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

The mitochondrial DNA of the cattle grub Hypoderma lineatum (de Villers) (Diptera: Oestridae) was completely sequenced. The entire molecule was 16 354 bp long and presented a heavy bias towards A + T, which accounted for 77.8% of the whole genome. Hypoderma lineatum genes were organized in the same order and orientation as in the mitochondrial genomes available for other species belonging to the Oestroidea superfamily and compared in this study [ Chrysomya putoria (Wiedemann), Cochliomyia hominivorax (Coquerel), Lucilia sericata (Meigen) and Dermatobia hominis (L.)], except for the occurrence of a 102-bp non-coding region partially present in other species. The complete sequence of H. lineatum will represent a useful dataset to evaluate the evolutionary pattern of mtDNA within Oestroidea by using molecular information in diagnostic, taxonomic and evolutionary studies. [ABSTRACT FROM AUTHOR]

Abstract: This study investigated genetic variability within the ‘eyeworm’Thelazia callipaeda (Nematoda: Thelazioidea) from Europe and Asia by polymerase chain reaction (PCR)-coupled sequencing and mutation scanning of the mitochondrial cytochrome c oxidase subunit 1 gene (cox 1). Eight different sequence variants of cox1 (haplotypes) were determined for the 50 individual adult specimens of T. callipaeda (from dogs, foxes or cats from Italy, Germany and the Netherlands and from dogs from China and Korea). Nucleotide variation (0.3–2%) was detected at 23 of 649 positions in the cox1. Six of these positions were invariable among all 37 individuals from Europe and among the 13 individuals from Asia (irrespective of host origin) but differed (five G↔A and one C↔T changes) between Europe and Asia. PCR-based single-strand conformation polymorphism (SSCP) analysis of the most variable portion (v-cox1) of the cox1 was validated (for a subset of samples) as a tool to rapidly screen for genetic (haplotypic) variability. The results for the SSCP analysis and sequencing were concordant, indicating that the mutation scanning approach provides a useful tool for investigating the population genetics and molecular ecology of T. callipaeda. [Copyright &y& Elsevier]

The subfamily Steganinae (Diptera, Drosophilidae) includes flies which display zoophilic feeding behaviour in the larval and/or adult stages, some of which act as vectors of Spirurida eyeworms, which infect both carnivores and humans. To date, the taxonomy and phylogeny of the subfamily Steganinae has been studied only superficially and many aspects of their systematics remain unresolved. Thus, the present study aimed to provide a molecular dataset to facilitate the identification and phylogenetic analysis of Steganinae species based on partial (∼ 700 basepairs) mitochondrial cytochrome c oxidase subunit 1 ( cox1) sequences. A total of 134 flies belonging to 13 species and eight genera of Steganinae were subjected to molecular and phylogenetic analyses. The mean nucleotide variation within the Steganinae subfamily was 8.1%, with a variation within genera for which more than one species was examined ranging from 1.6% (in Phortica spp.) to 21.8% (in Amiota spp.). Interspecific pairwise divergence ranged from 1.6% ( Phortica variegata vs. Phortica semivirgo) to 24.8% ( Cacoxenus indagator vs. Amiota alboguttata) and intraspecific variation ranged from 0% to 1%. Seventy of the 233 amino acids were variable, including 26 parsimony informative sites and 44 singleton sites, with some highly conserved residues identified within the genera Stegana and Amiota. Parsimony and maximum likelihood-based phylogenetic analyses provided strong support for the genus Phortica, phylogenetically distinct from the genus Amiota. Gitona distigma was placed in an unresolved position adjacent to the outgroup taxa, Drosophila yakuba and Drosophila melanogaster. The molecular data reported here represent the first molecular dataset based on cox1 of Steganinae flies and provide a base for further investigations into the evolutionary relationships among this little-studied subfamily. [ABSTRACT FROM AUTHOR]

Larvae belonging to five species of Hypoderma spp. (Diptera, Oestridae) cause myiasis in wild and domestic ruminants that is characterized by migrations within deep tissues. In China hypodermosis is one of the most important arthropod diseases affecting ruminants and, moreover, represents a significant zoonosis, with numerous reports of Hypoderma spp. affecting farmers. Recently, a sixth species, Hypoderma sinense Pleske, has been rediscovered but the endogenous migration pathway within the host body is completely unknown and it represents a major constraint for the control of larval infection. In December 2003 a total of 165 larval stages of Hypoderma spp. were collected from different anatomical sites of 40 yaks slaughtered at an abattoir in the province of Gansu, China. The morphological characters and size of the recovered larvae were used to infer migratory routes and 45 specimens were also subjected to a polymerase chain reaction (PCR) assay of cox1 mtDNA and amplicons sequenced. All the larvae molecularly processed were identified as H. sinense and sequence identity was confirmed by a PCR-restriction fragment length polymorphism (PCR-RFLP) tool carried out using BfaI and HinfI endonucleases. The finding of H. sinense larvae only in the oesophagus or both in oesophagus and subcutaneous tissue of 12 and 15 animals, respectively, indicates that H. sinense larvae migrate through the oesophagus similarly to Hypoderma lineatum (De Villers). The description of the endogenous life cycle of H. sinense will help to determine the timing of specific treatment programmes to guarantee the improvement of animal welfare and health, thus resulting in an increase in livestock production in China. [ABSTRACT FROM AUTHOR]

Abstract: Knowledge about Phortica variegata (Drosophilidae, Steganinae), the intermediate host of the eyeworm Thelazia callipaeda (Spirurida, Thelaziidae), is confined to experimental studies. To investigate the role P. variegata plays in the transmission of T. callipaeda under natural conditions, the population dynamics of these flies in the natural environment and their feeding preferences (on vegetables and/or animal lachrymal secretions) were examined. From April to November 2005, a total number of 969 (557 males and 412 females) P. variegata flies were collected weekly in a region of southern Italy with a history of canine thelaziosis. The flies were identified and dissected or subjected to a PCR assay specific for a region within the ribosomal ITS-1 DNA of T. callipaeda. The zoophilic preferences of P. variegata were assessed by collecting flies around the eyes of a person or around a fruit bait. Seven hundred and twenty flies (398 males and 322 females) were dissected under a stereomicroscope; 249 flies (158 males and 91 females) that died prior to the dissection were subjected to molecular investigation. Only P. variegata males were infected with larval T. callipaeda both at dissection (six, 0.83%) and with the specific PCR (seven, 2.81%), representing a total percentage of 1.34% flies infected. Interestingly, only males were collected around the eyes, compared with a male/female ratio of 1:4 around the fruit. This survey indicated that P. variegata males act as intermediate hosts of T. callipaeda under natural conditions in Europe. Both the zoophilic behaviour of P. variegata males on lachrymal secretions and their role as vector of T. callipaeda have been discussed as they represent a peculiarity in medical and veterinary entomology. The synchrony between the fly population dynamics and the biology of the nematode in the definitive host provides an interesting model for exploring the co-evolution of Thelazia spp. with their hosts. [Copyright &y& Elsevier]

Thelazia callipaedaRailliet and Henry (Spirurida: Thelaziidae), commonly called oriental eyeworm for its widespread presence in the Far East, has been recently found to affect dogs, cats and foxes in northern and southern Italy. Although the biology ofT. callipaedain the definitive hosts has been recently investigated, many doubts still remain about its biology in insect vectors. It has been suggested that more than one species of Diptera, namelyMusca domesticaLinnaeus (Diptera: Muscidae) andAmiota okadaiMaca (Diptera: Drosophilidae), may be involved in the transmission ofT. callipaedain China. The aim of the work described here was to verify the role ofM. domesticaas a vector ofT. callipaedaboth in experimental and natural conditions. A total of 310m. domestica(Group 1) were put in a cage and allowed to feed for 14 days around the eyes of a dog naturally infected byT. callipaeda. Ten flies were collected daily for 14 days. A total of 149 houseflies (Group 2) were fed withT. callipaedafirst stage larvae (L1) and dissected at 1, 2 and 7 days post-infection. From June to August 2003, flies were netted (Group 3) in two different sites every 10 days both from the environment and directly from the periocular region of dogs affected by thelaziosis.Muscaflies were examined for eyeworms by dissection and visual inspection of house flies (Groups 1 and 2) and using a molecular approach (Groups 1–3)viaa specific amplification of the ribosomal internal transcribed spacer 1 (ITS1) sequence ofT. callipaeda. On the whole, 180 pools ofM. domesticaflies were processed molecularly and all the experimentally infected flies (Groups 1 and 2) were found to be negative both at the visual dissection and at the molecular assay. Similarly, the 234M. domesticacollected from Group 3 were negative forT. callipaeda. The results clearly suggest thatM. domesticais unlikely to act as a vector ofT. callipaedain southern Europe, in contrast with a single previous report. [ABSTRACT FROM AUTHOR]

Abstract: Canine vector-borne diseases (CVBDs) are highly prevalent and increasing in distribution worldwide. A longitudinal study was conducted in southern Italy to determine the incidence of and protection against CVBD-causing pathogens in dogs treated with a combination of imidacloprid 10% and permethrin 50% (ImPer). One hundred eleven autochthonous young dogs were divided into group A (n =63) and group B (n =48), both groups containing dogs positive and negative for one or more CVBD-causing pathogens. Additionally, 10 naïve male beagles were introduced in each group in May 2008. Group A was treated with ImPer on day 0 and every 21±2 days whereas group B was left untreated. Blood and skin samples were collected at baseline (March–April 2008) and at the first, second and third follow-up times (July and October 2008 and April 2009). Bone marrow was sampled at baseline and at the third follow-up. Serological, cytological and molecular tests were performed to detect Anaplasma platys, Babesia spp., Bartonella spp., Dirofilaria immitis, Ehrlichia canis, Hepatozoon canis and Leishmania infantum. Ectoparasites (fleas, ticks, and sand flies) were monitored throughout the study. The baseline prevalence of CVBDs was 39.6% with 44 dogs positive for at least one pathogen. A. platys (27.5%) and Babesia spp. (15.6%) were the most prevalent species and co-infections with up to two pathogens were detected in 16 (14.7%) individuals. At the end of the evaluation period, there was a 90.7% reduction in overall CVBD incidence density rate (IDR) in group A, as following: 100% reduction in L. infantum; 94.6% in E. canis; 94.4% in Babesia spp.; and 81.8% in A. platys. Initially positive treated dogs showed significantly lower pathogen prevalence at the third follow-up than untreated ones. At the end of the evaluation period, 8 of the 10 untreated beagles were infected with at least one pathogen whereas one of the treated beagles was A. platys positive at a single time point (second follow-up). Overall efficacy against ticks was 97.9%. In October 2009, samples were collected from the remaining 83 dogs (44 from group A and 39 from group B) to investigate the annual incidence of CVBDs in the same, at this time untreated, dog population. A high year incidence for tick-borne diseases (78.1%) and for L. infantum (13.6%) was detected in dogs from group A, seven months after the treatment had been withdrawn. The results demonstrate that ImPer preventive treatment against arthropods protects autochthonous and naïve beagle dogs against CVBD-causing pathogens. [Copyright &y& Elsevier]