum with the typical dorsal spine
specimens of Cercopithifilaria bainae (Otranto et al. 2013; Solinas et al.; 2013). Sardinia Island is known as a holiday destination due to its beautiful beaches. Statistical data on the tourism income in Sardinia estimated more than 1.9 million arrivals in 2012 (1.9 % of Italy tourism), highlighting also the fact that since 2000 to 2012, the number of foreign people coming in Sardinia for their vacation has dramatically increased from 24 to 42 %. Interestingly, movement of tourists and of their pets have been recognised as a potential route for the introduction and spread of non-endemic pathogens (Genchi et al. 2011). Therefore, a constant surveillance of the epidemiology of the most important or emerging parasitoses in Sardinia, like cardiopulmonary filariosis and angiostrongylosis, is of importance considering also the insularity and the suitable climate that could favour their maintenance and spreading. This study aimed to investigate and update the epidemiology of canine heartworm, being that the last extensive survey was carried out in 2004, and to study, for the first time, the epidemiology and genetic make-up of Angiostrongylus vasorum in Sardinia.
Materials and methods From July 2012 to June 2013, a total of 146 faecal samples were obtained from dogs (72 females and 74 males) referred for routine vaccination or clinical visit at the Veterinary Teaching Hospital of the University of Sassari, Italy. For each Table 1 Multiple infections for filarial species in examined dogs
Fig. 1 First-stage larva (L1) of Angiostrongylus vasorum
Kind of infection
Number of positive
Prevalence (%)
D. immitis + D. repens D. immitis + A. reconditum; D. immitis + D. repens + A. reconditum Total
17 12 14
2.5 1.7 2
43
6.3
Parasitol Res Table 2 Prevalences per class of age and statistical analysis of data for D. immitis
Age groups (years)
Examined dogs (no.)
No. of positives
D. immitis (%)
Odds ratio
Statistical analysis
>0≤3 >3≤6 >6 Total
306 187 191 684
31 14 16 61
10.1 7.5 8.4 8.9
1.00 0.72 0. 81 1.00
χ2 for linear trend=0.579;
presence or absence of symptoms of canine filariosis by D. immitis was also performed. The identification of microfilariae was performed according to the morphometric keys as described by Euzeby (1981). L1 larvae (Angiostrongylus vasorum) and mfs (D. immitis) were examined and measured using a digital image processing system (LC micro Image Acquisition Software V.5.2, Olympus). Data collected during sampling were coded and made available for computerised analyses using an electronic spread sheet (Excel®, Microsoft Corp., Redmond, WA, USA). Data were then analysed using the statistical package Epi-Info (version 7.0, CDC/WHO, Atlanta, GA, USA).
sampled animal data on sex, age and presence/absence of respiratory symptoms and clinical signs were also recorded. Faecal samples were individually collected soon after emission and examined with the Baermann technique (Euzéby 1981) for recovering migrating broncho-pulmonary nematodes (BPN) L1 larvae. Briefly, about 5 g of faeces were placed in the centre of a cheesecloth layer forming a pouch put in a funnel filled with water and kept at room temperature. After 24–48 h, a few drops of Lugol’s solution were added to the sediment obtained after centrifugation and thereafter transferred onto a slide and microscopically examined at ×100 magnification for L1 recovery. Larvae were identified using morphometrical keys for Angiostrongylus vasorum (Moeramans et al. 2011; Traversa et al. 2010a). After light microscopy, DNA was extracted from positive sediments with L1 larvae with a commercial kit (High Pure PCR Template Preparation kit, Roche Diagnostics, Mannheim, Germany). Molecular characterization was performed for the mitochondrial cytochrome oxidase subunit 1 (coxI) and for the second ribosomal transcribed spacer region (ITS-2) as described by Jefferies et al. (2008). PCR products were then purified using ChargeSwitch ® PCR Clean-Up Kit (Invitrogen™) and sequenced (MWG Biotech/M-Medical, Milan, Italy). Sequences were then aligned using MEGA V.5.2.1 and compared with those available in the GenBank™ using BLAST analysis. In the same period, a parallel survey was carried out on 684 individual blood samples of dogs that were referred to local practitioners from all Sardinia provinces for routine health care examination. Blood samples were analysed using Knott’s technique (Lindsey 1965) for the detection of circulating microfilariae (mfs). Data on sex, attitude (i.e., hunting, guard, pet), and housing (i.e., outdoor or indoor) were acquired for all dogs. A clinical examination to ascertain the
Table 3 Prevalences (P) and odd ratio for D. immitis and the other filarial species for attitude
Attitude
Hunting Guard dogs Pets Total
Total
420 64 200 684
P=0.446
Results First-stage (L1) larvae of Angiostrongylus vasorum were found in 3.4 % (5/146) of examined dogs. No significative difference between sex was observed (2.7 % of females and 4.1 % of males; Yates corrected χ2 =0.00; P=0.975). L1 larvae of Angiostrongylus vasorum, presents a typical indentation of the cuticle (dorsal notch) and a bulging at the dorsal surface of the tail (dorsal spine) (Moeramans et al. 2011) with a mean length of 340 μm±4.3 (Figs. 1 and 2). Interestingly, all positive cases were found in dogs aged above 24 months except of a 3-months puppy. No significant difference have been observed between prevalences of dogs under or above 24 months aged (χ2 Yates corrected = 0.483; P = 0.487). Among Angiostrongylus vasorum positive animals, a 12year-old dog showed suggestive respiratory symptoms such as chronic cough, while two other dogs of 11 and 10 years old, respectively, presented severe pathological conditions such as cardiac failure and pulmonary hypertension.
D. immitis No.
P (%)
37 13 11 61
8.8 20.3 5.5
O. R.
1.00 2.21 0.60
D. repens No.
P (%)
36 12 13 61
8.7 20.3 6.7
O. R.
1.00 2.46 0.74
A. reconditum No.
P (%)
72 7 7 86
17.1 10.9 3.5
O. R.
1.00 0.59 0.18
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Biomolecular characterization of larvae allows us to confirm the morphological identification of the first-stage larvae of Angiostrongylus vasorum (GenBank accession numbers KF270685.1, KF270683.1). The alignment of our sequences with BLAST showed a homology of 100 % within our samples and with the Cox1 and ITS-2 sequences of An giostrongylus vasorum available in G enBank (EU493165.1, EU627597.1). In the epidemiological survey on filariosis, 20.9 % (143/ 684) of examined animals tested positive for at least one f i l a r i a l s p e c i e s . I n p a r t i c u l a r, m i c r o f i l a r i a e o f Acanthocheilonema reconditum were retrieved in 12.6 % (86/684) of samples, D. immitis in 8.9 % (61/684) and Dirofilaria repens in 8.9 % (61/684). Multiple infections were observed in 43 (6.3 %) dogs (Table 1). Prevalences grouped per sex for D. immitis were higher in males (9.5 %) than females (8.2 %) even if this difference was not statistically significant (χ2 =0.32; P=0.573). Stratification of data per class of age was not statistically significant (χ2 for linear trend=0.579; P=0.446) (Table 2). Regarding aptitude, even if guard dogs were more positive for D. immitis (20.3 %) compared to other evaluated categories, the difference was not statistically significant (χ2 for linear trend=1.005; P= 0.316) (Table 3). Microfilaremia for D. immitis was considerably higher in dogs living outdoor than indoor (10.3 vs 1.8 %; Yates corrected χ2 =7.13; P=0.007). Clinical signs as weakness, cough, wearing, weight loss, exercise intolerance, dyspnoea and tachypnea were reported in 6.6 % of examined dogs, and only 0.6 % of these animals were positive to D. immitis.
Discussion The present survey represents the first epidemiological survey on Angiostrongylus vasorum in Sardinia and allowed also the biomolecular characterization of this nematode in the island. Moreover, the paper updates data on the current distribution of D. immitis and other filarioids in dogs sampled across all the provinces. Particularly, prevalence found for Angiostrongylus vasorum (3.4 %) is consistent with those reported in Abruzzo (4.3 %) (Yates corrected χ2 =0.01; P=0.904) and in Latium (2 %) (Yates corrected χ2 =0.07; P=0.787) by Di Cesare et al. (2012) as well as in other European countries where this parasitoses is considered emergent, like Greece ( 1 . 1 % ) ( Ya t e s c o r r e c t e d χ 2 = 1 . 7 6 ; P = 0 . 1 8 4 ) (Papazahariadou et al. 2007). Sex and age do not seem to represent risk factors for these parasitoses. It seems therefore necessary to increase the attention of the vet practitioners on these parasitoses which should include Angiostrongylus vasorum in the differential diagnoses of dog’s cardiopulmonary diseases even in animals with few
or none respiratory symptoms, as suggested by Traversa et al. (2013). In addition, the research of larvae L1 of Angiostrongylus vasorum in dog’s faeces is not commonly performed by veterinarians in the island. Prevalence herein found for D. immitis shows that the parasitic pressure and the risk of infestation in the island are high, especially in guard dogs (27.9 %) and outdoor housed animals (10.5 %). These findings are in agreement with those reported by Giangaspero et al. (2013) in Apulia region. Age and sex do not seem to represent as risk factors for heartworm disease as previously described by Magi et al. (2011). Microfilaremia for filarial infections (20.9 %) is similar to that detected in Tuscany (23.2 %) by Magi et al. (2011). Particularly Acanthocheilonema reconditum (12.6 %; 86/ 684) seems to have a predominant role in Sardinia island and differs from the two other species for the intermediate hosts (fleas and ticks) (Scala et al. 2004; Genchi et al. 2005; Brianti et al. 2012) and the way of transmission, while D. repens is present with the same prevalences observed for D. immitis (8.9 %; 61/684). This scenario requires an implementation toward an accurate differential diagnosis especially between D. immitis and Acanthocheilonema reconditum that have similar length but differs for some morphological features and for clinical presentation and relevance. The morphometric analysis proved to be a useful, quick and cheap diagnostic tool and it represents the first step in the diagnosis of filarial infections. However, the discrimination between different species can be challenging in cases of mixed infections (Traversa et al. 2010b; Magi et al. 2011) or in cases with low parasitaemia (Magnis et al. 2013). This study shows that due to the high movements of tourists with pets in the island, mainly during spring and summer, the monitoring of cardiopulmonary parasites should be kept at the high level and included in differential diagnosis of dogs’ cardiopulmonary disease (Magnis et al. 2013). This scenario requires greater attention for the implementation of pharmacological prophylaxis, accurate diagnosis and correct treatment by the veterinary, for the protection of both animal and human health, taking into consideration the zoonotic potential of filarial species, particularly for D. repens and D. immitis (Genchi et al. 2011; Magi et al. 2011). In this sense, travelling dogs coming in Sardinia Island should be monitored for circulating mfs, treated with preventive drugs and reexamined for circulating mfs 6–7 months after they stay in the island (Genchi et al. 2011). In conclusion, the present work indicate that cardiopulmonary nematodes are present in Sardinia island where Angiostrongylus vasorum can be considered an emerging parasite while D. immitis confirmed a wide diffusion thus to include these parasites in the differential diagnosis of dogs cardiopulmonary disease (Di Cesare et al. 2011). Pharmacological prophylaxis should always be performed
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for travelling dogs coming in the island from other European and Italian regions, to prevent the risk of infection. Acknowledgements The research was funded by Regional Government of Sardinia, prot. CRP2 134 (L.R. 7, 2007).
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