Geographic distribution of the V1016G knockdown resistance mutation in Aedes albopictus: a warning bell for Europe.

BACKGROUND: Colonization of large part of Europe by the Asian tiger mosquito Aedes albopictus is causing autochthonous transmission of chikungunya and dengue exotic arboviruses. While pyrethroids are recommended only to reduce/limit transmission, they are widely implemented to reduce biting nuisance and to control agricultural pests, increasing the risk of insurgence of resistance mechanisms. Worryingly, pyrethroid resistance (with mortality < 70%) was recently reported in Ae. albopictus populations from Italy and Spain and associated with the V1016G point mutation in the voltage-sensitive sodium channel gene conferring knockdown resistance (kdr). Genotyping pyrethroid resistance-associated kdr mutations in field mosquito samples represents a powerful approach to detect early signs of resistance without the need for carrying out phenotypic bioassays which require availability of live mosquitoes, dedicated facilities and appropriate expertise. METHODS: Here we report results on the PCR-genotyping of the V1016G mutation in 2530 Ae. albopictus specimens from 69 sampling sites in 19 European countries. RESULTS: The mutation was identified in 12 sites from nine countries (with allele frequencies ranging from 1 to 8%), mostly distributed in two geographical clusters. The western cluster includes Mediterranean coastal sites from Italy, France and Malta as well as single sites from both Spain and Switzerland. The eastern cluster includes sites on both sides of the Black Sea in Bulgaria, Turkey and Georgia as well as one site from Romania. These results are consistent with genomic data showing high connectivity and close genetic relationship among West European populations and a major barrier to gene flow between West European and Balkan populations. CONCLUSIONS: The results of this first effort to map kdr mutations in Ae. albopictus on a continental scale show a widespread presence of the V1016G allele in Europe, although at lower frequencies than those previously reported from Italy. This represents a wake-up call for mosquito surveillance programs in Europe to include PCR-genotyping of pyrethroid resistance alleles, as well as phenotypic resistance assessments, in their routine activities.

Temperature is a common climatic descriptor of lachryphagous activity period in Phortica variegata (Diptera: Drosophilidae) from multiple geographical locations.

BACKGROUND: The drosophilid Phortica variegata is known as vector of Thelazia callipaeda, the oriental eyeworm native to Asia that has become an emergent zoonotic agent in several European regions. Unlike almost all other arthropod vectors of pathogens, only P. variegata males feed of lachrymal secretions of animals, ingesting first-stage larvae (L1) of the worm living in the orbital cavities of the host, and allowing with the same behaviour the introduction of infective L3. Despite the increased detection of T. callipaeda in many European countries, information about the length of the lachryphagous activity period of P. variegata and a deep knowledge of the environmental and climatic variables involved are still limited. METHODS: We herein present the results of a multicentre study involving five sites from four different countries (Italy, Spain, UK and USA) where canine thelaziosis is endemic and/or where it has already been ascertained the presence of P. variegata. Field data have been obtained on a fortnightly basis from mid-April to the end of November 2018 from a contemporary standardized sampling (same sampling effort and time of collection in all sites) of lachryphagous flies collected around the eyes of a human bait using an entomological net. These data have been associated to data collection of local climatic variables (day length, temperature, wind speed, barometric pressure and relative humidity). RESULTS: Overall, a total of 4862 P. variegata flies (4637 males and 224 females) were collected, with high differences in densities among the different sampling sites. Significant positive correlations were found between P. variegata male density and temperature and wind speed, while negative correlations were observed for barometric pressure and relative humidity. However, the above significant differences are confirmed in each sampling site separately only for the temperature. CONCLUSIONS: This multicentre study highlights that temperature is the major common environmental driver in describing the lachryphagous activity of P. variegata in Europe and USA and, therefore, the transmission risk of thelaziosis.

The southernmost foci of Dermacentor reticulatus in Italy and associated Babesia canis infection in dogs.

BACKGROUND: Two clustered clinical cases of canine babesiosis were diagnosed by veterinary practitioners in two areas of northeastern Italy close to natural parks. This study aimed to determine the seroprevalence of babesial infection in dogs, the etiological agents that cause canine babesiosis and the potential tick vector for the involved Babesia spp. METHODS: The study area was represented by two parks in northeastern Italy: Groane Regional Park (Site A) and the Ticino Valley Lombard Park (Site B). From March to May 2015 ticks were collected from the vegetation in three transects in each site. In the same period, blood samples were collected from 80 dogs randomly chosen from veterinary clinics and kennel located in the two areas. Morphological identification of the ticks was performed and six specimens were molecularly characterised by the amplification and sequencing of partial mitochondrial 12S rRNA, 16S rRNA and cox1 genes. For phylogenetic analyses, sequences herein obtained for all genes and those available from GenBank for other Dermacentor spp. were included. Dog serum samples were analysed with a commercial indirect fluorescent antibody test to detect the presence of IgG antibodies against Babesia canis. Ticks and blood samples were tested by PCR amplification using primers targeting 18S rRNA gene of Babesia spp. RESULTS: Ticks collected (n = 34) were morphologically identified as adults of D. reticulatus. Twenty-eight ticks were found in all transects from Site A and the remaining six were collected in Site B. Blast analysis of mitochondrial sequences confirmed the morphological identification of processed tick specimens by revealing a highest nucleotide similarity (99-100%) with those of D. reticulatus available in the GenBank database. The phylogenetic trees were concordant in clustering D. reticulatus in a monophyletic clade. Seven dogs (8.8%) had antibodies against B. canis, most of which (n = 6) came from Site A. Analysis of nucleotide sequences obtained from one tick and from one dog identified B. canis displayed a 100% similarity to those available in GenBank. CONCLUSIONS: This study morphologically and molecularly confirms the presence of D. reticulatus in Italy and links it, for the first time, with the occurrence of B. canis infection in dogs in this country.

Aberrant laryngeal location of Onchocerca lupi in a dog.

Onchocerca lupi (Spirurida, Onchocercidae) is an emerging vector-borne helminth that causes nodular lesions associated with acute or chronic ocular disease in dogs and cats. Since its first description in dogs in 1991, this zoonotic filarioid has been increasingly reported in Europe and the United States. An 8-year-old outdoor mixed-breed female dog from the Algarve (southern Portugal) was presented with a history of severe dyspnoea. Cervical and thoracic radiographs revealed a slight reduction in the diameter of the cervical trachea and a moderate increase in radiopacity of the laryngeal soft tissue. An exploratory laryngoscopy was performed, revealing filiform worms associated with stenosis of the thyroid cartilage and a purulent necrotic tissue in the larynx lumen. A single sessile nodule, protruding from the dorsal wall of the laryngeal lumen caused a severe reduction of the glottis and tracheal diameter. Fragments of the worms were morphologically and molecularly identified as O. lupi. Histological examination of the nodule showed a granulomatous reaction with sections of coiled gravid female nematodes. Following laryngoscopy, a tracheostomy tube was inserted to relieve dyspnoea and ivermectin (300 µg/kg, once a week, for 8 weeks) combined with prednisolone was prescribed. The dog showed a complete recovery. Although O. lupi has been isolated in human patients from the spinal cord, this is the first report of an aberrant migration of O. lupi in a dog. The veterinary medical community should pay attention to aberrant location of O. lupi and consider onchocercosis as a differential diagnosis for airway obstruction in dogs.

Ecology of phlebotomine sand flies and Leishmania infantum infection in a rural area of southern Italy.

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.

Spirocerca lupi infection in a dog from southern Italy: an "old fashioned" disease?

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.

Prevention of canine leishmaniosis in a hyper-endemic area using a combination of 10% imidacloprid/4.5% flumethrin.

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.

Therapeutic efficacy of milbemycin oxime/praziquantel oral formulation (Milbemax®) against Thelazia callipaeda in naturally infested dogs and cats.

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.

New insights into morphological and biological features of Capillaria aerophila (Trichocephalida, Trichuridae).

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.

Case report: First evidence of human zoonotic infection by Onchocerca lupi (Spirurida, Onchocercidae).

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.

Phlebotomine sand fly population dynamics in a leishmaniasis endemic peri-urban area in southern Italy.

A 2-year survey was carried out from May to November 2008 and 2009 to study the sand fly species composition, its seasonal phenology and density in Apulia region (southern, Italy). The study was conducted in a dog shelter located in a new residential urban district where Leishmania infantum is endemic. Sand flies were collected using sticky traps from May to November, at about 7-day intervals. Temperature and relative humidity were recorded daily. In December 2008, general environmental improvements (e.g., the ground was covered with gravel and the vegetation present inside the cages was removed to facilitate cleaning) were made in the study area. The most diffused species during the whole study period were Phlebotomus perniciosus (2008, n=248, 49.4%; 2009, n=254, 50.6%) followed by Phlebotomus neglectus (2008, n=76, 39.8%; 2009, n=115, 60.2%) and Phlebotomus papatasi (2008, n=5, 50.0%; 2009, n=5, 50.0%). Four specimens of Phlebotomus perfiliewi were collected only in the first year. The number of Sergentomyia minuta specimens collected increased considerably in the second (n=548, 86.2%) in comparison to the first year (n=88, 13.8%). The highest number of phlebotomine sand flies was collected in July and August when a mean temperature from 27.09 to 28.02°C and mean relative humidity from 47.28 to 56.36% were recorded. The variations in phlebotomine sand fly species diversity and abundance recorded in this study were related to climatic and environmental factors. Data here presented confirm that sand flies easily adapt to the urban environments and that the may represent a public health concern for L. infantum and other pathogen transmission also in similar urban environment of southern Europe.

Diagnosis of canine vector-borne diseases in young dogs: a longitudinal study.

Canine vector-borne diseases (CVBDs) pose a diagnostic challenge, particularly when a dog is coinfected with more than one pathogen. The purpose of this study was to generate information about the diagnosis of CVBDs in young dogs following their first exposure to flea, tick, sand fly, louse, and mosquito vectors. From March 2008 to May 2009, 10 purpose-bred young naive beagle dogs and a cohort of 48 mixed-breed dogs living in an area to which CVBD is endemic in southern Italy were monitored using different diagnostic tests (cytology, serology, and PCR). Overall, PCR detected the highest number of dogs infected with Anaplasma platys, Babesia vogeli, and Ehrlichia canis, whereas seroconversion was a more sensitive indicator of exposure to Leishmania infantum. For A. platys infection, combining blood and buffy coat cytology in parallel enhanced the relative sensitivity (SE(rel)) (87.3%). For B. vogeli, the best diagnostic combination was buffy coat cytology and serology used in parallel (SE(rel), 67.5%), whereas serology and PCR used in parallel (SE(rel), 100%) was the best combination for L. infantum. Overall, 12 (20.7%) dogs were coinfected; however, the percentage of new coinfections decreased from baseline (50%) to the first (33.3%) and second (16.6%) follow-up time points. Numbers of coinfections with A. platys and B. vogeli were significantly higher (P < 0.05) than coinfections with other pathogen combinations. The data generated in this study provide insights on the incidence of certain pathogens infecting young dogs in southern Italy, highlight important diagnostic testing limitations, and support the use of multiple diagnostic modalities when attempting to confirm a tick-borne infection in an individual dog or in a canine population.

Efficacy of an in-feed formulation containing ivermectin for the control of intestinal strongyles in captive zebras (Equus burchelli (Gray, 1824)).

The efficacy of medicated food pellets (containing 10mg of ivermectin per kg, UNIFEED, Veronesi, Verona, Italy) was evaluated for the control of intestinal strongyles in a group of captive zebras (Equus burchelli) at the Safari Park, Fasano (Apulia region, Brindisi Province, southern Italy). The egg reappearance period and the faecal egg counts in terms of eggs per gram of faeces were investigated. From day 0 until day +9, 30 zebras were fed with medicated pellets and pooled faecal samples were collected from the floor of paddocks. The drug showed an efficacy of 100% for up to 78 days post-treatment, with one exception. Strongyle eggs collected prior to the treatment were used to perform coprocultures and larvae harvested were molecularly identified as Cylicostephanus minutus and Cylicocyclus leptostomum with a reverse line blot hybridization assay. The administration of ivermectin with medicated food pellets was effective in controlling intestinal strongylosis in captive zebras. The opportunity to treat captive ungulates with an in-feed anthelmintic is discussed in relation to the fractious nature of these animals, which often impairs helminth control programs in zoo-parks.

Gastrointestinal parasites in mammals of two Italian zoological gardens.

The prevalence of gastrointestinal parasites was investigated in mammals housed in two of the main Italian zoological gardens: the Zoo Safari of Fasano (province of Bindisi, Apulia, Italy) and the Giardino Zoologico of Pistoia (Tuscany, Italy). In November 2007, fecal samples were collected at the Zoo Safari of Fasano (n = 96) and at the Giardino Zoologico of Pistoia (n = 60), from primates, carnivores, perissodactyls, artiodactyls and proboscideans. In most of the cases, the same animal species or genera were considered in both the zoos. One or more intestinal parasites were detected in 61.5% of the examined samples. However, very different percentages of protozoa- and helminth-positive samples were obtained in the animals from the two zoos, with an overall gastrointestinal parasitic infection of 77.1% in the Zoo Safari of Fasano and 36.7% in the Giardino Zoologico of Pistoia. Overall, 10% (Cryptosporidium sp.) and 43.3% (Toxocara cati, Strongyloides stercoralis, Toxascaris leonina, and hookworms) of carnivores, 66.7% (Cryptosporidium spp.) and 100% (Trichuris spp. and Strongyloides fülleborni) of primates, 25.0% (Eimeria spp., Cryptosporidium spp.), and 57.1% (Trichuris sp., Toxocara [Neoascaris] vitulorum, gastrointestinal strongyles, and Paramphistomidae) of artiodactyls were infected with protozoa and helminthes parasites, respectively. Only gastrointestinal strongyles were diagnosed in 16.7% of proboscideans and in 50.0% of perissodactyls examined. The results of this study suggest that zoonotic protozoans and gastrointestinal helminths are common in zoo mammals and that these animals may serve as a potential reservoir and transmit these parasites to humans. The different animal management in the two zoos indicates that the regular monitoring of parasitic diseases and the use of selective treatments can represent effective measures for the control of several gastrointestinal infections in zoological gardens.

Distribution and species-specific occurrence of cyathostomins (Nematoda, Strongylida) in naturally infected horses from Italy, United Kingdom and Germany.

A broad scale study was carried out in 2008 to evaluate the distribution and species-specific occurrence of cyathostomin populations in horse yards from Europe. In total 102 properties and 3123 horses were included in Italy (60 yards and 1646 animals), United Kingdom (22 yards and 737 animals) and Germany (20 yards and 740 animals). Individual faecal samples were examined with a McMaster technique while pooled samples were subjected to the microscopic examination of in vitro cultured larvae and to a Reverse Line Blot (RLB) assay able to molecularly identify the most diffused 13 species of cyathostomins. All yards were positive for the presence of cyathostomins both at the McMaster technique and at the microscopic examination of cultured larvae. One thousand and nine hundred thirty-one horses (61.8%) showed a positive faecal egg count, i.e. 1110 (67.4%), 463 (62.8%) and 358 (48.3%) from Italy, UK and Germany respectively. Out of the 1931 positive animals 1133 (36.3%) showed a faecal egg count per gram >150, specifically 694 (42.2%) from Italy, 237 (32.2%) from UK and 202 (27.3%) from Germany. The molecular results showed that all 13 species that can be detected by the RLB were found in each of the three countries, with a range of 3-13 species present in individual yards. The five most prevalent were Cylicocyclus nassatus, Cylicostephanus longibursatus, Cyathostomum catinatum, Cylicocyclus goldi and Cyathostomum pateratum. The relevance of these results and related biological and epidemiological features are discussed, together with their significance for both future studies of cyathostomins and further intervention programs aiming to control the spread of anthelmintic-resistant populations.

Clinical and laboratory monitoring of dogs naturally infected by Leishmania infantum.

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.

Thelazia callipaeda (Spirurida, Thelaziidae) in wild animals: report of new host species and ecological implications.

Thelazia callipaeda infects the eyes of carnivores and humans in Far Eastern Asiatic and European countries. Studies have demonstrated the occurrence of T. callipaeda in foxes from areas where canine thelaziosis is endemic. However, there is little information on the role of wild carnivores as hosts of this nematode. From May 2003 to May 2009, a total of 130 carcasses of red foxes (Vulpes vulpes; n=75), wolves (Canis lupus; n=2), beech martens (Martes foina; n=22), brown hares (Lepus europaeus; n=13), Eurasian badgers (Meles meles; n=10), and wild cats (Felis silvestris; n=8) were examined in an area of southern Italy where canine thelaziosis is highly prevalent. At necropsy, animals were examined and nematodes were collected from the conjunctival sacs of both eyes. All nematodes were morphologically identified and at least five specimens from each of the five host species were molecularly processed by PCR amplification and sequencing of a partial mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Five out of the six wild animal species examined were found to be infected with eyeworms. The overall infection rate, excluding the Eurasian badgers that were all negative, was 39.1%. All the 189 adult nematodes collected (intensity of infection=4+/-2.2) were morphologically identified as T. callipaeda. The molecular analysis confirmed that the only haplotype of T. callipaeda circulating in Europe (i.e., haplotype 1) is present in that area. The competence of red foxes, wolves, beech martens, brown hares, and wild cats as definitive hosts for T. callipaeda is discussed in relationship to their ecology and their likely exposure to the vector Phortica variegata in the study area. The role the wild fauna plays in maintaining and spreading eyeworm infection in humans and domestic animals is also discussed.

Faecal Cyathostomin Egg Count distribution and efficacy of anthelmintics against cyathostomins in Italy: a matter of geography?

BACKGROUND: In the framework of a trial carried out in 2008 in Europe to evaluate the efficacy of major parasiticides against horse cyathostomins, pre- and/or post-treatment Faecal Egg Counts (FEC) were evaluated in a total of 84 yards and 2105 horses from nine different regions from the South, the Center, the North-Center and North-East of Italy. Specifically, on the basis of FECs of the horses present in each property, 60 out of the 84 yards were enrolled for a Faecal Egg Count Reduction Test (FECRT) using fenbendazole, pyrantel, ivermectin and moxidectin. RESULTS: Of the 1646 horses bred in the 60 recruited yards, 416 animals had a FEC between 50 and 150 Eggs Per Gram (EPG) of faeces and 694 a FEC >150 EPG (i.e. with total of 1110 positive animals). Of the 1110 positive animals, those with the highest FECs (i.e. 988) were included in the FECRT. The FECRT for four anthelmintic compounds showed remarkable differences in terms of prevalence of reduced and equivocal efficacy against cyathostomins in the different areas of Italy. Administration of fenbendazole and pyrantel resulted in resistance present or suspected in about half of the yards examined while resistance to ivermectin was found in one yard from central Italy and suspected resistance was detected in three more yards, one in each the North, the Center and the South. Treatment with moxidectin was 100% effective in all yards examined. CONCLUSION: Cyathostomin populations in the South and the Center of Italy were more susceptible to fenbendazole and pyrantel than the populations present in the Center-North and North-Eastern areas of Italy. Fenbendazole and/or pyrantel were ineffective in almost all properties from the North of Italy. The reasons for such a difference among the Italian regions in terms of FECs and efficacy of antiparasitic drugs are discussed, together with the role that veterinarians, and horse owners and managers should have for effective worm control programs in this country.

Efficacy of Amitraz plus Metaflumizone for the treatment of canine demodicosis associated with Malassezia pachydermatis.

This case reports the efficacy of metaflumizone plus amitraz spot-on formulation (ProMeris Duo(R); Fort Dodge) against generalized demodectic mange. A two year-old male dog presented at clinical examination with poor general condition, diffused alopecia, crusted lesions, pruritus, skin scales and pustules. Demodex mites, Malassezia pachydermatis yeasts and bacteria were diagnosed. The dog was treated with cephalexin and topically with metaflumizone plus amitraz spot on formulation at two weeks intervals until two consecutive skin scrapings resulted negative for mites. The number of adult mites statistically decreased at follow-up with a reduction of approximately 42 and 94% at +14 and +28 days post treatment (p.t.) respectively. Nymphs and larvae could not be detected from +28 day p.t. while eggs were no longer present +42 day p.t. The dog was negative for both bacteria and M. pachydermatis at 14 days p.t., coinciding with improved general clinical conditions, recovering skin lesions and no further signs of pruritus. These results show that metaflumizone plus amitraz associated with the antibiotic therapy is highly effective for treating generalized demodectic mange and could also be effective toward controlling M. pachydermatis opportunistic infections.