Viviana Domenica Tarallo

Prevention of endemic canine vector-borne diseases using imidacloprid 10% and permethrin 50% in young dogs: a longitudinal field study.

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.

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.

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.

Efficacy of a slow-release imidacloprid (10%)/ flumethrin (4.5%) collar for the prevention of canine leishmaniosis

BackgroundThe 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).MethodsA 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).ResultsThree 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%.ConclusionsThe 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.

Hepatozoon canis infection in ticks during spring and summer in Italy

Hepatozoon canis is a common protozoan of dogs, being among the most prevalent tick-borne pathogens infecting dogs around the world. It is primarily transmitted by Rhipicephalus sanguineus, the brown dog tick. In this study we tested ticks collected from dogs and from the environment in order to track the origin of an outbreak of H. canis infection detected in October 2009 in a private dog shelter in southern Italy. Ticks from dogs (n = 267) were collected during the spring of 2009, whereas ticks from environment (n = 300) were found on sticky traps placed in the same shelter during the summer of 2009. All ticks were tested by PCR for the detection of a H. canis 18S ribosomal RNA gene fragment. Four (1.5%, one female and three males) ticks collected from dogs were PCR positive. None of the larvae collected from the environment were positive, but a relatively high infection rate (8.0%) was detected in nymphs. These findings point out that dogs became infected during the summer, when ticks were abundant and highly infected by H. canis. Moreover, this study suggests that castor oil sticky traps might be useful to collect engorged immature ticks in highly infested environments (e.g., dog shelters). This might be particularly interesting to evaluate the level of infection by certain pathogens in free-ranging ticks R. sanguineus, as done in the present study.

Feline and canine leishmaniosis and other vector-borne diseases in the Aeolian Islands: Pathogen and vector circulation in a confined environment

Vector-borne diseases (VBDs) are prevalently investigated in dogs. Studies on feline VBDs are scant, though feline leishmaniosis (FeL) is increasingly recognised as a disease of cats in endemic areas. Comprehensive investigations on the distribution of VBDs in populations of cats and dogs living in relatively small geographical areas, such as islands, are currently lacking. In this study the prevalence of Leishmania infantum and other VBD pathogens was assessed in cohorts of cats and dogs living in the Aeolian Islands. Autochthonous animals (330 cats and 263 dogs) of different age and sex were sampled. Blood and conjunctival samples were collected from cats and dogs for serological and molecular testing. Eighty-five (25.8%) cats were positive for L. infantum, 13 (3.9%) for Bartonella spp. and 1 (0.3%) for Hepatozoon felis. One-hundred and ten dogs (41.8%) were positive for L. infantum and three (1.1%) for Hepatozoon canis. The incidence of L. infantum infection in cats positive after one season of exposure to sand fly was 14.7%. Leishmania infantum prevalence and year incidence were higher in dogs than in cats (p=0.0001 and p=0.0003, respectively). Thirty-four cats (10.3%) scored positive for ticks (mean intensity rate of infestation, 2.03±1.4), which were identified to the species level as Ixodes ventalloi and Rhipicephalus pusillus. Conversely, Rhipicephalus sanguineus sensu lato (s.l.) was the only species identified in dogs (10.6%). A larger prevalence of infestation by Ctenocephalides felis was recorded in cats (n=91; 27.6%) than in dogs (n=33; 12.5%) (p=0.0001). In addition, one female Nosopsyllus fasciatus (syn. Ceratophyllus fasciatus) and one male Spilopsyllus cuniculi were also identified in flea-infected cats. VBDs are endemic in the Aeolian Islands being L. infantum the most prevalent vector-borne pathogen circulating between cats and dogs. The overall seroprevalence of FeL herein recorded is higher than that assessed, only by IFAT, in populations of cats in Greece and in Spain. Because L. infantum and VBDs are more commonly associated with dogs, the recognition of cats as hosts of different vector-borne pathogens is of paramount importance towards a better management of these diseases in both animals and humans.

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.

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®; 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.

Apparent tick paralysis by Rhipicephalus sanguineus (Acari: Ixodidae) in dogs

Certain tick species including Ixodes holocyclus can inoculate neurotoxins that induce a rapid, ascending flaccid paralysis in animals. Rhipicephalus sanguineus, the most widespread tick of dogs, is recognized as a vector of several pathogens causing diseases in dogs and humans. A single report suggests its role as cause of paralysis in dogs. This study presents the clinical history of 14 young dogs heavily infested by R. sanguineus (intensity of infestation, 63-328) in an endemic area of southern Italy. During May to June of 2011, dogs were presented at the clinical examination with neurological signs of different degrees (e.g., hind limb ataxia, generalized lethargy, and difficulty in movements). All animals were treated with acaricides and by manual tick removal but ten of them died within a day, displaying neurological signs. The other 4 dogs recovered within 3 days with acaricidal and supportive treatment. Twelve dogs were positive by blood smear examination for Hepatozoon canis with a high parasitemia, two also for Babesia vogeli and two were negative for hemoparasites. Low-grade thrombocytopenia, hypoalbuminemia, and pancytopenia were the haematological alterations most frequently recorded. Other causes of neurological disease in dogs were excluded and the diagnosis of tick paralysis by R. sanguineus was confirmed (ex juvantibus) by early and complete recovery of 4 dogs following acaricidal treatment and tick removal.

Multilocus molecular and phylogenetic analysis of phlebotomine sand flies (Diptera: Psychodidae) from southern Italy

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 GenBank™ 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.