This study describes a duplex real-time polymerase chain reaction (PCR) assay for the detection and differentiation between Dirofilaria immitis and Dirofilaria repens in dog blood and mosquitoes. Regions of a cytochrome oxidase 1 (cox1) mitochondrial DNA fragment and the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA were amplified from microfilariae and adult worm samples, using a sensitive SsoFast™ EvaGreen(®) based real-time PCR method coupled with melting-curve analysis. The limit of the real-time PCR in detecting microfilaria and adult worm DNA was also tested both in dog blood and in artificially infected microfilarial. Two peaks at different melting temperatures (T(m)) for D. immitis (mean ± SD=75.7 ± 0.3°C) and D. repens (mean ± SD=70 ± 0.7°C), respectively, were obtained for microfilarial and adult positive controls of both species when examined separately and together. The real-time PCR protocol was also efficient in detecting microfilarial and adult DNA of both species when tested in samples spiked with DNA from Aedes albopictus, in Aedes aegypti experimentally infected by D. repens and in Culex pipiens naturally infected by D. repens and D. immitis. The high sensitivity of real-time PCR confirmed its reliability in detecting small amounts of genomic DNA either in dog blood or mosquitoes (2.5 pg/μl and 3 × 10(-1)pg/μl for D. immitis and D. repens, respectively). This assay is proposed as a tool for the epidemiological surveillance of the two most important Dirofilaria species in areas where they are endemic and sympatric.

The present study reports the applicability of a multiplex PCR for the simultaneous detection and differentiation of common filarioids infecting dogs, i.e., Dirofilaria immitis, Dirofilaria repens, Acanthocheilonema reconditum and Cercopithifilaria sp. Amplicons of different sizes (i.e., 170 bp, 480 bp, 590 bp and 300 bp, respectively) of regions within the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were amplified on a single-step multiplex PCR using a mix of species-specific forward primers coupled with a single reverse primer. Experiments were carried out by amplifying genomic DNA extracted from blood or skin samples test-positive for microfilariae (mff). The number of mff present in each blood sample was quantified (from 800 to 25,000 mff/ml for A. reconditum and D. repens, respectively) and mixed blood samples were tested for the simultaneous detection of DNA from these mff. Specific amplicons for blood-circulating mff of A. reconditum, D. immitis and D. repens and for those whose adults are localized in skin (i.e., A. reconditum and Cercopithifilaria sp.) were simultaneously detected on agarose gel up to a dilution of 250 mff/ml for D. repens. The specific identity of the amplicons was confirmed by sequencing. The multiplex PCR assay reported herein represents a new tool for the molecular detection and differentiation of canine filarioids in blood and skin samples.

Members of the genus Malassezia are lypophilic and/or lipid-dependent, unipolar budding yeasts that can become pathogenic under the influence of particular predisposing factors (e.g., changes in the cutaneous microenvironment and/or alterations in host defences). This genus comprises at least 14 species, which have been identified traditionally based on their morphology and biochemical features. However, phenetic characteristics often do not allow the identification or delineation of closely related Malassezia spp., such that molecular tools need to be used to assist in fundamental studies of the epidemiology and ecology of Malassezia as well as aspects of the pathogenesis and disease caused by members of this genus. This article briefly reviews the morphological and biochemical methods commonly used for the identification of Malassezia as well as DNA technological methods that have been established for the specific identification of members of this genus and the diagnosis of their infections. New avenues for the development of improved molecular-diagnostic methods to overcome diagnostic limitations and to underpin fundamental investigations of this interesting group of yeasts are proposed.

This study investigates sequence variation in mitochondrial cytochrome c oxidase subunit 1 gene within Cercopithifilaria sp. recorded recently in Italy. Fourteen sequence types (haplotypes) were characterized for 163 (7.7%) amplicons from 2111 Genomic DNA samples prepared from skin samples from dogs and from Rhipicephalus sanguineus (ticks) from different geographical areas of the Mediterranean basin (i.e., Italy, Spain and Greece). The most prevalent sequence types represented haplotypes I (70.5%) and X (16.0%), followed by haplotype VIII (4.9%) and other 11 haplotypes (8.6%). Three haplotypes (II, V and VI) were found exclusively in ticks. The overall intraspecific nucleotide variation among pcox1 haplotypes ranged from 0.4 to 3.5% (mean = 1.6%), whereas a mean interspecific difference of 9.5% was detected as compared with other onchocercids. Phylogenetic analysis of the nucleotide sequence data showed a clustering of Cercopithifilaria sp. with the other Cercopithifilaria species (with strong statistical support) to the exclusion of other onchocercids. The number of haplotypes identified here might be explained by complex ecology and transmission patterns as well as the high mutation rate of mitochondrial DNA and/or inbreeding associated with hosts and their vectors.

Cardiopulmonary infections by Angiostrongylus chabaudi affect domestic and wild felids but, due to limited information on the biology of this nematode, its pathogenicity remains unclear. This article describes the histopathological alterations associated with Angiostrongylus infection in a wildcat from Bulgaria, and reviews current literature on this feline angiostrongylid. Nematodes were isolated from lung lavage and faecal samples of a road killed wildcat in Southern Bulgaria. The morphological identification of parasite larvae as A. chabaudi was confirmed by molecular analysis of part of the 18S ribosomal RNA gene. Upon histopathological examination, severe granulomatous pneumonia, ranging from multifocal to coalescing, and pulmonary vascular lesions were observed. Extensive alveolar collapse, alveolar emphysematous changes, parenchymal haemorrhages and small artery wall hyperplasia were observed in the parenchyma adjacent to the granulomas. Histopathological examination revealed the presence of cross-sections of adult female parasites within the lumen of the pulmonary artery branches, the intima altered markedly by subendothelial proliferation and oedematous changes. This study compliments current knowledge of the pathogenesis of feline angiostrongylosis by A. chabaudi in wildcats, as well as of the distribution of this little-known parasite.

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).

In April 2008, whole blood samples were collected from 36 dromedary camels in Sokoto, North-western Nigeria. Following PCR and reverse line blotting, twenty-two samples (61%) resulted positive for Ehrlichia/Anaplasma spp. and three (8%) for Theileria/Babesia spp., with three (8%) cases of co-infections being found. Both sequence and BLAST analyses identified Ehrlichia/Anaplasma spp. and Theileria/Babesia spp. positive cases as Anaplasma platys and Theileria ovis, respectively. This is the first report of the detection of A. platys and T. ovis in camels from sub-Saharan Africa. The epidemiological relevance of this finding is enhanced by the close living of these animals with both dogs and small ruminants. The high prevalence detected for A. platys suggests a possible role of camels as carriers of this infection.

Capillaria aerophila, Aelurostrongylus abstrusus, Angiostrongylus vasorum and Dirofilaria immitis are cardiopulmonary nematodes affecting dogs and cats and presently emerging in several countries. The results obtained in 2009 - 2010 during a study aiming to investigate the occurrence of these nematodes in regions from Central (Marche and Abruzzo regions--Sites A and B, respectively) and southern (Apulia--Site C) Italy are here reported. A total of 534 and 436 individual faecal samples collected from dogs and cats were examined, together with 471 and 34 faecal environmental samples taken from dog shelters and catteries. One hundred and ninety-two individual blood samples were also collected from dogs. Faeces were examined using copromicroscopical flotations and Baermann technique, whereas blood samples were tested by Knott's method. Eggs of C. aerophila were detected in 1.48 % and 20 % (Site A), 8.67 % and 2.71 % (Site B), and 16.67 % and 0 % (Site C) of individual and environmental canine samples. C. aerophila was found in 2.90 % (Site A), 3.03 % (Site B) and 14.29 % (Site C) of individual cats. Larvae of A. vasorum were found in 0.96 % and 2.48 % of individual and environmental samples from Site B, respectively, while those of A. abstrusus in 1.82 % (Site A) and 9.96 % (Site B) of individual faeces. Microfilariae of Dirofilaria immitis, identified on the basis of key morphological and morphometric features, were detected in 2.56 % of samples collected from Site B. Despite the small sample size, these results indicate that cardiopulmonary nematodes occur in Central and southern Italy, thus they should be included in the differential diagnosis of pet cardiorespiratory diseases. Larger surveys are necessary to gain more information on the diffusion of these parasites, especially for C. aerophila and A. vasorum, for which the actual distribution is poorly known.

Infections with Onchocerca lupi nematodes are diagnosed sporadically in the United States. We report 8 cases of canine onchocercosis in Minnesota, New Mexico, Colorado, and Florida. Identification of 1 cytochrome c oxidase subunit 1 gene haplotype identical to 1 of 5 from Europe suggests recent introduction of this nematode into the United States.

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.

Despite the widespread distribution of Cercopithifilaria bainae among canine and tick populations worldwide, this filarioid is currently considered of ‘minor importance’ in veterinary medicine, particularly when compared to related filarioids, such as Dirofilaria immitis and Dirofilaria repens. To date, only a single case of dermatological alterations possibly associated to infection by C. bainae had been reported in a dog. In the present study, we describe the first case of systemic alterations associated to C. bainae infection in a dog suffering from diffused chronic polyarthritis. The animal had a previous history of reluctance to move and stiff gait and displayed multiple joint pain during manipulation of limbs. No biochemical, haematological and X-ray alterations were detected; microfilariae were observed in the synovial fluids collected from the joints. In spite of the morphological and molecular identification of these microfilariae as C. bainae, the dog did not respond to multiple microfilaricidal treatments with milbemicyn oxyme. The potential role of C. bainae in the pathogenesis of this clinical condition is discussed. Given the potential pathogenicity of this parasite, improved knowledge of this little known tick-borne nematode is warranted in order to assist the development of novel and effective treatment strategies.

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 aim of this study was to investigate the anatomical distribution of microfilariae of a recently described Cercopithifilaria sp. and the relationship with the preferred attachment sites of its vector, the brown dog tick Rhipicephalus sanguineus. Skin samples from 20 dogs were collected from eight anatomical sites and soaked twice in saline solution at 37°C. All samples were also molecularly processed for the specific amplification of partial cytochrome c oxidase 1 gene. Microfilariae were unevenly distributed on the body, with higher frequencies on interscapular region (n=13; 68.4%) and on the head (n=9; 47.4%). Larval abundance in dogs was positively correlated with the number of positive anatomical sites with a higher percentage of skin samples scoring positive at the microscopic examination of the first sediment (n=47; 30.9%) than of the second (n=8; 5.3%). Histological examination revealed that microfilariae were scattered in the dermis, in association with inflammatory cells. Molecular analysis of skin samples showed a lower frequency of positive sites (n=37; 24.3%) when compared to the microscopic examination (n=53; 34.9%) with five haplotypes sequenced. The results here presented suggested a close co-evolution of this filarioid with its vector

Phlebotomine sand flies (Diptera, Psychodidae) are vectors of Leishmania spp., among which Leishmania infantum is recognized as the main agent of human and canine leishmaniosis (CanL) in the Mediterranean area. In this study, females of Phlebotomus spp. (P. perniciosus, P. neglectus and P. papatasi) and Sergentomyia minuta were collected in a dog shelter of southern Italy, where CanL is endemic, and examined for Leishmania DNA. In total, 32 out of 56 of Phlebotomus spp. insects (57.1%) were found positive for L. infantum DNA by quantitative PCR (qPCR), with a mean parasite load of 1.9 x 103 promastigotes/ml among 23 positive P. perniciosus and 2.1 x 103 promastigotes/ml among five positive P. neglectus. Four P. papatasi, a species known to be refractory to L. infantum development, were also found positive. Among 216 S. minuta specimens examined, 25 (11.6%) scored positive for Leishmania tarentolae by conventional nested PCR; two (16.7%) of them were also positive for lizard blood, which is in agreement with the feeding preference of this phlebotomine species. Nine S. minuta (4.2%) were positive for L. infantum by qPCR, with a mean parasite load of 1.62 × 102 promastigotes/ml. The detection of L. infantum DNA in S. minuta may suggest that this species could acquire the protozoan, occasionally feeding on infected dogs. Further investigations need to clarify the potential role that S. minuta may have in the transmission of L. infantum to receptive mammal hosts.

causes canine infectious cyclic thrombocytopenia (CICT). The vector role of Rhipicephalus sang-uineus group ticks has been only suggested, but definitive evidence is lacking. Thisstudy aimed to detect and quantify A. platys DNA in infected dogs and in their respec-tive ticks through a quantitative real-time PCR assay. From March to May 2009, bloodand tick samples from dogs residing in a CICT-endemic area were collected and molec-ularly analysed. Differences (p < 0.05) were detected in the bacterial load betweentick nymphs (3.5 × 10−2± 2.5 × 10−2) and adults (female: 1.2 × 10−1± 1.1 × 10−1; male:9.3 × 10−2± 1.2 × 10−2) and between unengorged (1.1 × 10−1± 2.8 × 10−2) and partially orfully engorged ticks (1.9 × 10−1± 2.7 × 10−2). No difference was found between ticks col-lected from positive (1.1 × 10−1± 0.6 × 10−1) and negative (1.2 × 10−1± 0.3 × 10−1) dogs(p > 0.05). The mean bacterial load detected in positive dogs was lower than that in theirrespective ticks (p > 0.05). This study provides circumstantial evidence of the putative roleof Rhipicephalus sp. I as a vector of this pathogen.

Background: Hepatozoon canis is a widespread tick-borne protozoan affecting dogs. The diagnosis of H. canis infection is usually performed by cytology of blood or buffy coat smears, but this method may not be sensitive. Our study aimed to evaluate the best method to achieve a parasitological diagnosis of H. canis infection in a population of receptive young dogs, previously negative by cytology and exposed to tick infestation for one summer season. Results: A total of 73 mongrel dogs and ten beagles younger than 18 months of age, living in an animal shelter in southern Italy where dogs are highly infested by Rhipicephalus sanguineus, were included in this study. In March- April 2009 and in October 2009, blood and bone marrow were sampled from each dog. Blood, buffy coat and bone marrow were examined by cytology only (at the first sampling) and also by PCR for H. canis (second sampling). In March-April 2009, only one dog was positive for H. canis by cytological examination, whereas in October 2009 (after the summer season), the overall incidence of H. canis infection by cytological examinations was 43.9%. Molecular tests carried out on samples taken in October 2009 showed a considerably higher number of dogs positive by PCR (from 27.7% up to 51.2% on skin and buffy coat tissues, respectively), with an overall positivity of 57.8%. All animals, but one, which were positive by cytology were also PCR-positive. PCR on blood or buffy coat detected the highest number of H. canis-positive dogs displaying a sensitivity of 85.7% for both tissues that increased up to 98% when used in parallel. Twenty-six (74.8%) out of the 28 H. canis-positive dogs presented hematological abnormalities, eosinophilia being the commonest alteration observed. Conclusions: The results suggest that PCR on buffy coat and blood is the best diagnostic assay for detecting H. canis infection in dogs, although when PCR is not available, cytology on buffy coat should be preferred to blood smear evaluation. This study has also demonstrated that H. canis infection can spread among young dogs infested by R. sanguineus and be present in the majority of the exposed population within 6 months.

BACKGROUND: Filarioids belonging to the genus Cercopithifilaria (Spirurida: Onchocercidae) have been described in dogs in association with Rhipicephalus sanguineus group ticks, which act as their biological vectors. This study represents the first investigation on Cercopithifilaria spp. in dogs from Portugal. FINDINGS: Dogs (n = 102) from the Algarve region (south of Portugal) were sampled by skin snip collection and tissues were left to soak overnight in saline solution. Sediments were observed under a light microscope and the detected microfilariae identified according to their morphology. Twenty-four dogs (23.5%) were found infected with at least one species of Cercopithifilaria, namely C. bainae (9.8%), C. grassii (3.9%) and Cercopithifilaria sp. II sensu Otranto et al., 2013 (13.7%). Results were confirmed by molecular amplification of partial cytochrome c oxidase subunit I and 12S rRNA genes and sequence analysis. Co-infections with more than one Cercopithifilaria species were detected in 3.9% of the animals. CONCLUSIONS: This is the first report of Cercopithifilaria spp. in dogs from Portugal. The estimated level of infection with C. bainae, C. grassii and Cercopithifilaria sp. II suggests that these filarioids are prevalent in the canine population of southern Portugal.

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: The efficacy of a slow-release insecticidal and repellent collar containing 10% imidacloprid and 4.5% flumethrin (Seresto, Bayer Animal Health) in preventing Leishmania infantum infection was evaluated in a large population of dogs living in a hyper-endemic area of Sicily (Italy). Methods: A total of 219 dogs, negative for L. infantum were enrolled in a multicentre, controlled study. Dogs were divided into two homogeneous groups, defined as G1 (n = 102) and G2 (n = 117). Before the start of the sand fly season, dogs in G1 were treated with the collar while animals in G2 were left untreated, serving as negative controls. Dogs were serially sampled on day D90, D180, D210 and D300 in order to assess Leishmania infection by IFAT, PCR on skin (D210-D300) and bone marrow (D300) and cytology on bone marrow aspirate (D300). Results: Three dogs (2.9%) in G1 and 41 (40.2%) in G2 became positive for L. infantum in at least one of the diagnostic tests employed in the study. The number of seropositive dogs in G2 increased in the course of the study from 15 (D90) to 41 (D300), with some of them also positive in other diagnostic tests. Eight (19.6%) of the seropositive dogs in G2 showed an increase in antibody titers ranging from 1:160 to 1:1,280. At the last follow-up, some of dogs in G2 displayed overt clinical signs suggestive of leishmaniosis. The mean incidence density rate at the final follow-up was 4.0% for G1 and 60.7% for G2, leading to a mean efficacy of the collar in protecting dogs at both sites of 93.4%. Conclusions: The slow-release collar tested in this study was shown to be safe and highly effective in preventing L. infantum infection in a large population of dogs. Protection conferred by a single collar (up to eight months) spanned an entire sand fly season in a hyper-endemic area of southern Italy. The regular use of collars, at least during the sand fly season, may represent a reliable and sustainable strategy for the prevention of leishmaniosis in dogs living in or travelling to an endemic area.

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.

Metastrongyloids of cats are emerging pathogens that may cause fatal broncho-pulmonary disease. Infestation of definitive hosts occurs after ingestion of intermediate or paratenic hosts. Among metastrongyloids of cats, Troglostrongylus brevior and Troglostrongylus subcrenatus (Strongylida: Crenosomatidae) have recently been described as agents of severe broncho-pulmonary disease. Here, we provide, for the first time, observational evidence suggesting the direct transmission of T. brevior from queen cat to suckling kittens. This new knowledge will have a significant impact on current scientific information of this parasite and shed new light into the biology and epidemiology of metastrongyloid nematodes.

Given the spread of Aedes albopictus from northern to southern Italy, and the lack of updated data on Dirofilaria infections, this study was carried out to assess the infection risk for dogs and cats in Apulia region. During a 2-year study, 175 A. albopictus female specimens and samples of blood from 427 dogs (309 privately owned dogs and 118 shelter dogs) and 12 cats were collected. All blood samples were subjected to a modified Knott method, to a test for the detection of circulating Dirofilaria immitis antigen, and to a Dirofilaria species-specific real-time PCR for the simultaneous detection of D. immitis and Dirofilaria repens, targeting on partial cytochrome oxidase subunit 1 and internal transcribed spacer-2, respectively. Two abdomen and one thorax pools from A. albopictus were positive for D. immitis, with minimum infection rates of 1.14 and 0.51, respectively, and a probability of a single positive specimen to be infected of P = 0.6 % (95 % confidence interval (CI) = 0.12-1.73). Out of 439 examined subjects, 22 (5.0 %) tested positive for Dirofilaria spp. in at least one diagnostic test. A specific D. immitis infestation rate of 3.5 % was found among the privately owned dogs, while shelter dogs tested positive only for D. repens with a prevalence of 3.4 %; one cat tested molecularly positive for D. immitis. There was a significantly higher rate of positivity among guard dogs for D. immitis (odds ratio, 6.24, 95 % CI, 1.26-25.28; P < 0.05). The increasing risk of D. immitis infection in southern Italy is supported by the noteworthy positivity of A. albopictus populations and the cat. Our data highlight the usefulness to include filarioid infestation in routine diagnosis.

BACKGROUND: Thelazia callipaeda eyeworms are transmitted by the non-biting insect vector Phortica variegata in Europe and infest the conjunctiva(s) of several mammalians, including dogs and humans. Infested hosts might remain asymptomatic or display clinical manifestations characterized by variable degrees of severity. METHODS: From July to November 2011, nine dogs were detected with eyeworms at two veterinary clinics in Chaves and Bragança (North of Portugal). Nematodes collected from dogs were morphologically and molecularly characterized at species level. RESULTS: Nematodes were identified as T. callipaeda. The number of worms collected from each dog ranged from three to 76 (average = 17.9 ± 26.8) and was not associated with the severity of clinical signs. Ocular discharge and conjunctivitis were observed in all dogs and ocular pruritus occurred in six of them. Polymerase chain reaction and sequencing of a portion of target cytochrome c oxidase subunit 1 gene further identified all nematodes as haplotype 1. CONCLUSIONS: This is the first report of T. callipaeda and associated ocular disease in dogs from Portugal, suggesting that thelaziosis should be included in the differential diagnosis of canine ocular affections. The risk of the infestation spreading from Spain and France to Portugal, through domestic dogs or wild mammals, is realistic.

Little detailed information is available on the association of Malassezia pachydermatis genotypes and the extent of skin damage that they cause. In the present study, isolates of M. pachydermatis, recovered from the skin of healthy dogs and dogs with dermatitis in Brazil, were characterized on the basis of partial sequencing of the large subunit (LSU), first internal transcribed spacer (ITS-1) and chitin synthase 2 gene (chs-2). The determination of phospholipase production was also included in the investigations. The severity of lesions and hyperpigmentation of dogs with skin disease were evaluated. For each locus, two main sequence types were designated as genotypes A and C. Two other minor sequence types (A2(I)-C2(I)) were also recorded and defined for the ITS-1. Genotype A isolates were the most prevalent, being recovered from healthy and diseased animals. No significant difference was detected among genotypes or ITS-1 sequence types and grades of skin damage or hyperpigmentation in the dogs with skin lesions. The number of M. pachydermatis isolates that produced phospholipase was statistically higher for diseased dogs than for strains found in healthy animals. The present study reveals that multiple genetic variants of M. pachydermatis occur in dogs and that the distribution patterns of particular genotypes on the skin of dogs in Brazil might be related to environmental and ecological factors which maintain distinctive genotype assemblages in specific geographical areas.

Abstract Eucoleus aerophilus (syn. Capillaria aerophila) is a trichuroid nematode affecting domestic and wild carnivores and, sometimes, humans. This parasite has a worldwide distribution and may cause significant clinical disease in pet animals. The present paper investigates the sequence variation in partial mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of E. aerophilus isolates from pets and wild animals from different countries. Forty-four egg pools of E. aerophilus were collected from dogs, cats and foxes from Italy, while seventeen adult stages of E. aerophilus were obtained from red foxes and beech martens from Portugal, Romania, Serbia and UK. Fifteen different haplotypes were characterized and five were shared between pets in Italy and wildlife from Europe. The remaining haplotypes were either confined only in hosts or countries, or in a given host from a country. The phylogenetic analysis showed that all haplotypes clustered as a monophyletic group with a strong nodal support, indicating that all sequence types represented E. aerophilus. The results here presented have implications for a better understanding of the epidemiology, phylo-geography and clinical impact of E. aerophilus. In particular, the geographic distribution of E. aerophilus haplotypes in different host species and geographic regions, and their variation in terms of pathogenic impact and zoonotic role, warrant further investigations.

In order to evaluate infection occurrence and the potential zoonotic role of horse isolates of Giardia 25 duodenalis, 431 individual fecal samples were genetically characterized by PCR tests –coupled sequencing 26 and phylogenetic analysis. Thirty-seven (8.6%) animals resulted infected by different Assemblage. The 27 presence of sub-Assemblage was assessed by characterizing the b-giardin gene for 16 of the 37 positive 28 horses. Ten isolates showed 99.6% to 100% homology with the sub-Assemblage described as B1–2 and 29 B1–6, three Assemblage A showed 99.8% homology with sub-Assemblage A1, while one Assemblage E 30 displayed 98.8% homology with sub-Assemblage E3. Furthermore, one isolate characterized as Assem- 31 blage A showed 99.6% homology with the sub-Assemblage B1–2 and one characterized as E was 100% 32 identical with sub-Assemblage B1–6. These results demonstrate the presence of both animal and human 33 sub-Assemblage of G. duodenalis in horses from Italy. Epidemiological and sanitary implications are 34 discussed.

Hepatozoon canis infection in ticks during spring and summer in Italy

With the exception of Aelurostrongylus abstrusus, feline lungworms have been poorly studied. Information on their distribution is patchy and mostly limited to case reports. In this study, the occurrence of feline lungworms and co-infecting gastrointestinal parasites has been investigated in 12 European countries (i.e. Austria, Belgium, Bulgaria, France, Greece, Hungary, Italy, Portugal, Romania, Spain, Switzerland and the United Kingdom). An average of 10 domestic cats, with regular outdoor access, was sampled each month for 12months, and freshly passed faeces were collected. Stools were processed using a McMaster assay and a quantitative Baermann-Wetzel method. Animals positive for lungworms and/or gastrointestinal parasites were treated with a formulation containing fipronil, (S)-methoprene, eprinomectin, and praziquantel (Broadline®, Merial), and re-sampled 28days post-treatment. The association between lungworm infection and risk factors was analysed using statistical medians/means and the efficacy of the treatment against each lungworm species was assessed. Of 1990 cats sampled, 613 (30.8%) were positive for at least one parasite, while 210 (10.6%) were infected by lungworms. The prevalence of lungworm infection varied between the sampled sites, with the highest recorded in Bulgaria (35.8%) and the lowest in Switzerland (0.8%). None of the cats from Austria or the United Kingdom were infected by lungworms. Aelurostrongylus abstrusus was the species most frequently detected (78.1%), followed by Troglostrongylus brevior (19.5%), Eucoleus aerophilus (14.8%) and Oslerus rostratus (3.8%). The overall efficacy of the treatment was 99% for A. abstrusus and 100% for T. brevior, O. rostratus and E. aerophilus. Data presented provide a comprehensive account of the diagnosis, epidemiology and treatment of feline lungworms in Europe, as well as of the occurrence of co-infections by gastrointestinal parasites.

In the past years, canine and human cases of infestation by Dirofilaria repens (Spirurida, Onchocercidae) have been increasingly reported in several European countries. Subcutaneous dirofilariosis by D. repens may either be asymptomatic in dogs or may be characterized by subcutaneous nodules and other symptoms. Information on the periodicity of D. repens microfilariae in naturally infested animals is scant, and this might impair the accurate diagnosis of subcutaneous dirofilariosis and appropriate control plans. In the present study, eight dogs infested with D. repens were sampled twice daily at 12-h intervals for ten consecutive days, and the dog with the highest mean value of microfilariaemia was further sampled every 4 h for four consecutive days. The blood was microscopically and molecularly examined for microfilariae, and, additionally, negative samples were also subjected to a real-time PCR to evaluate the level of circulating DNA. The results demonstrated significant variations in circadian rhythms of D. repens larvae, with higher values of microfilariae per milliliter in the evening samples. A significant variation was also found at the individual level for the dogs with the highest values of microfilariaemia. All samples which were negative at the light microscopy and positive at the real-time PCR displayed levels of circulating parasite DNA <1 microfilaria per milliliter. Biological and clinical implications have been here discussed.

Capillaria aerophila, a trichuroid nematode causing pulmonary infections in wild and domestic carnivores, is occasionally and potentially poorly recognized in infections of humans due to clinicopathological mimicry and a lack of accurate, robust laboratory diagnostics. The present work evaluated the efficiency of a DNA-based assay amplifying a partial cytochrome c oxidase subunit 1 (cox1) gene of C. aerophila in the diagnosis of lung capillariosis. Fecal samples from 34 dogs and 10 cats positive at parasitological examination for C. aerophila and other endoparasites (i.e., other lungworms, whipworms, roundworms, hookworms, tapeworms, and/or coccidia) and from 44 animals negative for C. aerophila but positive for other endoparasites were molecularly examined. Of the 44 samples positive for C. aerophila at copromicroscopy, 43 scored positive (i.e., 33/34 dogs and 10/10 cats) in seminested PCR, resulting in a sensitivity of 97 to 100%. Samples that were copromicroscopy negative for C. aerophila although positive for other endoparasites never produced a PCR product or nonspecific amplicons. The specific PCR amplification of C. aerophila (i.e., specificity of 100%) was confirmed by a nucleotide sequence analysis of the cox1 amplicons. The potential implications of the molecular diagnosis of lung capillariosis are discussed.

The Rhipicephalus sanguineus group includes some of the most widespread dog ticks, whose identification currently represents a difficult task due to the morphological similarities of these ixodid species. Recently, following the morphological and molecular characterization of tick specimens collected from dogs in all continents, Rhipicephalus sanguineus sensu lato (s.l.), Rhipicephalus turanicus and three different operational taxonomic units (namely Rhipicephalus sp. I-III) have been identified. Therefore, a comprehensive molecular study has been herein carried out to detect selected canine vector-borne pathogens (i.e., Anaplasma platys, Cercopithifilaria spp., Ehrlichia canis and Hepatozoon canis) in ticks belonging to the R. sanguineus group. A total of 204 tick specimens from dogs were examined and identified as R. sanguineus s.l. (. n=. 81), R. turanicus (. n=. 17), Rhipicephalus sp. I (. n=. 66), Rhipicephalus sp. II (. n=. 37), and Rhipicephalus sp. III (. n=. 3). PCR assays were performed to detect mitochondrial and ribosomal target genes of Cercopithifilaria spp., A. platys, E. canis and H. canis. Out of 204 specimens examined, 2.5%, 7.4% and 21.6% scored positive to A. platys, H. canis and Cercopithifilaria spp., respectively. In addition, co-infections with two pathogens (i.e., Cercopithifilaria bainae and A. platys or H. canis) were detected in four tick specimens. This study suggests a relationship between ticks belonging to the R. sanguineus group and the geographical distribution of A. platys, H. canis and Cercopithifilaria spp. However, the role of each representative tick species within the R. sanguineus group in the transmission of these canine pathogens needs to be further investigated.

Dermatophytes are fungi that invade and propagate in the keratinized skin of mammals, including humans, often causing contagious infections. The species of medical concern belong to the genera Microsporum, Trichophyton, Epidermophyton (in their anamorphic state) and Arthroderma (in their telomorphic state), which were traditionally identified based on their morphology and biochemical characters. Nonetheless, limitations linked to the differentiation of closely related agents at species and strains level have been recently overcome by molecular studies. Indeed, an accurate identification of dermatophytes is pivotal for the establishment of effective control and prevention programs as well as for determining the most appropriate and effective antifungal therapies to be applied. This article reviews the DNA techniques and the molecular markers used to identify and to characterize dermatophyte species, as well as aspects of their phylogeny and evolution. The applications of typing molecular strain to both basic and applied research (e.g., taxonomy, ecology, typing of infection, antifungal susceptibility) have also been discussed.

BACKGROUND: Dirofilaria immitis and Dirofilaria repens are transmitted by bloodsucking culicid mosquitoes belonging to Culex, Aedes, Ochlerotatus, Anopheles and Mansonia genera. The detection of filariae in mosquitoes for assessing distribution of vectors and/or of pathogens in a given area (also known as "xenomonitoring"), when based on individual dissection of wild-caught female mosquitoes is time consuming and hardly applicable in large epidemiological surveys. Our study aimed to evaluate the recently developed duplex real-time PCR for screening large number of culicids and to assess their positivity for D. immitis and D. repens in an area where both species are endemic. METHODS: A duplex real-time PCR was used to detect and differentiate D. immitis and D. repens in mosquitoes collected in six provinces of the Veneto region using 43 carbon dioxide-baited traps under the frame of an entomological surveillance program to monitor the vectors of West Nile disease. From early May till October 2010, unfed female mosquitoes (n = 40,892) were captured in 20 selected sites. RESULTS: Mosquitoes were identified as Culex pipiens, Ochlerotatus caspius, Aedes vexans and Culex modestus and grouped into 995 pools according to species, day and site of collection (from minimum of 1 to maximum of 57). Out of 955 pools, 23 (2.41 %) scored positive for Dirofilaria spp. of which, 21 (2.2 %) for D. immitis and two (0.21 %) for D. repens. An overall Estimated Rate of Infection (ERI) of 0.06 % was recorded, being higher in Och. caspius and Ae. vexans (i.e., 0.18 % and 0.14 %, respectively). At least one mosquito pool was positive for Dirofilaria spp. in each province with the highest ERI recorded in Vicenza and Padova provinces (i.e., 0.42% and 0.16 %, respectively). Mosquitoes collected in all provinces were positive for D. immitis whereas, only two (i.e., Padova and Rovigo) provinces scored positive for D. repens. All mosquito species, except for Cx. modestus, were positive for D. immitis, whereas D. repens was only found in Cx. pipiens. CONCLUSIONS: The results suggest that both Dirofilaria species are endemic and may occur in sympatry in the examined area. The molecular approach herein used represents a powerful tool for surveillance programs of D. immitis and D. repens in the culicid vectors towards a better understanding of the epidemiology of the infections they cause and their seasonal transmission patterns.

BACKGROUND: The taxonomic status of the brown dog tick (Rhipicephalus sanguineus sensu stricto), which has long been regarded as the most widespread tick worldwide and a vector of many pathogens to dogs and humans, is currently under dispute. METHODS: We conducted a comprehensive morphological and genetic study of 276 representative specimens, which belonged to different species (i.e., Rhipicephalus bursa, R. guilhoni, R. microplus, R. muhsamae, R. pusillus, R. sanguineus sensu lato, and R. turanicus) collected from Europe, Asia, Americas, and Oceania. After detailed morphological examination, ticks were molecularly processed for the analysis of partial mitochondrial (16S rDNA, 12S rDNA, and cox1) gene sequences. RESULTS: In addition to R. sanguineus s.l. and R. turanicus, three different operational taxonomic units (namely, R. sp. I, R. sp. II, and R. sp. III) were found on dogs. These operational taxonomical units were morphologically and genetically different from R. sanguineus s.l. and R. turanicus. Ticks identified as R. sanguineus s.l., which corresponds to the so-called "tropical species" (=northern lineage), were found in all continents and genetically it represents a sister group of R. guilhoni. R. turanicus was found on a wide range of hosts in Italy and also on dogs in Greece. CONCLUSIONS: The tropical species and the temperate species (=southern lineage) are paraphyletic groups. The occurrence of R. turanicus in the Mediterranean region is confirmed. A consensual re-description of R. sanguineus s.s. and R. turanicus will be necessary to solve the taxonomic problems within the so-called R. sanguineus group.

Dermal microfilariae found in a dog from Sicily, Italy, were characterized morphologically and genetically and differentiated from those of all the other blood microfilariae commonly found in dogs. In particular, the microfilariae were short (mean length of 186.7 μm), presented a body flattened dorso-ventrally and a rounded head, bearing a tiny cephalic hook. The genetic identity of microfilariae herein studied was also assessed by molecular amplification, sequencing and analyzing of multiple ribosomal ITS-2 and mitochondrial (cox1 and 12S) target genes. Both morphologic and genetic characterization as well as the molecular phylogenetic history inferred using sequences of a barcoding dataset were concordant in supporting the identification of Cercopithifilaria at the genus level. Surprisingly, microfilariae here examined were well distinct from Cercopithifilaria grassii (Noè, 1907), from northern Italy, and resembled those of a species described in Brazil, Cercopithifilaria bainae Almeida & Vicente, 1984. This paper provides evidence for the existence of a Cercopithifilaria species infesting a dog from Sicily and also presents a PCR protocol on skin samples as a tool for further epidemiological studies, which could provide evidence on the aetiology and the natural history of this filarial species.

This study reports a combined analysis of mitochondrial and ribosomal DNA target regions of phlebotomine sand flies (Diptera: Psychodidae) from the Mediterranean region. A ∼900 bp long fragment of the mitochondrial DNA encompassing regions within cytb and nd1 gene and the complete ITS2 ribosomal region (∼500 bp) were sequenced and characterized for Phlebotomus perniciosus, Phlebotomus perfiliewi, Phlebotomus neglectus, Phlebotomus papatasi, and Sergentomyia minuta, captured in two sites of southern Italy. From one to eight mitochondrial haplotypes and from one to three ITS2 sequence types were found for the examined specimens according to the different sand fly species. The mean interspecific difference in the mitochondrial sequences was of 16.1%, with an overall intraspecific nucleotide variation from 0.1 to 2.8%. A higher interspecific difference (mean 25.1%) was recorded for the ITS2 sequence, with an overall intraspecific nucleotide variation up to 4.9%. The sequence types alignment of ITS2 region showed that all phlebotomine specimens possessed a split 5.8S rRNA, consisting of a mature 5.8S rRNA and a 2S rRNA separated by a short transcribed spacer. Phylogenetic analysis of the Phlebotomus spp. sequences, herein determined and of those available in GenBankTM were concordant in clustering P. neglectus, P. perfiliewi and P. papatasi with the same species collected from different geographic areas of the Mediterranean basin in four main clades for mtDNA and ITS2, respectively. This study demonstrates the utility of multilocus sequencing, provides a dataset for the molecular identification of the most prevalent phlebotomine sand flies in southern Europe and defines the phylogenetic relationships among species examined.

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.

Three lipid-dependent Malassezia isolates (here named 114A, 114B and 114C) recovered from a dog with skin lesions were phenotypically and genotypically characterized. All presented ovoid cells and buds formed on a narrow base. Most of the results from physiological tests were consistent with those of Malassezia furfur. The phylogenetic analysis of ITS-1 and LSU nucleotide sequences was concordant in placing all three clinical Malassezia isolates close to M. furfur. However, the phylogenetic data on the chs-2 sequence revealed that clinical isolate 114A is distinct from M. furfur and was closely affiliated to the sequence of M. pachydermatis with high nodal support. In particular, lipid-dependent isolates 114A displayed chs-2 sequences similar (100%) to that of the non-lipid dependent species Malassezia pachydermatis. The presence of the genetic and physiological polymorphisms detected in these three isolates of M. furfur could have resulted from a process of adaptation of this anthropophilic species to a new host.

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.

Background: Leishmania infantum is a widespread parasite that affects dogs and humans worldwide. It is transmitted primarily by phlebotomine sand flies, but recently there has been much discussion on the role of the brown dog tick, Rhipicephalus sanguineus, as a potential vector for this protozoan. Recent laboratory and field investigations have contributed to this hypothesis, but a proof of the vector capacity of R. sanguineus has yet to be provided. Following a recent study suggesting that L. infantum passes transovarially from the female tick to her progeny the current study provides new evidence of the transovarial transmission of L. infantum in R. sanguineus. Methods: Engorged females of R. sanguineus were collected from the environment in a dog shelter of southern Italy, where canine leishmaniosis is endemic. In the laboratory, 97 females that successfully laid eggs, their eggs and the originated larvae were subjected to DNA extraction and then tested by a TaqMan-based real time PCR targeting a fragment of the kinetoplast DNA (kDNA) of L. infantum. Results and conclusions: L. infantum kDNA was detected in engorged females, their eggs and originating larvae, with a parasite load ranging from 1.8 × 10-4 to 10.0 × 100. Certainly, the current study provides further evidence on the passage of L. infantum from R. sanguineus females to their offspring. The observation of promastigote forms in larvae is necessary to definitively confirm this hypothesis, which would raise interesting questions about the possible role of ticks in the maintenance of L. infantum infection among dogs in certain areas.

Ocular thelaziosis by Thelazia callipaeda is an emerging disease that affects primarily dogs, but also cats, foxes and other wild carnivores, as well as humans. Three clinical cases of unilateral conjunctivitis caused by Thelazia nematodes were detected in Border Collie, a dog breed intolerant to the macrocyclic lactones. Animals came from southwestern Spain, on the border with Portugal. Eight worms were collected and identified molecularly as T. callipaeda by amplification and sequencing of partial cytochrome c oxidase subunit 1 gene. Oral treatment with mebendazole 20 mg/kg (Telmin®) was effective in curing the infection.

The life cycles of filarioids of dogs presenting dermal microfilariae have been little studied. Following the recent retrieval of dermal microfilariae identified as Cercopithifilaria sp. in a dog from Sicily (Italy), this study was designed to assess the role of the brown dog tick Rhipicephalus sanguineus as an intermediate host of this filarial species. An experimental tick infestation was performed on an infected dog using 300 nymphs of R. sanguineus. Engorged nymphs were collected and examined by both microscopic dissection and molecular analysis at five time points (i.e., the same day of tick detachment and 10, 20, 30 and 50 days post-detachment) to detect the presence and developmental stage of filariae in the ticks. A total of 270 engorged nymphs were collected from the dog and developing filarioid larvae detected in 10 (5%) out of 200 ticks dissected. Infective third-stage larvae were observed in 4 (2%) of the all dissected ticks, 30 days post-detachment. Twelve (6.6%) out of 181 samples molecularly tested were positive for Cercopithifilaria sp. This study demonstrates that nymphs of R. sanguineus feeding on a dog naturally infected by Cercopithifilaria sp. can ingest microfilariae, which develop up to the third infective stage thus suggesting that this tick species might act as an intermediate host of this little known canine filarioid.

Background: Danube Delta Biosphere Reserve is one of the most interesting regions in Europe from an epidemiological point of view due to its great biodiversity, local climatic conditions and various types of habitats. Moreover, there is no data regarding the ectoparasite communities of dogs from this area. In this frame, the aims of our study were to establish the tick communities parasitizing dogs and to provide new data regarding seasonal dynamics of a neglected tick species, Rhipicephalus rossicus. Methods: A survey was carried out in order to gather information regarding tick species attaching to domestic dogs from a steppic region of southeastern Romania and to establish their seasonal dynamics. The research was conducted from 1 December 2012 to 30 November 2013, on 8 dogs from Iazurile, a locality from the west-central part of the Danube Delta Biosphere Reserve. In total, 384 examinations were made, each dog being checked for tick infestation 4 times per month, for one year. Results: The 893 ticks found belonged to six species: R. rossicus (95.6%), Dermacentor reticulatus (3.2%), Ixodes ricinus (0.5%), Hyalomma marginatum (0.3%), Rhipicephalus sanguineus sensu lato (s.l.) (0.2%) and Ixodes crenulatus (0.1%). From the 91 positive examinations, R. rossicus was found in 80 (87.9%). Single species infestation occurred in 84 examinations. In 7 out of 91 positive examinations mixed infestation were found. No ticks were found in December, January and September. Conclusions: For R. rossicus, high frequency and intensity were observed in May, June and July. The activity peaks for D. reticulatus were in spring and autumn. Our results highlight that within the range of R. sanguineus s.l., the most common dog tick worldwide, selected dog populations may be predominantly infested by closely related species, like in our case, R. rossicus.

In addition to Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae), referred to asthe feline lungworm, Troglostrongylus brevior (Strongylida: Crenosomatidae) has recentlybeen identified as an agent of bronco-pulmonary infestations in cats. These two parasiteshave a similar biology, share ecological niches, potentially co-infesting cats, but are dif-ficult to be differentiated due to the morphological similarities of their first-stage larvae(L1). This paper describes a molecular tool, based on single-step duplex polymerase chainreaction (duplex-PCR) on the ribosomal internal transcribed spacer 2 region (ITS-2) for thesimultaneous detection and differentiation of T. brevior and A. abstrusus. L1 of both specieswere collected from faecal samples, morphologically identified, and single larval specimensisolated. An aliquot of faeces was used as a test sample for a case of mixed natural infes-tation. The duplex-PCR was performed using species-specific forward primer sets for theITS-2 region (i.e., A. abstrusus: 220 bp; T. brevior: 370 bp). The detection limit of the molec-ular assay was also assessed by serial dilutions of DNA from single larvae of both species(from ∼4.0 to 4.0 × 10−5g/l). The duplex-PCR carried out on individual DNA sampleswas able to detect as low as 5.2 × 10−3g/l of DNA for A. abstrusus, 4.9 × 10−3g/l for T.brevior, and as low as 4.0 × 10−3g/l for samples containing both species. Species-specificbands of the expected sizes and two bands were simultaneously amplified from the fae-cal sample containing both species. The phylogenetic analyses of the ITS-2 sequences hereexamined and those available for other metastrongyloids were concordant in clusteringthem with those of other Troglostrongylus brevior and A. abstrusus sequences available inGenBank database. This molecular approach proved to be effective and highly sensitivefor the simultaneous detection of the two lungworms species and it might be used formolecular epidemiological studies and for monitoring therapeutic protocols.

Following the recent description of microfilariae of a Cercopithifilaria sp. in a dog from Sicily, Italy, (herein after referred to as Cercopithifilaria sp. I), numerous skin samples were collected from dogs in the Mediterranean region. In addition to Cercopithifilaria sp. I (185·7±7·2 μm long), microfilariae of 2 other species were identified, namely Cercopithifilaria grassii (651·7±23·6 μm long) and a yet undescribed microfilaria, Cercopithifilaria sp. II (264·4±20·2 μm long, with evident lateral alae). The morphological differentiation among the 3 species of dermal microfilariae was confirmed by differences in cytochrome c oxidase subunit 1 and ribosomal 12S sequences examined (mean level of interspecific pairwise distance of 11·4%, and 17·7%, respectively). Phylogenetic analyses were concordant in clustering these with other sequences of Cercopithifilaria spp. to the exclusion of Dirofilaria spp., Onchocerca spp. and Acanthocheilonema spp. Dermal microfilariae collected (n=132) were morphologically identified as Cercopithifilaria sp. I (n=108, 81·8%), Cercopithifilaria sp. II (n=17, 12·9%), whereas only 7 (5·3%) were identified as C. grassii. Mixed infestations were detected in all sites examined. The great diversity of these neglected filarioids in dogs is of biological interest, considering the complex interactions occurring among hosts, ticks and Cercopithifilaria spp. in different environments.

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.

After Thelazia callipaeda infection in dogs and cats were reported in Spain, a human case of thelaziosis in this country was reported, suggesting zoonotic transmission. The active reproductive status of this nematode in situ indicates that humans are competent hosts for this parasite.

Background: Ticks may transmit a large variety of pathogens, which cause illnesses in animals and humans, commonly referred to as to tick-borne diseases (TBDs). The incidence of human TBDs in Italy is underestimated because of poor surveillance and the scant amount of studies available. Methods: Samples (n = 561) were collected from humans in four main geographical areas of Italy (i.e., northwestern, northeastern, southern Italy, and Sicily), which represent a variety of environments. After being morphologically identified, ticks were molecularly tested with selected protocols for the presence of pathogens of the genera Rickettsia, Babesia, Theileria, Candidatus Neoehrlichia mikurensis, Borrelia and Anaplasma. Results: Ticks belonged to 16 species of the genera Argas, Dermacentor, Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus, with Ixodes ricinus (59.5%) being the species most frequently retrieved, followed by Rhipicephalus sanguineus sensu lato (21.4%). Nymphs were the life stage most frequently retrieved (41%), followed by adult females (34.6%). The overall positivity to any pathogen detected was 18%. Detected microorganisms were Rickettsia spp. (17.0%), Anaplasma phagocytophilum (0.8%), Borrelia afzelii (0.5%), Borrelia valaisiana (0.3%), C. N. mikurensis (0.5%) and Babesia venatorum (0.6%). Conclusions: Results indicate that people living in the Italian peninsula are at risk of being bitten by different tick species, which may transmit a plethora of TBD causing pathogens and that co-infections may also occur.

The present study reports two simple molecular approaches allowing a rapid identification of the most prevalent species of phlebotomine sand flies in the Mediterranean region. A PCR protocol for the amplification of ITS2 ribosomal region and a PCR-RFLP on a mitochondrial DNA fragment (cytb-nd1) were settled in order to identify and discriminate among Phlebotomus perniciosus, Phlebotomus neglectus, Phlebotomus perfiliewi, Phlebotomus papatasi and Sergentomyia minuta. The ITS2 regions showed a certain degree of interspecific variability, which led to PCR amplicons of different sizes, i.e., 450, 490, 460, 480 and 530 bp for P. perniciosus, P. neglectus, P. perfiliewi, P. papatasi, and S. minuta, respectively. Analogously, the digestion of a mitochondrial DNA amplicon with Ase I enzyme showed five different restriction profiles, which allowed the unequivocal differentiation of the sand fly species examined. These methods might represent useful tools for a molecular large scale screening of phlebotomine sand fly species caught in areas where leishmaniasis is endemic, in order to plan appropriate epidemiological surveillance programs for both Leishmania spp. and their vectors.

Background:Aelurostrongylus abstrusus is currently regarded as the main metastrongyloid infesting domestic cats, whereas the reports of Troglostrongylus spp. in domestic and wild felids largely remain anecdotic. This paper reports on pulmonary infestation caused by Troglostrongylus brevior and Troglostrongylus subcrenatus in two kittens and describes, for the first time, associated clinical presentations and pathological features. Morphometrical, molecular and phylogenetic analyses have also been conducted to differentiate here the examined Troglostrongylus species from A. abstrusus, towards a clearer delineation of metastrongyloids affecting cats. Methods Two kittens were referred for respiratory distress and hospitalized with a diagnosis of severe aelurostrongylosis, based on the presence of metastrongyloid larvae in the faeces. Despite prompt treatment, kittens died within 48 hours. Both kittens were submitted to necropsy to determine the cause of death. Results At necropsy, nematode species were found in the trachea, bronchi and bronchioles and were associated with respiratory signs (i.e., dyspnoea, polypnea, severe coughing and nasal discharge). Morphology and measurements of adult parasites found allowed the unequivocal identification of T. brevior and T. subcrenatus, even if first stage larvae were rather similar to those of A. abstrusus. Briefly, T. brevior and T. subcrenatus larvae were shorter in length and lacking the typical knob-like terminal end of A. abstrusus. Molecular and phylogenetic analyses corroborated morphological identification and provided data on mitochondrial and ribosomal DNA genes of T. brevior. Conclusions Data presented here indicate that T. brevior and T. subcrenatus cause major respiratory distress in domestic cats. Consequently, these two species should be included, along with A. abstrusus, in the differential diagnosis of cat bronchopulmonary affections and, treatment protocols need to be evaluated. Thorough research on the biology, epidemiology and control of troglostrongylid infestations in domestic cats are advisable to implement current knowledge on these neglected metastrongyloids.

Onchocerca lupi infection is reported primarily in symptomatic dogs. We aimed to determine the infection in dogs from areas of Greece and Portugal with reported cases. Of 107 dogs, 9 (8%) were skin snip–positive for the parasite. DNA sequences of parasites in specimens from distinct dog populations differed genetically from those in GenBank.