Aberrant ocular infection by Angiostrongylus vasorum in a dog Lia Riccardo Paolo1 Traversa Donato2 D’Anna Nunzio3 Giannelli Alessio1, Dantas-Torres Filipe1,4, Otranto Domenico1 Dipartimento di Medicina Veterinaria, Università degli Studi di Bari, Italy; 2Facoltà di Medicina Veterinaria, Università degli Studi di Teramo, Italy; 3 Clinica Veterinaria Roma Sud, Roma, Italy; 4 Department of Immunology, Aggeu Magalhães Research Centre, Recife-PE, Brazil Introduction Canine angiostrongylosis caused by Angiostrongylus vasorum (Strongylida: Angiostrongylidae) is an emerging mollusc-borne parasitosis causing cardio-respiratory signs and coagulopathies. The nematode resides in the pulmonary arteries and in the right side of the heart of dogs that become infected by the ingestion of gastropod intermediate hosts (i.e., slugs and snails). This helminthic disease is an emerging issue in several European countries, including Italy (Traversa et al., 2013, Parasitol Res, 112:2473-2480). Although A. vasorum may localize in other districts (i.e., spleen, pancreas, kidneys, liver, skeletal muscles, brain), aberrant ocular migrations have been reported only sporadically (Raillet & Henry, 1913, Bullettin de la Societe Centrale de Medicine Veterinarie. 67:209-215; Henry & Lesbouyries, 1927, Bullettin de la Societe Centrale de Medicine Veterinarie, 80:263-265; Rosenlund et al., 1993, Eur J. Compan Anim. Pract., 3:31-33; King et al., 1994, J. Small Anim. Pract., 35:326-328; Parry et al., 1994, Can. Vet. J., 32:430-431; Payen, 2004, International Veterinary Ophthalmology Meeting; Munich. p. 125; Manning, 2007, Vet Rec., 160:625-627). The present study describes an unusual case of ocular angiostrongylosis in a dog living in central Italy. Materials and methods A 5 month-old mixed-breed dog was referred to a private veterinary clinic in Roma for an ophthalmological examination, during which corneal oedema and episcleral hyperaemia in the right eye was observed. After a diagnosis of anterior uveitis, the ultrasound examination of the eye showed a blood clot in the anterior chamber. Fifteen days after the visit, the hyphema disappeared but a free-swimming nematode was retrieved in the anterior chamber. The nematode was extracted surgically and morphologically and morphometrically identified (Costa et al., 2003, Revue Med. Vet., 154:9-16). Topical and systemic antibiotics and steroids were administered for the following 3 weeks and dog recovered without any complication. Results The nematode (105 mm in length and 0.29 mm in width at the middle portion; anterior and posterior ends of 0.109 mm and 0.176 mm in length, respectively) was identified as a male A. vasorum. Briefly, the nematode presented a smooth cuticle and a slender body, attenuated at the end and enlarged anteriorly where a small buccal aperture was observed. The posterior end was ventrally curved, with a short copulatory bursa. Spicules measured 0.404 mm and 0.388 mm in length. Conclusions Several clinical signs have been associated to A. vasorum, but the presence of larvae and pre-adult stages within the eyes are an occasional finding. Ocular angiostrongylosis has been sporadically described in dogs from France, United Kingdom, Denmark and Canada. This is the first report of A. vasorum in the eye of a dog from Italy. Practitioners working in areas where A. vasorum is endemic should include canine angiostrongylosis not only in the differential diagnosis of canine cardio-respiratory diseases but also of ocular diseases.

The present study was carried out in a protected wooded area, which is part of the Parco Regionale Gallipoli Cognato Piccole Dolomiti Lucane, one of the most important ecological reserves in southern Italy. From April 2010 to April 2011, 212 birds, comprising 22 species from 12 families, were captured and examined for ticks. A total of 75 (35.4 %) birds were found infested by ticks, with 451 ticks being collected. All ticks were identified as Ixodes ricinus, of which 241 (53.4 %) were larvae and 210 nymphs (46.6 %). The highest intensity of infestation was found in April 2010, when 117 ticks were retrieved on 25 birds. No ticks were found on birds (n = 5) netted in December 2010. High infestation rates were recorded on blackbirds (Turdus merula) (90 %; 29 birds examined) and on mistle thrushes (Turdus viscivorus) (100 %; 2 birds examined). The highest intensity of infestation by larvae was found on wrens (5.6 larvae/bird) and by nymphs on mistle thrushes (11.5 nymphs/bird). Temperature and number of hours of light showed to influence the activity of larvae and nymphs. These data support the notion that birds may be responsible for the heterogeneous distribution of I. ricinus in Europe, thus playing a role in the epidemiology of certain tick-borne pathogens.

The brown dog tick Rhipicephalus sanguineus (sensu stricto) is reputed to be the most widespread tick of domestic dogs worldwide and has also been implicated in the transmission of many pathogens to dogs and humans. For more than two centuries, Rh. sanguineus (s.s.) was regarded as a single taxon, even considering its poor original description and the inexistence of a type specimen. However, genetic and crossbreeding experiments have indicated the existence of at least two distinct taxa within this name: the so-called "temperate" and "tropical" lineages of Rh. sanguineus (sensu lato). Recent genetic studies have also demonstrated the existence of additional lineages of Rh. sanguineus (s.l.) in Europe and Asia. Herein, we assessed the biological compatibility between two lineages of Rh. sanguineus (s.l.) found in southern Europe, namely Rhipicephalus sp. I (from Italy) and Rhipicephalus sp. II (from Portugal).

Canine angiostrongylosis is an increasingly reported disease in Europe which can be fatal if left untreated. The wide range of clinical presentation along with the absence of pathognomonic alterations can make the diagnosis challenging; thus any additional information that may provide clues to an early diagnosis may be of value, in order to ensure adequate anthelmintic treatment. Aim of the study was to assess a clinicopathological scoring systemassociated with natural Angiostrongylus vasorum infection diagnosed in canine patients during clinical practice, to clinically and paraclinically monitor infected dogs after treatment, and to monitor the presence of L1 larvae in faecal samples by Baermann’s test.Of the total 210 enrolled animals A. vasoruminfection was diagnosed in 7 dogs.These dogs were clinically and paraclinically investigated andmonitored after specific treatment. Further 3 symptomatic dogs were retrospectively included in the monitoring. Results suggest that the computed scoring system can help to increase the clinical suspicion of infection particularly in asymptomatic dogs before the onset of potentially lethal lesions. Data of faecal monitoring suggested that treatment may control parasite burden but be unable to eradicate infection. Thus, a continued faecal monitoring after treatment is advisable for identification of still infected or reinfected dogs.

In the past decades, cases of canine ocular onchocercosis have been reported worldwide, particularly in the United States and Europe. Onchocerca lupi, originally described from a wolf, has been implicated in some of these cases, and its zoonotic role has been hypothesized on the basis of the reexamination of two cases of human ocular onchocerciasis. In the present study, we describe, for the first time, the occurrence of O. lupi in the subconjunctival region of the human eye in a patient from Turkey. The nematode was identified as O. lupi based on its morphology and molecular phylogenetic analysis of partial cox1 and 12S ribosomal DNA genes. The results suggest that O. lupi should be considered in the differential diagnosis of other eye parasitic infections in humans. The role of dogs as natural hosts of O. lupi and the vectors of this zoonotic parasite need to be investigated.

Cercopithifilaria rugosicauda (Spirurida, Onchocercidae) is a subcutaneous filarial nematode of the European roe deer (Capreolus capreolus) transmitted by Ixodes ricinus (Acari, Ixodidae). At the necropsy of a roe deer from the Parco Regionale di Gallipoli Cognato (Basilicata region, southern Italy), two female nematodes of C. rugosicauda were found. Following the necropsy, seven skin snips were sampled from different body regions and 96 I. ricinus ticks were collected. In addition, 240 ticks were collected by dragging in the enclosure where the roe deer lived. Samples were examined for the presence of C. rugosicauda larvae and assayed by PCR targeting cytochrome c oxidase subunit 1 (cox1, ∼300 bp) and 12S rDNA (∼330 bp) gene fragments. Female nematodes, microfilariae from skin samples and eight third stage larvae (L3) from ticks were morphologically and molecularly identified as C. rugosicauda. Phylogenetic analyses clustered this species with other sequences of Cercopithifilaria spp. This study represents the first report of C. rugosicauda in a roe deer and ticks from Italy and provides new morphological and molecular data on this little known nematode.

The clinical evolution of Leishmania infantum infection in dogs is largely influenced by the host's individual immune response. Few studies have investigated the time course and clinical evolution of the infection both under experimental and natural conditions. In the present investigation, the time course of L. infantum infection was studied by monitoring clinical and laboratory features in naturally infected dogs sheltered in southern Italy. Twenty-three dogs that had one or more positive diagnostic tests for L. infantum were enrolled in the study and followed up every 4. months. A clinical score was assigned at each visit after assessing the presence of clinical signs suggestive of leishmaniosis. L. infantum-infected dogs were classified into three different categories based on their clinical score and serological and parasitological test results. Based on data from diagnostic tests and clinical scores, the time course of infection was defined as transient asymptomatic infection (11 dogs), persistent asymptomatic infection (2 dogs), and symptomatic infection (8 dogs). Two dogs were lost after the first sand fly season. The results of the present study provide a framework for assessing the clinical status of L. infantum infection in dogs and suggest that infected animals should be monitored over time to expedite therapeutic decisions and plan appropriate control interventions. © 2009 Elsevier Ltd.

angiostrongylosis is an emerging potentially fatal disease in Europe. Aim of the study- clinical monitoring of twelve naturally Angiostrongylus vasorum naturally infected dogs under treatment. Materials and methods- twelve dogs referred to the Clinical Unit of the Veterinary Faculty of Bari (Italy) were enrolled in the study. Diagnosis was made by means of larval morphological identification on faecal samples analysed by Baermann test and/or direct microscopic observation. A blood sample was collected for haematology, biochemical analysis, serum protein electrophoresis and coagulation profile. Thoracic radiographs and echocardiography was performed. Specific therapy was administered choosing fenbendazole (Panacur Ò, Intervet, Animal Health) and/or Im/Mox (AdvocateÒ spot-on, Bayer) based on the severity of symptoms. The study monitoring was stopped at normalization of clinico-pathological alterations and/or at the end of treatment. During monitoring clinical examination was performed once a week in symptomatic dogs, while laboratory and radiographic controls were performed according to owner availability. Results-the owner complaint was heterogeneous: dyspnea, polypnea and hemoptysis, gradual distension of the abdomen, epistaxis and acute unilateral hyphema, severe coughing. Five cases were presented for routine control visit and one case for ocular specialistic examination. Thoracic auscultation was normal in seven cases, while in the other animals rales, wheezing, muffling of heart sounds and loud bronchovescicular sounds were registered. Thoracic radiographs showed pathological findings in all dogs except for one. Circular areas of radiopacity were observed in four cases. The complete blood count showed anemia from mild to moderate in five cases, leucocytosis in four cases and eosinophilia in six cases. The biochemical examination showed non-specific and non-uniform alterations. Primary hemostasis disorders characterized by thrombocytopenia were detected in three cases, associated or not to alterations of clotting profile. Serum protein electrophoresis showed a variable increase in β globulin fraction in all animals except for two asymptomatic dogs. In general clinical pictures significantly improved in all symptomatic dogs after 1-2 weeks from treatment starting. The reverse to normal of clinico-pathological findings and radiographic abnormalities were variable. Fenbendazole was given in monotherapy in five cases for 21 days. The Im/Mox was administered in monotherapy in five cases and in combination with fenbendazole in two other cases. Conclusions - The reported cases show different clinical presentations associated with the presence of A. vasorum in dogs suggesting that angiostrongylosis should be considered as a possible etiological cause of several clinical conditions. Moreover the occasional detectionof L1 in the feces of five dogs referred for a routine control visit suggest that fecal samples (direct or Baermann test) can reveal asymptomatic subjects (Koch et al., 2009). The detection of L1 in a dog of 12 years reveals the receptivity to infection also in old dogs, although a greater risk of infection is reported in young subjects (Chapman et al., 2004; Barutzki and Schaper, 2009). Thoracic radiographic findings are described in the course of angiostrongylosis with the possibility of abnormalities also in asymptomatic dogs (Boag et al., 2004) as it was in the cases described herein.. The particular aspect of the circular/serpiginous radiopacity described in four cases have not been previously reported; these lesions could be attributed to fistulas created during larval migration from the pulmonary capillaries into the alveoli or to bronchoectasiae. A. vasorum infection can be fatal, thus an early diagnosis and appropriate treatment is auspicable. Although none of clinico-pathological findings are specific, a combination of these findings should increase the index of suspicion also in asymptomatic dogs.

Angiostrongylus vasorum has been recently reported in central and southern Italy. Although a wide range of clinical signs have been described in association with A. vasorum respiratory disease and the haemorrhagic diatheses predominate. The aim of the present study is to report the different clinical presentations, laboratory and imaging findings of 8 cases of angiostrongilosis. Diagnosis was made by means of larval morphological identification; faecal samples were analysed by the Baermann test and direct microscopic observation. Different treatment protocols were used, and animals weekly monitored by larval detection in faecal samples. These case reports associate different clinical presentations with the presence of A. vasorum infection in dogs and also suggest that angiostrongylosis should be considered as a possible aetiological cause of several clinical conditions. Although A. vasorum can be fatal a prompt diagnosis and appropriate treatment may lead to a complete clinical resolution, as it was in the cases described herein

Over the past 20 years,Thelazia callipaeda(the oriental eyeworm) has become endemic in Europe, infecting domestic and wild carnivores and humans. The vector of this nematode, the fruit flyPhortica variegata, has recently been discovered in the United States, and its vector competence is demonstrated forT. callipaedain this article, therefore representing a potential new threat for infection of carnivores and humans in the United States.

Crenosoma vulpis is a metastrongyloid nematode primarily associated with respiratory tract infections of red foxes in North America and Europe. Sporadic cases have also been reported in domestic dogs. The present study aimed to provide morphological, molecular, and epidemiological data on the geographical distribution of this nematode throughout Italy. From 2012 to 2014, 12 of the 138 foxes examined, three dogs and one badger scored positive for C. vulpis. Forty adults were isolated from foxes and the badger, whereas first-stage larvae were detected in the three dogs. All specimens were morphologically identified as C. vulpis, and 28 nematodes were also molecularly characterized by sequencing mitochondrial (12S ribosomal DNA (rDNA)) and nuclear (18S rDNA) ribosomal genes. Four haplotypes were identified based on the 12S rDNA target gene, with the most representative (78.5 %) designated as haplotype I. No genetic variability was detected for the 18S rDNA gene. The molecular identification was consistent with the distinct separation of species-specific clades inferred by the phylogenetic analyses of both mitochondrial and ribosomal genes. Data herein reported indicates that C. vulpis has a wide distribution in foxes from southern Italy, and it also occurs in dogs from southern and northern regions of the country. Practitioners should consider the occurrence of this nematode in the differential diagnosis of canine respiratory disease, particularly in dogs living close to rural areas where foxes are present.

The fruit fly Phortica variegata (Drosophilidae: Steganinae) feeds on the ocular secretions of animals and humans, and has been described as an intermediate host of the eye worm Thelazia callipaeda (Spirurida: Thelaziidae) in Italy. Despite the increased detection of T. callipaeda in many European countries, information about its vector role in natural conditions is still limited. In the Iberian Peninsula, thelaziosis caused by T. callipaeda has been reported in dogs, cats, red foxes, wild rabbits and humans.

Phlebotomine sand flies are insects of major medico-veterinary significance in the Mediterranean region, as they may transmit pathogens to animals and humans, including viruses and protozoa. The present study was conducted in southern Italy, in an area where visceral leishmaniasis caused by Leishmania infantum is endemic. Insects were collected monthly during two consecutive years using light traps set in five different ecologic contexts (i.e., a stonewall near a woodhouse, a tree near volcanic rocks in a high-altitude area, a tree trunk in a meadow habitat, a sheep stable, and a chicken coop) and weekly in one site (the garage of a private house). A total of 13,087 specimens were collected and six species identified (i.e., Phlebotomus perfiliewi, Phlebotomus perniciosus, Phlebotomus neglectus, Phlebotomus papatasi, Phlebotomus mascittii, and Sergentomyia minuta), representing 75% of the total number of phlebotomine species found in Italy. P. perfiliewi was the most abundant species, comprising 88.14% of the specimens identified. The greatest species diversity and abundance was recorded in human dwellings and in animal sheds. Sand flies were active from June to October, peaking in July-August in 2010 and July-September in 2011. Part of the females (n=8865) was grouped into 617 pools (range, 1-10 insects each) according to species, feeding status, day and site of collection. A total of four pools (10 non-engorged specimens each) and one engorged female of P. perfiliewi were positive for L. infantum. This study confirms that phlebotomine vectors in southern Italy are highly adapted to human-modified environments (e.g., animal sheds) and that P. perfiliewi is a major vector of L. infantum in some regions of southern Italy.

Background: Tick-borne diseases comprise a group of maladies that are of substantial medical and veterinary significance. A range of tick-borne pathogens, including diverse species of bacteria and protozoa, can infect both dogs and humans. Hence, the control of tick infestations is pivotal to decrease or prevent tick-borne pathogen transmission. Therefore, different commercial products with insecticidal, repellent or both properties have been developed for use on dogs. Recently, a collar containing a combination of imidacloprid 10% and flumethrin 4.5% has proven effective to prevent tick and flea infestations in dogs under field conditions and the infection by some vector-borne pathogens they transmit under laboratory-controlled conditions. Methods: From March 2011 to April 2012, a field study was conducted in a private shelter in southern Italy to assess the efficacy of the imidacloprid/flumethrin collar against tick and flea infestations and to determine if this strategy would decrease tick-borne pathogen transmission in young dogs. A total of 122 animals were enrolled in the study and randomly assigned to group A (n = 64; collared) or group B (n = 58; untreated controls). Dogs were examined monthly for ticks and fleas and systematically tested for selected tick-borne pathogens. Results: Compared to controls, the collar provided overall efficacies of 99.7% and 100% against tick and flea infestation, respectively. The overall efficacy for the prevention of tick-borne pathogens (i.e., Anaplasma platys and Babesia vogeli) was 91.6%. Conclusions: This study demonstrates that the imidacloprid/flumethrin collar is efficacious against flea and tick infestation as well as tick-borne pathogen transmission to dogs under field conditions.

It is acknowledged that data from field studies on tick ecology might be biased by collection methods, but actually comparative studies are still limited. Herein we assessed whether the efficiency of flagging and dragging varies according to tick developmental stage, species, season and habitat. Ticks were collected in three sites bordered by an oak forest. The abundance of ticks collected by each collection method varied according to tick species, developmental stage, season, and habitat. Flagging was in general more efficient in collecting adult ticks, especially in spring and winter. Females were more frequently collected by flagging in the meadow and grassland habitats and males in the man-made trail. Flagging collected significantly more adults of Dermacentor marginatus, Hyalomma marginatum, Haemaphysalis inermis and Ixodes ricinus. Flagging was more efficient in collecting D. marginatus and I. ricinus in spring, and H. inermis and I. ricinus females in both spring and winter. In summer and autumn tick abundances were generally similar, with the exception of D. marginatus female in autumn. Flagging was more efficient in collecting D. marginatus adults in the meadow habitat and in the man-made trail, and I. ricinus adults in the meadow and grassland habitats. Dragging was more efficient in grassland for R. turanicus. Our results suggest that variations in terms of collection method performance are associated to factors linked to tick behaviour, habitat characteristics, and climate. Field studies employing these collection methods should take this into account to avoid misleading conclusions about tick population dynamics and tick-borne pathogen transmission risk.

INTRODUCTION: Drug resistance in cyathostomins is a potential threat for horse health, welfare and production. While resistance to benzimidazoles (BZs) is worldwide recognized and to tetrahydropirimidines (THPs) is increasing, it is up until recently that the first evidence of reduced efficacy of macrocyclic lactones (MLs) has been reported. Recent studies indicated a reduction in egg reappearance period (ERP) after treatment with ivermectin (IVM) in different countries, while moxidectin (MOX) is still highly effective in Europe (von Samson-Himmelstjerna et al., 2007, Vet. Parasitol. 144,74-80; Lyons et al., 2011, Parasitol. Res. 108,1315-1319; Traversa et al., 2009, Parasit. Vectors 2:S2; Traversa et al., 2012, Vet. Parasitol., 188, 294-300). This study investigated the ERP after treatment with IVM or MOX in horses infected with cyathostomins in Italy. MATERIALS AND METHODS: Ten farms have been selected in Italy on the basis of a history either of <100% efficacy of MLs or of frequent use of IVM or MOX. In each farm ten horses have been selected on the basis of a minimum pre-treatment faecal egg count (FEC) of 50 (range 50 -2425) per gram of faeces (EPG). Horses were randomly allocated to one of two treatment groups based on pre-treatment FEC and treated either with IVM or MOX on Day 0. Individual faecal samples were taken from all animals on Day 14, 28, 42 and 56 for IVM and MOX, and 70 and 84 for MOX. All samples were examined using a modified McMaster technique with a sensitivity of 25 EPG and the efficacy for each treatment was calculated based on the arithmetic mean (AM) FEC before and at each time point after treatment with the following formula: %reduction= 100x (AMpre-treatment - AMpost-treatment)/AMpre-treatment. RESULTS: Efficacy at Day 14 was high for both IVM and MOX in all sites. The ERP was as expected for MOX except on one farm, in which decreased ERP was found for both MLs. Decreased ERP and efficacy was found in another farm on Day 56 for IVM, while decreased ERP and efficacy was detected in tail (i.e. on Day 84) for MOX on a third farm. CONCLUSIONS: The reduced cyathostomin ERP was found for IVM and/or MOX in 3 farms of this study, consistently with the results of a similar study carried out in Belgium and the Netherlands which demonstrated reduced ERP for IVM and MOX (Geurden et al. 2014, Vet. Parasitol., submitted). The duration of the ERP varies with anthelmintic class and worm populations, thus effective molecules must be evaluated separately in each herd to characterize local properties. Given that a reduction in the ERP is possibly indicative of early anthelmintic resistance, appropriate use of anthelmintics in worm control strategies, including reduced treatment frequency or selective treatments, along with maintenance of refugia, should be implemented to delay the occurrence of drug resistance in horse cyathostomins.

Aedes albopictus has spread during the last few decades all over the world. This has increased significantly the risk of exotic arbovirus transmission (e.g. chikungunya, dengue, and Zika) also in temperate areas, as demonstrated by the Chikungunya 2007 and 2017 outbreaks in northeastern and central Italy. Insecticides are an important tool for limiting the circulation of these mosquito-borne viruses. The aim of the present study was to address the gap in current knowledge of pyrethroid insecticide resistance of European Ae. albopictus populations, focusing on populations from Italy, Albania and Greece.

Background: Capillaria plica (syn. Pearsonema plica) is a nematode that resides in the urinary bladder and rarely in ureters or in the kidney pelvis of various carnivores, especially foxes and dogs. Urine sedimentation technique is actually the only diagnostic tool that permits the identification of C. plica eggs, but its sensitivity is low and when an infection is suspected (or when it is necessary to confirm treatment efficacy) more than one examination of urine sediment should be performed. The present paper reports a clinical case of natural C. plica infection in a dog from southern Italy. In addition, two new techniques, FLOTAC and Mini-FLOTAC, were used for the diagnosis of C. plica in dog urine and compared with the technique of sedimentation. Results: Using FLOTAC with fresh urine and sodium chloride as flotation solution, were obtained the best results for the diagnosis of C. plica in dog urine in term of eggs counted (mean eggs per 10 ml of urine = 70.3 FLOTAC vs 40.3 Mini FLOTAC vs 32.8 sedimentation) and coefficient of variation (CV%) (6.2 FLOTAC vs 13.4 Mini-FLOTAC vs 32.9 sedimentation). Conclusions: The FLOTAC was the more sensitive method, but also the Mini-FLOTAC could be a valid alternative diagnostic method because gave better results than the classical sedimentation and can be used in place of the FLOTAC in laboratories where the centrifugation step cannot be performed. © 2014 Maurelli et al.; licensee BioMed Central Ltd.

Canine angiostrongylosis is a cardiopulmonary disease emerging in Europe which can be fatal if left untreated. An early diagnosis and appropriate treatment is auspicable not only in symptomatic dogs because it may lead to a complete clinical resolution but also in asymptomatic dogs to reduce the risk of parasite importation in new areas suitable for parasite establishment. The aim of the present work is to investigate the different clinical and paraclinical findings in both symptomatic and asymptomatic dogs naturally infected by Angiostrongylus vasorum. Twelve dogs were included in the study. Diagnosis was made by means of larval morphological identification on faecal samples. Pathological paraclinical findings were registered both in symptomatic and asymptomatic dogs. In particular, the increase in b globulin fraction at serum protein electrophoresis and radiographic alterations were reported as usefull findings to increase the suspicion of infection in asymptomatic dogs.

Hepatozoon canis infection in ticks during spring and summer in Italy

Onchocerca lupi, a zoonotic nematode infecting the eyes of carnivores, has been increasingly reported in dogs from Europe and the USA. In order to improve the current status of knowledge on this neglected filarioid, diagnostic imaging tools (i.e., ultrasound scan, computed tomography and magnetic resonance imaging) are herein used to diagnose canine onchocercosis in two dogs, which scored positive for O. lupi microfilariae at the skin snip test and to assess the anatomical location of the nematode within the ocular apparatus. Results indicate that ultrasound tools are useful to address the diagnosis of O. lupi in dogs and to evaluate the localization of nodules or cysts containing the adult nematode.

Background: Cercopithifilaria bainae is a filarioid parasite that infects dogs, being transmitted by Rhipicephalus sanguineus group ticks in many countries of the Mediterranean basin. This study assessed the incidence density rate (IDR) of infection by C. bainae in dogs and the probability of co-infection with other tick-borne pathogens (i.e., Anaplasma platys, Babesia vogeli and Hepatozoon canis), in an area of high endemicity in southern Italy. Methodology/Principal Findings: From March 2011 to October 2012, a field study involving 58 young dogs naturally exposed to tick infestation was conducted. Skin and blood samples obtained from each dog six times during an 18-month period were tested for C. bainae by parasite detection within skin snip sediments, with subsequent confirmation through PCR and DNA sequencing. Dogs examined monthly for ticks and A. platys, B. vogeli and H. canis were microscopically and/or molecularly diagnosed and after the first and the second summer seasons, the IDR for positive animal-month at risk was 3.8% and 1.7% in November 2011 and October 2012, respectively. All 58 C. bainae-infected dogs were simultaneously infected with at least one other tick-borne pathogen. After the first summer season (assessment in November 2011), a C. bainae-infected dog had a 33% probability of being infected with H. canis or A. platys, whereas after the second tick season (assessment in October 2012) the probability of co-infection was 78%, 22% and 11% for H. canis, A. platys and B. vogeli, respectively. Conclusions: Our data indicate that tick-infested dogs are at risk of acquiring infection by C. bainae. In addition, the detection of C. bainae microfilariae indicates a prior tick exposure and, should stimulate testing for other tick-borne disease causing pathogens.

The present study aimed to identify ticks collected from road-killed wildlife species retrieved in several localities of southern Italy and to assess the presence of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Rickettsia spp. DNA in ticks. Collections were carried out from January 2000 to December 2009 on wild animals found dead within the territories of 11 municipalities from three regions (i.e., Apulia, Basilicata, and Calabria). In total, 189 carcasses of wild animals belonging to 10 species were checked for tick infestation, and 40 animals belonging to seven species were found parasitized. One hundred and twenty-five ixodid ticks (11 larvae, 14 nymphs, 77 males, and 23 females) were collected and identified as belonging to nine species, namely Dermacentor marginatus, Haemaphysalis erinacei, Hyalomma marginatum, Ixodes acuminatus, Ixodes canisuga, Ixodes hexagonus, Ixodes ricinus, Rhipicephalus bursa, and Rhipicephalus turanicus. None of the 36 tick specimens tested by PCR was positive for tick-borne pathogens. The results add new information on the tick fauna associated with wild animals in Italy, reporting new tick-host associations. Further field studies are still needed to ascertain the suitability of certain wildlife species as hosts for some tick species, particularly for those implicated in the transmission of pathogens to domestic animals and humans. Finally, from a conservation perspective, it would be interesting to assess whether these wild animals (e.g., Lepus corsicanus) are exposed to tick-borne pathogens, investigating the possible implications for their health and behavior.

Angiostrongylus vasorum is a nematode which causes a potentially fatal chronic parasitic pneumonia in dogs. A. vasorum infection is considered endemic in various parts of Europe (Koch and Willesen, 2009) and in Italy it is recently assumig increasing importance. Aim- To monitor A. vasorum natural infection in dogs under treatment by direct microscopy and modified Baermann technique on faecal samples. Materials and methods- Owned dogs presented at the Clinical Unit of the Veterinary Faculty of Bari, Italy, and positive to A. vasorum infection were included in the study. The diagnosis was reached by means of L1 detection on direct faecal smear and/or using the modified Baermann technique. Faecal samples positive to nematodes mobile larvae were sent to the Parasitology Unit for morphological identification. The animals were treated and clinically and parasitologically monitored. Asymptomatic dogs were treated with an imidacloprid/moxidectin spot-on formulation (Im/Mox) at T0, T+15,T+30. Symptomatic dogs were treated with fenbendazole 25 mg/kg/ bid/os for 21 days. Treatment was weekly monitored using Baermann test on three days faecal pool both in symptomatic and asymptomatic animals for one month, than once a month till possible. When infection persisted a second-line treatment was considered. In symptomatic animals clinical examination was performed once a week till clinical recovery than twice a month, while asymptomatic animals were revaluated only after the third spot-on treatment. Results- Ten dogs were enrolled in the study. Signalment, clinical signs at presentation, therapy, clinical remission time and results of faecal monitoring are reported in table. Briefly 5 dogs were treated with fenbendazole and the others with Im/Mox. In three dogs a second-line treatment was needed and fenbendazole 25 mg/kg/die for 21 days associated to Im/Mox was used. In these three dogs a long term monitoring was possible. Conclusions- Given its proven efficacy (Chapman et al., 2004) fenbendazole has been used in symptomatic patients, whereas Im/Mox, more recently proposed (Willesen et al., 2007) was used for asymptomatic dogs. All dogs treated with fenbendazole (dog 6-10) resulted negative at Baermann after 1-2 weeks of treatment but a long term monitoring was available only in two dogs (dog 6,7) showing negative results. A highly variable response was registered in dogs treated with Im/Mox (dog 1-5). Two dogs (dog 1 and 4) reached negative results 1-2 weeks after first spot-on administration and persisted negative for 16 and 20 weeks respectively. It is reported that larval excretion may continue for over 3 weeks, even if anthelmintic treatment was successful (Schnyder et al., 2010). Differently one case (dog 5) slowly reached negative results (8 weeks after the first spot-on) but it reverse newly positive two times during monitoring. Other two cases (dog 2 and 3) resulted persistently positive for 12 weeks. The last three dogs were treated with the second-line treatment. Cases 2 and 3 achieved negative results so slowly (8 and 20 weeks after second-line treatment) to make difficult a direct association with treatment efficacy, anyway they persisted negative in a long term follow up. Case 5 showed negative results 4 weeks after second-line treatment but unexpectedly it reverse positive a second time. Reinfection or infection not controlled? The question is open, we can only make assumptions. It is already known that anthelmintic treatments may not completely eliminate adult worms but be able to sterilise them leading to a reduced pathogenicity of A. vasorum infection (in: Schnyder et al., 2010). In experimental studies (Schnyder et al., 2010) on dogs treated with Im/Mox spot-on and untreated, eggs and larvae were only present in untreated dogs. The highly variable response registered in this study suggests that in natural conditions several factors could affect the response to treatment probably associated to the host and/or to the parasite

Background Rhipicephalus sanguineus sensu lato ticks are widespread worldwide due to their adaptability to survive under different environmental conditions. They may act as vectors of a wide range of pathogens to humans and animals and their control is based on the use of chemical products on dogs and in the environment. Alternative control strategies, such as the use of entomopathogenic fungi as bio-control agents have also been investigated. The ability of native strains of Beauveria bassiana sensu lato in causing mortality in different tick species (e.g., Amblyomma cajennense and Rhipicephalus microplus) has been demonstrated. However, limited studies have assessed the use of B. bassiana for the control of R. sanguineus s.l. and none of them have employed native strains of this fungus. Here we investigated the pathogenicity of a native strain of B. bassiana (CD1123) against all developmental stages of R. sanguineus s.l.. Methods Batches of eggs, larvae, nymphs and adult ticks were immersed in a suspension of 107 conidia/ml of B. bassiana s.l., isolated from a R. sanguineus s.l. engorged female. All treatment and control groups were observed for 20 days, and the biological parameters (i.e., mortality, hatching, moulting percentage, pre-oviposition period, oviposition period and rate, eggs production efficiency, reproductive efficiency and fitness indexes) were assessed. Results The effect of the B. bassiana strain tested herein on eggs, larvae, nymphs and adults showed a significantly higher mortality than those of the control groups (p < 0.05) at 5 days post-infection. No infected eggs hatched and no infected larvae moulted. Only 15% of infected nymphs moulted into adults. All biological parameters of treated groups differed significantly (p < 0.001) from those of control groups. Conclusions This study demonstrates that a suspension containing 107 conidia/ml of a native B. bassiana strain is highly virulent towards all life-cycle developmental stages of R. sanguineus s.l. and may be of potential interest as a biological control agent against these ticks.

Capillaria aerophila is a trichuroid nematode affecting the respiratory system of dogs, cats, wild carnivores and, occasionally, humans. Animals become infected by ingesting larvated eggs or earthworms, which act as facultative intermediate hosts. The aim of this work is to present new insights into morphological and biological features of this neglected lungworm. Typical features of C. aerophila eggs, differentiating them from those of most known trichuroid whipworms (i.e. size, asymmetry of bipolar plugs and a wall with a network of anastomosing ridges), were detected upon light and scanning electron microscopy. Eggs of C. aerophila were used for in vitro development. Light microscopy showed typical features of C. aerophila eggs: size, asymmetry of bipolar plugs and a wall with a network of anastomosing ridges. All these features were confirmed upon SEM, in that C. aerophila eggs showed an outer densely striated and net-like shell. Eggs of T. vulpis, used for a comparative analysis, were bigger than those of C. aerophila and showed a thick and smooth wall at both light and scanning electron microscopy. Eggs started to develop after 35 days from shedding and mobile larvae were observed in the eggs after two months. The results of this study provide key information on the biological cycle of C. aerophila and present key morphological characters for the identification of eggs in faeces.

Species ranked within the genus Baylisascaris (Ascaridida, Ascarididae) have been implicated in clinical and subclinical intestinal diseases in their natural hosts (e.g., raccoons and bears) as well as in life-threatening larva migrans syndromes in a number of incidental hosts, including humans. Following the diagnosis of Baylisascaris transfuga infestation in two captive polar bears, living in the zoo park of Pistoia (Tuscany, Italy), nematodes (n=300; both sexes) have been characterized by morphological and molecular methods by sequencing and analysing ribosomal (large ribosomal DNA (28S) and internal transcribed spacer region 1 and 2 (ITSs)) and mitochondrial (cytochrome c oxidase subunit 1 (cox1) and cytochrome c oxidase subunit 2 (cox2)) target regions. In addition, seven faecal samples were collected from the animal enclosure and submitted to copromicroscopic and molecular examination. All nematodes were morphologically identified as B. transfuga and their main distinctive features are here presented. No variation in size and nucleotide polymorphisms was detected within each target sequence among all samples analysed. These data contribute to facilitate an accurate diagnosis of this little known nematode infestation in order to apply appropriate anthelmintic strategies.

Strongyloidiasis caused by Strongyloides stercoralis is a zoonotic disease potentially fatal to dogs and humans. Data on the epidemiology of canine strongyloidasis is scant, likely because of inadeguate diagnostics currently employed for the detection of dog infections by Strongyloides. To our knowledge, polymerase chain reaction (PCR) has not been applied to dogs either for epidemiological surveys or for comparison of diagnostic methods. We reported the clinical presentation and outcome of 5 dogs with S. stercoralis infection diagnosed by the detection of larvae in fecal samples. In addition, a qPCR routinely used in human medicine has been used for the diagnosis of canine strongyloidasis. Dogs were hosted in a single shelter in the province of Bari in which a history of diarrhea, weight loss, reduced appetite and respiratory symptoms was reported. The five dogs were positive for S. stercoralis by coproscopy on direct faecal smears and via the Baermann method. All of them, but one, were treated with fenbendazole (Panacur®, Intervet, Animal Health, 50mg/kg, PO daily for five days), or with a combination of fenbendazole and moxidectin plus imidacloprid spot-on (Im/Mox; Advocate® spot-on, Bayer). Following treatment all dogs resulted negative at Baermann in at least 12 days. Anyway, failure of treatment was documented in two dogs by means of reverted Baermann positive results throughout the study and/or post-mortem detection of adult parasites. PCR was positive in all pre-treatment fecal samples and in all fecal debris. One dog resulted negative at Baermann test one week after treatment and showed positive PCR results. This study documents, for the first time, the presence of S. stercoralis infection in sheltered dogs from southern Italy. Treatment with fenbendazole alone or combined with moxidectin plus imidacloprid could be not effective against the infection. PCR routinely used for S. stercoralis diagnosis in humans showed good performance in dog and could be important not only for diagnostic purpose but also to evaluate the efficacy of treatments. Finally, data indicate that S. stercoralis infection may represent an issue for sheltered animals and a potential risk for personnel working in the large number of municipal shelters present in Southern European countries.

Background: This study was aimed at investigating the distribution of a Cercopithifilaria sp. sensu Otranto et al., 2011 with dermal microfilariae recently identified in a dog from Sicily (Italy). A large epidemiological survey was conducted by examining skin samples (n = 917) and ticks (n = 890) collected from dogs at different time points in Italy, central Spain and eastern Greece. Results: The overall prevalence of Cercopithifilaria sp. in the sampled animal populations was 13.9% and 10.5% by microscopy of skin sediments and by PCR on skin samples, respectively. Up to 21.6% and 45.5% of dogs in Spain were positive by microscopical examination and by PCR. Cumulative incidence rates ranging from 7.7% to 13.9% were estimated in dogs from two sites in Italy. A low level of agreement between the two diagnostic tests (microscopical examination and PCR) was recorded in sites where samples were processed in parallel. Infestation rate as determined by tick dissection (from 5.2% to 16.7%) was higher than that detected by PCR (from 0% to 3.9%); tick infestation was significantly associated with Cercopithifilaria sp. infestation in dogs from two out of four sites. Developing larvae found in ticks were morphometrically studied and as many as 1469 larvae were found in a single tick. Conclusions: Our data suggest that, in addition to the most common species of filarioids known to infest dogs (i. e., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum), Cercopithifilaria sp. with dermal microfilariae should be considered due to its widespread distribution in southern Europe and high frequency in tick-exposed dogs.

Troglostrongylus brevior is a neglected feline lungworm species, which has been increasingly reported in the Mediterranean area, although scant data are available on the respiratory alterations it causes in cats. Therefore, we describe the gross and histological lesions of a 20-week old kitten that succumbed due to the onset of a fulminant respiratory failure. At necropsy, a catarrhal exudate was observed in the airways, along with nematodes in the trachea and bronchi. The lungs were processed for histological examination and serial pulmonary sections were performed. A total of 14 nematodes were collected, being all morphologically and molecularly identified as T. brevior. Lungworms were histologically localized within the bronchial lumen, surrounded by an eosinophilic infiltrate. The presence of T. brevior in the airways has been histologically documented for the first time and its life-threatening potential is discussed.

ATP-binding cassette (ABC) transporters have been shown to be involved in pesticide detoxification in arthropod vectors and are thought to contribute to the development of drug resistance. Little is currently known about the role they play in ticks, which are among the more important vectors of human and animal pathogens. Here, the role of ABC transporters in the transport of fipronil and ivermectin acaricides in the tick Rhipicephalus sanguineus (Ixodida: Ixodidae) was investigated. Larvae were treated with acaricide alone and acaricide in combination with a sub-lethal dose of the ABC transporter inhibitor cyclosporine A. The LC50 doses and 95% confidence intervals (CIs) estimated by mortality data using probit analysis were 67.930p.p.m. (95% CI 53.780-90.861) for fipronil and 3741p.p.m. (95% CI 2857-4647) for ivermectin. The pre-exposure of larvae to a sub-lethal dose of cyclosporine A reduced the LC50 dose of fipronil to 4.808p.p.m. (95% CI 0.715-9.527) and that of ivermectin to 167p.p.m. (95% CI 15-449), which increased toxicity by about 14- and 22-fold, respectively. The comparison of mortality data for each separate acaricide concentration showed the synergic effect of cyclosporine A to be reduced at higher concentrations of acaricide. These results show for the first time a strong association between ABC transporters and acaricide detoxification in R.sanguineus s.l.

Background: Dogs are the main reservoir hosts of Leishmania infantum, the agent of human zoonotic visceral leishmaniosis. This study investigated the efficacy of a polymer matrix collar containing a combination of 10% imidacloprid and 4.5% flumethrin as a novel prophylactic measure to prevent L. infantum infections in young dogs from a hyper-endemic area of southern Italy, with a view towards enhancing current control strategies against both human and canine leishmaniosis. Methodology/Principal Findings: The study was carried out on 124 young dogs, of which 63 were collared (Group A) while 61 were left untreated (Group B), from March-April 2011 until March 2012. Blood and skin samples were collected at baseline (April 2011) and at the first, second, third and fourth follow-up time points (July, September 2011 and November 2011, and March 2012, respectively). Bone marrow and conjunctiva were sampled at baseline and at the fourth follow-up. Serological, cytological and molecular tests were performed to detect the presence of L. infantum in the different tissues collected. At the end of the trial, no dog from Group A proved positive for L. infantum at any follow-up, whereas 22 dogs from Group B were infected (incidence density rate = 45.1%); therefore, the combination of 10% imidacloprid and 4.5% flumethrin was 100% efficacious for the prevention of L. infantum infection in young dogs prior to their first exposure to the parasite in a hyper-endemic area for CanL. Conclusions: The use of collars containing 10% imidacloprid and 4.5% flumethrin conferred long-term protection against infection by L. infantum to dogs located in a hyper-endemic area, thus representing a reliable and sustainable strategy to decrease the frequency and spread of this disease among the canine population which will ultimately result in the reduction of associated risks to human health.

Canine spirocercosis caused by Spirocerca lupi is a life-threatening helminthic disease featured by severe clinical signs and potential development of oesophageal neoplasia. This infection is considered fairly frequent in Europe but almost unknown in Italy, from where only few reports have been published in local journals at the beginning of the XXI century. In the present study, an autochthonous case of canine spirocercosis in a 2-year-old dog from southern Italy is described. The animal was admitted to a private veterinary clinic in the municipality of Potenza (Basilicata region) due to persistent dyspnoea, vomiting, and regurgitation. At the abdominal ultrasound, a mass (1.3 × 2 cm) was observed and, thereafter, surgically removed from the apical part of the stomach. A female specimen of S. lupi was morphologically identified during the histological examination of the nodule and its eggs were detected in the faeces. In addition, the morphological identification was confirmed by molecular amplification and sequencing of partial cox2 gene sequence of S. lupi. Veterinarians should be aware of the potential risks derived from infection with this spirurid, which requires specific diagnostic and preventive measures.

Background: Thelazia callipaeda (Spirurida, Thelaziidae), also known as “oriental eyeworm”, is a small nematode parasite that lives in the conjunctival sac of domestic and wild carnivores, rabbits and even humans, causing mild (e.g., conjunctivitis, epiphora, and ocular discharge) to severe (e.g., keratitis, and corneal ulcers) ocular disease. This study reports, for the first time, the occurrence of T. callipaeda infection in the Balkan regions (i.e., Bosnia and Herzegovina and Croatia), it provides genetic evidence on the origin of the infection in that area and discusses potential expansion pathways in the near future. Methods: This survey was conducted in two Western Balkan countries, Bosnia and Herzegovina and Croatia. At necropsy, from January 2011 to April 2014, a total of 184 carcasses of red foxes were examined throughout the study area and worms were collected from the conjunctival sac. In the same period, worms were also collected during clinical examination from the conjunctival sac of four dogs and a cat from Bosnia and Herzegovina and two dogs from Croatia. All nematodes collected were morphologically identified and molecularly characterized by sequencing of partial cox1 gene. Results: T. callipaeda was observed in 51 (27.71%) foxes and the highest prevalence (50.0%) was in the region of East Bosnia. Beside the 4 cases of hyperemia (7.84%), most of the infected animals had no signs of ocular infection (n = 47, 92.15%). A total of 417 adult nematodes collected (364 from foxes, 51 from dogs, 2 from cat) were morphologically and molecularly identified as T. callipaeda haplotype 1. Conclusion: This is the first report of autochthonous cases of T. callipaeda infection in red foxes, dogs and cat in Bosnia and Herzegovina and Croatia and data presented here suggest that reports of thelaziosis in other Balkan areas are, as yet, not diagnosed most likely due to the lack of awareness of practitioners. In addition, data regarding the spread of the infection in Europe over the last ten years suggests that an increasing pattern in the distribution of this disease in domestic and wild animals should be expected in the future.

BACKGROUND: Over the last few decades, canine and feline thelaziosis caused by Thelazia callipaeda eye worms has gained the attention of the veterinary community due to the spread of this ocular infestation in geographical areas previously regarded as non endemic. The therapeutic efficacy of milbemycin oxime/praziquantel tablets (Milbemax®) against T. callipaeda was tested in naturally infested dogs and cats. METHODS: From January 2009 to July 2011 a placebo controlled and randomized field study was conducted in T. callipaeda endemic areas of Switzerland (CH) and Italy (ITA) involving client-owned animals. Dogs (n = 56) and cats (n = 31) were physically examined at enrolment Day 0 (D0) and twice afterwards (D7 and D14). Infested animals were orally treated with Milbemax® or with placebo tablets on D0 and, if an animal was found still infested with T. callipaeda, also on D7. On D14 nematodes were flushed from the conjunctiva, identified and counted. RESULTS: Out of 56 dogs, 43 were included in the statistical analysis, whereas 13 were excluded because the products under investigation were not administered with food, as required by the label. On D7 and D14, 72.7% and 90.9% of treated dogs were eye worm free, whereas in the placebo group 95.2% and 76.2% still harbored nematodes, resulting in a mean percentage worm count reduction for the Milbemax® group of 86.1% and 96.8%, respectively. Both results were significantly higher (p = 0.0001) than the placebo group. Out of the 31 cats included in the study at D7 and D14, 53.3% and 73.3% treated with Milbemax® were free of T. callipaeda, while 81.3% and 73.3 in the placebo group were still harbouring eye worms, resulting in a mean percentage worm count reduction for the treated group of 62.2% and 80.0%, respectively. Both results were significantly higher (p = 0.0106 and p = 0.0043) than the placebo group. CONCLUSIONS: The commercial formulation of milbemycin oxime at the minimal dose of 0.5 mg/kg and 2 mg/k in dogs and cats, respectively, showed a high therapeutic efficacy in curing T. callipaeda infestations. The advantages of an oral application are additionally increased by the large spectrum of activity of praziquantel and milbemycin oxime against Cestodes and Nematodes infesting dogs and cats.

Introduction and aim of the study - The clinical evolution of Leishmania infantum infection in dogs is largely influenced by the host's individual immune response. Few studies have investigated the time-course and clinical evolution of the infection both under experimental and natural conditions. In the present investigation, the time-course of L infantum infection was studied by monitoring clinical and laboratory features in naturally infected animals. Material and methods - The study starts from 218 dogs resulted negative to diagnostic tests for L infantum on March 2005, of which 23 dogs positivized to one or more diagnostic tests between November 2005 and March 2006.The infected animals were monitored serologically (IFAT), parasitologically (lymph-node smear microscopical examination, PCR on dermal tissue) and clinically, through a clinical-pathological score, every 4 months till March 2007. Results - After assessing the diagnostic tests over time and the clinical scores, the time course of infection was defined as transient asymptomatic infection (II dogs), persistent asymptomatic infection (2 dogs), patent symptomatic infection (8 dogs). Discussion -The results of this study confirm the high variability of the L infantum infection progression in endemic areas and underline the difficulty to propose a clear method to define the clinical status of the infection with single-time samples. So it's advisable to monitor infected animals over time in order to make easier any clinical interpretation or the beginning of a possible therapy.