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ABSTRACT: Here, we present an investigation on the spatial distribution of the bacterial symbiont Candidatus Midichloria mitochondrii within Ixodes ricinus, by whole mount fluorescence in situ hybridization (FISH). M. mitochondrii is a peculiar, recently discovered bacterium that resides in the mitochondria of female ticks. We applied a rapid and specific FISH protocol with oligonucleotide probes targeted on the 16S rRNA of M. mitochondrii, 12S rRNA of tick mitochondria, and a probe revealing active mitochondria. In this report that represents the first application of whole mount FISH on ticks, we observed strong, specific fluorescence signals in all the examined life stages, as the optimized protocol allowed us to overcome the autofluorescence interference of the cuticle. Cellular localization and quantification of the symbionts were also assessed with electron microscopy and specific real-time PCR, respectively.
Ticks and Tick-borne Diseases 12/2012;
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ABSTRACT: According to Bergey's Manual of Systematic Bacteriology, the Rickettsiales are '…bacteria with typical gram-negative cell walls and no flagella'. The recently sequenced genome of 'Candidatus Midichloria mitochondrii', a divergent lineage within the order Rickettsiales capable of invading mitochondria in ixodid ticks, revealed the presence of 26 putative flagellar genes. Open questions in relation to this observation are whether these genes are expressed and whether they possess the domains expected for the flagellar function. Here we show that: (a) the putative flagellar proteins of 'Ca. M. mitochondrii' actually possess the conserved domains and structural features required for their function in a model bacterium; (b) the seven flagellar genes of 'Ca. M. mitochondrii' that have been tested are expressed at the RNA level; and (c) the putative flagellar cap gene of this bacterium (FliD) is expressed at the protein level, and can be stained within the bacterium and at its surface. Beside the specific questions that we have addressed that relate to the first evidence, to our knowledge, for a flagellar apparatus in a member of the order Rickettsiales, we present here novel tools (recombinant protein and antibodies) that will facilitate the study of 'Ca. M. mitochondrii'.
Microbiology 03/2012; 158(Pt 7):1677-83. · 3.06 Impact Factor
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ABSTRACT: 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.
Veterinary Parasitology 11/2011; 185(2-4):181-5. · 2.58 Impact Factor
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Lorenzo Domenis,
Simone Peletto,
Luciano Sacchi,
Emanuela Clementi, Marco Genchi,
Lucia Felisari,
Carla Felisari,
Patrizia Mo,
Paola Modesto,
Fabio Zuccon,
Chiara Campanella,
Cristiana Maurella,
Cristina Guidetti,
Pier Luigi Acutis
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ABSTRACT: The aim of this study was to determine the prevalence of sarcosporidiosis in semi-intensively bred cattle in northwestern Italy. A diagnostic protocol was setup in which infected animals were identified by rapid histological examination of the esophagus, diaphragm, and heart and the detected Sarcocystis spp. were subsequently typed using conventional electron microscopy in combination with molecular techniques. Sarcosporidia cysts were detected in 78.1% of the animals and were seen most often in the esophagus. The cattle is intermediate host for Sarcocystis hominis (final host, humans and some primates), Sarcocystis cruzi (final host, domestic and wild canids), and Sarcocystis hirsuta (final host, wild and domestic cats).All these three species of Sarcocystis were identified, variously associated, with the following prevalence: S. cruzi (74.2%), S. hirsuta (1.8%), and S. hominis (42.7%). Furthermore, a new S. hominis-like (prevalence 18.5%), characterized by hook-like structures of villar protrusion and a different sequence of the 18S rRNA gene, was identified. The cattle sheds testing positive for zoonotic Sarcocystis were assessed for risk factors contributing to the maintenance of the parasite's life cycle. Significant associations emerged between consumption of raw meat by the farm owner, mountain pasturing, and absence of a sewerage system on the farm and cattle breed. Our study demonstrates that sarcosporidiosis may constitute a public health problem in Italy and indicates several issues to be addressed when planning surveillance and prevention actions. The applied diagnostic approach revealed that cattle can harbor a further type of Sarcocystis, of which life cycle and zoonotic potential should be investigated.
Parasitology Research 05/2011; 109(6):1677-87. · 2.15 Impact Factor
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ABSTRACT: Climatic changes, together with an increase in the movement of dogs across Europe, have caused an increase in the geographical range of Dirofilaria infections. The present paper is focuses on northeastern European countries, where survey data have shown an increase of Dirofilaria repens infections both in animals and humans. A growing degree day-based forecast model has been developed to predict the occurrence. The model is based on evidence that there is a threshold of 14 °C below which Dirofilaria development will not proceed in mosquitoes, there is a requirement of 130 growing degree-days (GDDs) for larvae to reach infectivity, and there is a maximum life expectancy of 30 days for a mosquito vector. The output of this model predicted that the summer temperatures (with peaks in August) are sufficient to facilitate extrinsic incubation of Dirofilaria even at latitudes of 56 °N and longitudes of 39 °E. Despite the fact that both Dirofilaria immitis and D. repens have the same temperature requirement for extrinsic incubation in mosquitoes, empirical data has shown that D. repens is the main cause of dirofilarial infections in both humans and animals. Clinical signs are absent in most canine infections with D. repens. Furthermore, diagnosis is problematic and in-clinic serological tests, such as those for D. immitis, do not exist. Therefore, most infections go undiagnosed, allowing the infection to spread undetected.
Veterinary Parasitology 01/2011; 176(4):295-9. · 2.58 Impact Factor
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ABSTRACT: A female Rottweiler dog was presented with a history of intermittent vomiting and diarrhoea, dysorexia, weakness, and weight loss. Haemocytometry and biochemistry values were within normal ranges except for electrolyte analyses, that demonstrated hyponatremia and hyperkalemia with a decreased sodium/potassium ratio. A diagnosis of hypoadrenocorticism was suspected. Basal and post-ACTH stimulation cortisolemia were within the normal values. Electrocardiography was normal, and thoracic radiography showed no significant modifications. On abdominal ultrasonography, adrenal glands appeared normal, while the bowel was distended, and several thin linear hyperechoic objects floating in the lumen were observed. Two adult female whipworms (Trichuris vulpis) were collected following bowel irrigation. Anthelmintic treatment against the parasite was curative.
Journal of Parasitology Research 01/2011; 2011:682039.
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Claudia Damiani,
Irene Ricci,
Elena Crotti,
Paolo Rossi,
Aurora Rizzi,
Patrizia Scuppa,
Aida Capone,
Ulisse Ulissi,
Sara Epis, Marco Genchi, [......],
Angray Kang,
Bessem Chouaia,
Cheryl Whitehorn,
Guelbeogo W Moussa,
Mauro Mandrioli,
Fulvio Esposito,
Luciano Sacchi,
Claudio Bandi,
Daniele Daffonchio,
Guido Favia
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ABSTRACT: The symbiotic relationship between Asaia, an α-proteobacterium belonging to the family Acetobacteriaceae, and mosquitoes has been studied mainly in the Asian malaria vector Anopheles stephensi. Thus, we have investigated the nature of the association between Asaia and the major Afro-tropical malaria vector Anopheles gambiae. We have isolated Asaia from different wild and laboratory reared colonies of A. gambiae, and it was detected by PCR in all the developmental stages of the mosquito and in all the specimens analyzed. Additionally, we have shown that it localizes in the midgut, salivary glands and reproductive organs. Using recombinant strains of Asaia expressing fluorescent proteins, we have demonstrated the ability of the bacterium to colonize A. gambiae mosquitoes with a pattern similar to that described for A. stephensi. Finally, fluorescent in situ hybridization on the reproductive tract of females of A. gambiae showed a concentration of Asaia at the very periphery of the eggs, suggesting that transmission of Asaia from mother to offspring is likely mediated by a mechanism of egg-smearing. We suggest that Asaia has potential for use in the paratransgenic control of malaria transmitted by A. gambiae.
Microbial Ecology 10/2010; 60(3):644-54. · 2.91 Impact Factor
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ABSTRACT: Wolbachia is the most widespread bacterial endosymbiont in insects. It is responsible for a variety of reproductive alterations of the hosts. Wolbachia is transmitted through the germline from mother to offspring and, in rare cases, between individuals. This implies that acquired properties (through symbiosis with Wolbachia) can become heritable. We investigated the transovarial inheritance of Wolbachia in two phylogenetically distant insects, Drosophila melanogaster and Zyginidia pullula. We detected in both systems bacteriocyte-like cells, densely packed with Wolbachia endosymbionts, at the tip of the ovarioles. Bacteriocytes are cells specialized to harbour bacteria, typical of mutualistic insect symbiosis. Our observations of bacteriocyte-like cells harbouring Wolbachia in the ovary emphasize the plasticity of the female reproductive system of insects, which maintains its function while some cells are densely colonized by bacteria. In summary, there is evidence from different insects that bacteria which behave as parasites of reproduction are harboured by cells resembling bacteriocytes, which appear to mediate transmission of the bacteria to the progeny. It seems a valid hypothesis that the bacteriocyte-like cells that we observed are not the result of a co-evolution of host and symbiont, considering that Wolbachia is not an obligatory symbiont in Drosophila and Zyginidia.
Tissue and Cell 10/2010; 42(5):328-33. · 1.04 Impact Factor
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Veterinary Clinical Pathology 06/2010; 39(2):255-6. · 1.56 Impact Factor
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ABSTRACT: A study was carried out to assess the efficacy of a moxidectin microsphere sustained release (SR) injectable formulation (Guardian SR Iniettabile, Fort Dodge) for the prevention of Dirofilaria repens infection in experimentally infected dogs. On day 0, 18 Beagle dogs, 9 male dogs and 9 female dogs, weighing 12-16 kg were ranked in ascending order of body weight (b.w.) and blocked into pairs. Within each pair, dogs were allocated to Group 1 or Group 2 at random. On the same day, dogs in Group 1 were injected with 0.05 ml/kg b.w. of saline solution and dogs in Group 2 were injected with moxidectin SR at the label dose 0.17 mg/kg b.w. (0.05 ml/kg b.w.). Six months after moxidectin SR or saline injection, on day 180, each dog in the two groups was challenged with 50 infective larvae of D. repens collected from laboratory-reared, experimentally infected Aedes aegypti. Dogs were humanely euthanized on day 380 of the study, approximately 7 months from D. repens challenge. At necropsy, no worms were found in dogs treated with moxidectin SR (Group 2) while adult worms were found in saline-treated dogs (total 90; 38 males and 52 females; arithmetic mean 10, standard error 0.96, median 9, range 7-15) (Group 1 vs Group 2 P<0.001). In this experimental study, moxidectin SR injectable showed full efficacy (100%) lasting at least 6 months, and is able to prevent subcutaneous D. repens patent infection throughout the entire transmission season in Europe.
Veterinary Parasitology 02/2010; 170(1-2):167-9. · 2.58 Impact Factor
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Veterinary Clinical Pathology 01/2010; 39(2):255 - 256. · 1.56 Impact Factor
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Elena Crotti,
Claudia Damiani,
Massimo Pajoro,
Elena Gonella,
Aurora Rizzi,
Irene Ricci,
Ilaria Negri,
Patrizia Scuppa,
Paolo Rossi,
Patrizia Ballarini,
Noura Raddadi,
Massimo Marzorati,
Luciano Sacchi,
Emanuela Clementi, Marco Genchi,
Mauro Mandrioli,
Claudio Bandi,
Guido Favia,
Alberto Alma,
Daniele Daffonchio
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ABSTRACT: Bacterial symbionts of insects have been proposed for blocking transmission of vector-borne pathogens. However, in many vector models the ecology of symbionts and their capability of cross-colonizing different hosts, an important feature in the symbiotic control approach, is poorly known. Here we show that the acetic acid bacterium Asaia, previously found in the malaria mosquito vector Anopheles stephensi, is also present in, and capable of cross-colonizing other sugar-feeding insects of phylogenetically distant genera and orders. PCR, real-time PCR and in situ hybridization experiments showed Asaia in the body of the mosquito Aedes aegypti and the leafhopper Scaphoideus titanus, vectors of human viruses and a grapevine phytoplasma respectively. Cross-colonization patterns of the body of Ae. aegypti, An. stephensi and S. titanus have been documented with Asaia strains isolated from An. stephensi or Ae. aegypti, and labelled with plasmid- or chromosome-encoded fluorescent proteins (Gfp and DsRed respectively). Fluorescence and confocal microscopy showed that Asaia, administered with the sugar meal, efficiently colonized guts, male and female reproductive systems and the salivary glands. The ability in cross-colonizing insects of phylogenetically distant orders indicated that Asaia adopts body invasion mechanisms independent from host-specific biological characteristics. This versatility is an important property for the development of symbiont-based control of different vector-borne diseases.
Environmental Microbiology 10/2009; 11(12):3252-64. · 5.84 Impact Factor
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ABSTRACT: A study was carried out to assess the efficacy and the economic profit of prophylactic treatment against Isopsora suis with toltrazuril or with a sulfamethazine/trimethoprim combination in piglets from an intensive pig farm. Thirty-one litters were included in study. Eight litters were treated once with toltrazuril (20 mg/kg b.w.) at 3 days of age (Toltra group); 8 litters were treated with 2 ml/animal of a [corrected] sulphonamide combination (sodium sulfamethazine 250 [DOSAGE ERROR CORRECTED] mg and trimethoprim 50 [DOSAGE ERROR CORRECTED] mg/kg b.w.) for 3 consecutive days starting at 3 days of age (Sulfa group), and 15 litters were untreated (control group). Counts of oocyst per gram on pooled feces sampled from each litter were carried out on Days 7, 14, 21 and 28 and diarrhea was registered daily from pooled samples. Piglets were weighed on Days 1, 7 and 28 and mean weight gain (WG) and daily weight gain (DWG) were evaluated. The economic profit of treatment was evaluated comparing the WG of piglets of each treatment group from the day of birth to Day 28. On Days 14, 21 and 28, toltrazuril showed a better efficacy in controlling fecal oocyst output, diarrhea and weight gain compared with sulphamidic treatment (P<0.001). The budgeting analysis showed a return of economic benefit of euro 0.915 per toltrazuril-treated piglets and an additional cost of euro 1.155 per sulphonamide-treated piglets.
Veterinary Parasitology 06/2009; 163(4):362-5. · 2.58 Impact Factor
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ABSTRACT: Climatic changes, together with an increase in the movement of cats and dogs across Europe, have caused an increase in the geographical range of several vector borne parasites like Dirofilaria, and in the risk of infection for animals and humans. The present paper reviews the effects of climate and other global drivers on Dirofilaria immitis and Dirofilaria repens infections in Europe and the possible implications on the transmission and control of these mosquito-borne nematodes. In the last several years, growing degree day (GDD)-based forecast models, which use wide or local scale temperature data, have been developed to predict the occurrence and seasonality of Dirofilaria in different parts of the world. All these models are based on the fact that: there is a threshold of 14 degrees C below which Dirofilaria development will not proceed; and there is a requirement of 130 GDD for larvae to reach infectivity and a maximum life expectancy of 30 days for a vector mosquito. The output of these models predicts that the summer temperatures (with peaks in July) are sufficient to facilitate extrinsic incubation of Dirofilaria even at high latitudes. The global warming projected by the Intergovernmental Panel on Climate Change suggests that warm summers suitable for Dirofilaria transmission in Europe will be the rule in the future decades and if the actual trend of temperature increase continues, filarial infection should spread into previously infection-free areas. These factors not only favour incubation of Dirofilaria, but also impact on mosquito species. Recent findings have also demonstrated that Aedes albopictus is now considered to be an important, competent vector of Dirofilaria infections. This mosquito species could spread from southern to northern European countries in the near future, changing the epidemiological patterns of dirofilariosis both in humans and animals.
Veterinary Parasitology 04/2009; 163(4):286-92. · 2.58 Impact Factor
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ABSTRACT: A study was conducted to assess the duration and the outcome (self-cure or death) of feline heartworm infection and the life expectancy of infected cats. To be included in the study, cats had to be positive for heartworm antibody (Ab) and heartworm antigen (Ag) and had to demonstrate the presence of worms by echocardiography. Self-cure was defined as (1) negative for heartworm Ag and (2) no further visualization of worms by echocardiography. Of the 1962 eligible cats, 364 (18.5%) were positive for heartworm Ag and 131 were positive for heartworm Ag and for echocardiography diagnosis (prevalence 6.7%). None of the cats was microfilaremic. Of 43 asymptomatic cats included into the follow-up study with owners' consent, 34 (79%) self-cured and nine (21%) died. Eleven (26%) cats remained asymptomatic and self cured within 21-48 months, 23 (53%) showed symptoms but self-cured within 18-49 months, 6 (14%) died within 8-41 months of follow-up and 3 (7%) suddenly died after 38-40 months, which was related to heartworm infection. The probability for death or sudden death increased significantly with age at diagnosis, but no difference was detected by gender, survival time after diagnosis, or the presence or absence of symptoms. The presence/absence of symptoms showed significant interaction with the age at diagnosis (i.e., symptomatic cats showed increasing duration of heartworm infection along with age at diagnosis compared to that for asymptomatic cats. Heartworm-infected cats survived significantly longer than heartworm-negative cats affected by hypertrophic cardiomyopathy, chronic renal failure, or neoplastic diseases.
Veterinary Parasitology 10/2008; 158(3):177-82. · 2.58 Impact Factor
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ABSTRACT: A study was carried out to compare the efficacy of two injectable formulations of ivermectin, Ivomec,(1) Merial (IVM reference) and Ivogell,(2) Intervet (IVM generic) in the treatment of psoroptic mange (Psoroptes ovis) in Charollais feedlot cattle. A total of 22 animals were ranked in order of the severity of mange and allocated to 11 replicates of 2 animals each. Within each replicate, one animal was randomly allocated to IVM reference product treatment (Group 1) and one to IVM generic (Group 2). Animals were treated on Day 0 and on Day 8 at the recommended dosage of 200 microg ivermectin/kg bodyweight. The pharmacokinetics profiles (pK) of both IVM formulations were evaluated in plasma samples taken from 6 cattle randomly chosen per group on Day 0, before treatment, and then at 6, 12, 24 hours and daily from Day 2 to Day 7 after the treatment on Day 0. Additionally, the severity of mange lesions was assessed and mites were counted in skin scrapings on Days 0, 8, 15 and 25. Animals were weighed on Day 0 and 25 and body weight and average daily gains (ADG) were evaluated. No statistical differences were found between the cattle of the two groups in any pK parameters, although the mean IVM plasma concentrations in cattle treated with the IVM reference product were consistently higher than those found in cattle treated with the generic compound. By Day 25, all animals in Group 1 had recovered clinically and parasitologically from psoroptic mange while cattle from Group 2 still had mange lesions and, in two animals, living mites were found in the skin scrapings; these differences were significant (P<0.001). The mean body weight of the two groups was significantly different on Day 25 (P<0.01) when animals in Group 1 weighed 20 kg more than those in Group 2. In conclusion, despite similarities in their pharmacokinetic profiles and formulations, the clinical efficacy of the two injectable formulations of IVM differed significantly in their therapeutic efficacy against psoroptic mange in feedlot cattle up to 25 days after treatment: this difference in response was reflected in an incomplete clinical and parasitological response in Group 2 and a slower growth rate.
Veterinary Parasitology 09/2008; 158(1-2):110-6. · 2.58 Impact Factor
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ABSTRACT: Throughout the end of March to beginning of May 2006, 212 owned cats and 608 owned dogs from a heavy endemic area for canine heartworm (HW) disease in northern Italy have been examined to assess HW infection prevalence. Both cats and dogs were clinically examined and blood samples were taken from each animal to be examined for HW antibody (Ab). Ab-positive cats were further examined for circulating microfilariae, HW antigens (Ag) and by echocardiography (ECHO) to assess the presence of adult worms. Dogs were clinically examined and blood samples taken from each animal were examined for circulating microfilariae and for HW Ag. Ten cats (4.7%) were found Ab positive. Of these, 6 cats were Ag positive (2.6%) and in 4 (1.8%) the worms were visualized by ECHO. HW prevalence in dogs was 36% (221/608). One hundred and seventy-six (29%) were both microfilaraemic and Ag positive, 40 (7%) had occult infections (no circulating microfilariae) and 7 (1%) were microfilaraemic but Ag negative. Upon owners' consent, 132 cats (including cats Ab and/or Ag and ECHO positive) were prophylactically treated against HW disease with an imidacloprid/moxidectin spot-on combination (10% imidacloprid/1% moxidectin) monthly administered for 6 months. Cats were re-examined for HW infection in November, 1 month after the last drug administration, and in May-June 2007, 7-8 months after the last treatment. All 122 cats found HW negative before treatment, were found negative at the two examinations at the end of study. The 4 cats Ab positive, 2 cats Ab and Ag positive and 1 Ab, Ag and ECHO positive at the beginning of treatment were found negative. Throughout the treatment, transitory hypersalivation and generic signs of annoyance were reported by owners in 6 cats (4.5%). All signs regressed spontaneously.
Veterinary Parasitology 07/2008; 154(1-2):67-70. · 2.58 Impact Factor
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Guido Favia,
Irene Ricci,
Claudia Damiani,
Noura Raddadi,
Elena Crotti,
Massimo Marzorati,
Aurora Rizzi,
Roberta Urso,
Lorenzo Brusetti,
Sara Borin, [......], Marco Genchi,
Silvia Corona,
Ilaria Negri,
Giulio Grandi,
Alberto Alma,
Laura Kramer,
Fulvio Esposito,
Claudio Bandi,
Luciano Sacchi,
Daniele Daffonchio
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ABSTRACT: Here, we show that an alpha-proteobacterium of the genus Asaia is stably associated with larvae and adults of Anopheles stephensi, an important mosquito vector of Plasmodium vivax, a main malaria agent in Asia. Asaia bacteria dominate mosquito-associated microbiota, as shown by 16S rRNA gene abundance, quantitative PCR, transmission electron microscopy and in situ-hybridization of 16S rRNA genes. In adult mosquitoes, Asaia sp. is present in high population density in the female gut and in the male reproductive tract. Asaia sp. from An. stephensi has been cultured in cell-free media and then transformed with foreign DNA. A green fluorescent protein-tagged Asaia sp. strain effectively lodged in the female gut and salivary glands, sites that are crucial for Plasmodium sp. development and transmission. The larval gut and the male reproductive system were also colonized by the transformed Asaia sp. strain. As an efficient inducible colonizer of mosquitoes that transmit Plasmodium sp., Asaia sp. may be a candidate for malaria control.
Proceedings of the National Academy of Sciences 06/2007; 104(21):9047-51. · 9.68 Impact Factor
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ABSTRACT: The authors report the efficacy of an injectable, moxidectin sustained release (SR) formulation for the prevention of canine heartworm infection in endemic areas in northern and central Italy. Three field trials were carried out on a total of 324 dogs. Two hundred forty-three dogs were treated with moxidectin SR 6 months apart and 81 dogs (positive controls) with moxidectin tablets given monthly for 5 consecutive months during the risk season each year throughout the study. Results of testing for microfilariae and circulating adult female antigens were negative for all the experimentally treated dogs at the 6, 7, 11 and 19 months after the last injection. No adverse reactions to moxidectin SR were observed but a moderate pain at palpation and swelling (5-6 cm) at the injection site after the first treatment. In the study areas, prevalence of Dirofilaria immitis infection calculated by testing dogs which had no preventive treatment in the previous transmission season ranged from 33 to 63%. This study confirms the efficacy and safety of injectable, moxidectin SR formulation in the prevention of heartworm infection in dogs and demonstrates that the prophylactic efficacy lasts for the full season and strongly suggests that the product gives 1-year protection.
Veterinary Parasitology 01/2003; 110(1-2):85-91. · 2.58 Impact Factor
