1,720,998 research outputs found
A duplex real-time polymerase chain reaction assay for the detection of and differentiation between Dirofilaria immitis and Dirofilaria repens in dogs and mosquitoes.
No full textThis 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 SsoFastTM 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
A multiplex PCR for the simultaneoous detection of species of filarioids infesting dogs.
No full textThe present study reports the applicability of a multiplex PCR for the simultaneous detection and differentiation of common filarioids infecting dogs, i.e., Dirofilaria immitis, Dirofilaria repens, Acanthocheilonema reconditum and Cercopithifilaria sp. Amplicons of different sizes (i.e., 170 bp, 480 bp, 590 bp and 300 bp, respectively) of regions within the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were amplified on a single-step multiplex PCR using a mix of species-specific forward primers coupled with a single reverse primer. Experiments were carried out by amplifying genomic DNA extracted from blood or skin samples test-positive for microfilariae (mff). The number of mff present in each blood sample was quantified (from 800 to 25,000 mff/ml for A. reconditum and D. repens, respectively) and mixed blood samples were tested for the simultaneous detection of DNA from these mff. Specific amplicons for blood-circulating mff of A. reconditum, D. immitis and D. repens and for those whose adults are localized in skin (i.e., A. reconditum and Cercopithifilaria sp.) were simultaneously detected on agarose gel up to a dilution of 250 mff/ml for D. repens. The specific identity of the amplicons was confirmed by sequencing. The multiplex PCR assay reported herein represents a new tool for the molecular detection and differentiation of canine filarioids in blood and skin samples
Towards a rapid molecular identification of the common phlebotomine sand flies in the Mediterranean region
No full textThe present study reports two simple molecular approaches allowing a rapid identification of the most prevalent species of phlebotomine sand flies in the Mediterranean region. A PCR protocol for the amplification of ITS2 ribosomal region and a PCR-RFLP on a mitochondrial DNA fragment (cytb-nd1) were settled in order to identify and discriminate among Phlebotomus perniciosus, Phlebotomus neglectus, Phlebotomus perfiliewi, Phlebotomus papatasi and Sergentomyia minuta. The ITS2 regions showed a certain degree of interspecific variability, which led to PCR amplicons of different sizes, i.e., 450, 490, 460, 480 and 530 bp for P. perniciosus, P. neglectus, P. perfiliewi, P. papatasi, and S. minuta, respectively. Analogously, the digestion of a mitochondrial DNA amplicon with Ase I enzyme showed five different restriction profiles, which allowed the unequivocal differentiation of the sand fly species examined. These methods might represent useful tools for a molecular large scale screening of phlebotomine sand fly species caught in areas where leishmaniasis is endemic, in order to plan appropriate epidemiological surveillance programs for both Leishmania spp. and their vectors
Comparative analyses of mitochondrial and nuclear genetic markers for the molecular identification of Rhipicephalus spp.
No full textThe genus Rhipicephalus (Acari: Ixodidae) comprises a large number of vectors of pathogens of substantial medical and veterinary concern; however, species identification based solely on morphological features is often challenging. In the present study, genetic distance within selected Rhipicephalus species (i.e., Rhipicephalus bursa, Rhipicephalus guilhoni, Rhipicephalus muhsamae, Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus), were investigated based on molecular and phylogenetic analyses of fragments of the mitochondrial 16S, 12S and cytochrome c oxidase subunit 1 (cox1) genes, as well as of the whole sequences of the ribosomal internal transcribed spacer-2 (ITS-2) region. Mean values of inter-specific genetic distance (e.g., up to 12.6%, 11.1% and 15.2%), as well as of intra-specific genetic distance (e.g., 0.9%, 0.9% and 1%), calculated using the Kimura-2 parameter substitution model with uniform rates among sites for 16S, 12S and cox1 genes, respectively, confirmed the differentiation of the rhipicephaline species herein examined. The molecular identification was also supported by the distinct separation of species-specific clades inferred from the phylogenetic analyses of all mitochondrial sequences. Conversely, little interspecific divergence was detected amongst ribosomal ITS-2 sequences (i.e., up to 2.8%) for species belonging to the R. sanguineus complex, which resulted in the ambiguous placement of selected R. sanguineus s.l. and R. turanicus sequences in the corresponding phylogenetic tree. Results from this study confirm the suitability of mtDNA markers for the reliable identification of ticks within the Rhipicephalus genus and provide a framework for future studies of taxonomy, speciation history and evolution of this group of ticks
Rhipicephalus turanicus, a new vector of Hepatozoon canis
No full textThe distribution of Hepatozoon canis mainly encompasses areas where its main tick vector, Rhipicephalus sanguineus sensu lato, is present. However, the detection of this pathogen in dogs, foxes and golden jackals well outside the areas inhabited by this tick species reinforced the hypothesis that additional ixodids are involved in the life cycle and transmission of this protozoon. The present study provides, for the first time, data supporting the sporogonic development of H. canis in specimens of Rhipicephalus turanicus collected from a naturally infected fox from southern Italy. The epidemiological role of R. turanicus as a vector of H. canis is discussed, along with information on the potential use of cell cultures for the experimental infection with H. canis sporozoites. The in vitro infection of canine leucocytes by sporozoites from ticks is proposed as a potential tool for future in-depth studies on the biology of H. canis
Sarcocystis bertrami in skeletal muscles of donkeys (Equus africanus asinus) from Southern Italy
No full textAmong the protozoa of the genus Sarcocystis (Apicomplexa; Sarcocystidae), Sarcocystis bertrami (syn. Sarcocystis fayeri) is an obligate intracellular parasite of donkeys and horses with worldwide distribution. Here, we report the detection of S. bertrami in naturally infected donkeys from southern Italy and describe their structure by light microscopy (LM) and transmission electron microscopy (TEM). Protozoal cysts were detected both morphologically and molecularly in skeletal muscles of 28.57% (40/140) donkeys. Mature cysts of S. bertrami were found in skeletal muscle measuring 31–102 μm long and 19–83 μm wide with radially striated thick cyst wall. The high prevalence of infected donkeys suggests that dogs, the definitive hosts of S. bertrami, are contaminating environment with environmentally resistant sporocysts. Considering the increased consumption of raw donkey meat results also suggest a potential risk for human health
Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis.
No full textBACKGROUND:
Dirofilaria immitis and Dirofilaria repens are transmitted by bloodsucking culicid mosquitoes belonging to Culex, Aedes, Ochlerotatus, Anopheles and Mansonia genera. The detection of filariae in mosquitoes for assessing distribution of vectors and/or of pathogens in a given area (also known as "xenomonitoring"), when based on individual dissection of wild-caught female mosquitoes is time consuming and hardly applicable in large epidemiological surveys. Our study aimed to evaluate the recently developed duplex real-time PCR for screening large number of culicids and to assess their positivity for D. immitis and D. repens in an area where both species are endemic.
METHODS:
A duplex real-time PCR was used to detect and differentiate D. immitis and D. repens in mosquitoes collected in six provinces of the Veneto region using 43 carbon dioxide-baited traps under the frame of an entomological surveillance program to monitor the vectors of West Nile disease. From early May till October 2010, unfed female mosquitoes (n = 40,892) were captured in 20 selected sites.
RESULTS:
Mosquitoes were identified as Culex pipiens, Ochlerotatus caspius, Aedes vexans and Culex modestus and grouped into 995 pools according to species, day and site of collection (from minimum of 1 to maximum of 57). Out of 955 pools, 23 (2.41 %) scored positive for Dirofilaria spp. of which, 21 (2.2 %) for D. immitis and two (0.21 %) for D. repens. An overall Estimated Rate of Infection (ERI) of 0.06 % was recorded, being higher in Och. caspius and Ae. vexans (i.e., 0.18 % and 0.14 %, respectively). At least one mosquito pool was positive for Dirofilaria spp. in each province with the highest ERI recorded in Vicenza and Padova provinces (i.e., 0.42% and 0.16 %, respectively). Mosquitoes collected in all provinces were positive for D. immitis whereas, only two (i.e., Padova and Rovigo) provinces scored positive for D. repens. All mosquito species, except for Cx. modestus, were positive for D. immitis, whereas D. repens was only found in Cx. pipiens.
CONCLUSIONS:
The results suggest that both Dirofilaria species are endemic and may occur in sympatry in the examined area. The molecular approach herein used represents a powerful tool for surveillance programs of D. immitis and D. repens in the culicid vectors towards a better understanding of the epidemiology of the infections they cause and their seasonal transmission patterns
Cercopithifilaria sp. II in Vulpes vulpes: new host affiliation for an enigmatic canine filarioid
No full textCercopithifilaria bainae and Cercopithifilaria grassii (Spirurida, Onchocercidae) are filarioids inhabiting the skin of dogs worldwide. The microfilariae of a third species namely, Cercopithifilaria sp. II sensu Otranto et al. 2013, have been morphologically and molecularly characterized but scientific knowledge of this parasite is minimal. The first case of infection of a red fox (Vulpes vulpes) with the filarioid Cercopithifilaria sp. II is herein described in Castro Marim, Portugal. Microfilariae from skin sediment of the fox’s ear were morphological characterized, and the identification was confirmed molecularly in samples from skin sediment, skin samples, and from Rhipicephalus sanguineus group ticks collected from the animal (99% homology with Cercopithifilaria sp. II). Studies should evaluate if red foxes might play a role in the maintenance and distribution of Cercopithifilaria sp. II infection in dog populations
An assessment of genetic variability in the mitochondrial cytochrome c oxidase subunit 1 gene of Cercopithifilaria sp. (Spirurida, Onchocercidae) from dog and Rhipicephalus sanguineus populations
No full textThis study investigates sequence variation in mitochondrial cytochrome c oxidase subunit 1 gene within Cercopithifilaria sp. recorded recently in Italy. Fourteen sequence types (haplotypes) were characterized for 163 (7.7%) amplicons from 2111 Genomic DNA samples prepared from skin samples from dogs and from Rhipicephalus sanguineus (ticks) from different geographical areas of the Mediterranean basin (i.e., Italy, Spain and Greece). The most prevalent sequence types represented haplotypes I (70.5%) and X (16.0%), followed by haplotype VIII (4.9%) and other 11 haplotypes (8.6%). Three haplotypes (II, V and VI) were found exclusively in ticks. The overall intraspecific nucleotide variation among pcox1 haplotypes ranged from 0.4 to 3.5% (mean = 1.6%), whereas a mean interspecific difference of 9.5% was detected as compared with other onchocercids. Phylogenetic analysis of the nucleotide sequence data showed a clustering of Cercopithifilaria sp. with the other Cercopithifilaria species (with strong statistical support) to the exclusion of other onchocercids. The number of haplotypes identified here might be explained by complex ecology and transmission patterns as well as the high mutation rate of mitochondrial DNA and/or inbreeding associated with hosts and their vectors
The spread of zoonotic Thelazia callipaeda in the Balkan area
No full textBackground: Thelazia callipaeda (Spirurida, Thelaziidae), also known as “oriental eyeworm”, is a small nematode
parasite that lives in the conjunctival sac of domestic and wild carnivores, rabbits and even humans, causing mild
(e.g., conjunctivitis, epiphora, and ocular discharge) to severe (e.g., keratitis, and corneal ulcers) ocular disease. This
study reports, for the first time, the occurrence of T. callipaeda infection in the Balkan regions (i.e., Bosnia and
Herzegovina and Croatia), it provides genetic evidence on the origin of the infection in that area and discusses
potential expansion pathways in the near future.
Methods: This survey was conducted in two Western Balkan countries, Bosnia and Herzegovina and Croatia. At
necropsy, from January 2011 to April 2014, a total of 184 carcasses of red foxes were examined throughout the
study area and worms were collected from the conjunctival sac. In the same period, worms were also collected
during clinical examination from the conjunctival sac of four dogs and a cat from Bosnia and Herzegovina and
two dogs from Croatia. All nematodes collected were morphologically identified and molecularly characterized by
sequencing of partial cox1 gene.
Results: T. callipaeda was observed in 51 (27.71%) foxes and the highest prevalence (50.0%) was in the region of
East Bosnia. Beside the 4 cases of hyperemia (7.84%), most of the infected animals had no signs of ocular infection
(n = 47, 92.15%). A total of 417 adult nematodes collected (364 from foxes, 51 from dogs, 2 from cat) were
morphologically and molecularly identified as T. callipaeda haplotype 1.
Conclusion: This is the first report of autochthonous cases of T. callipaeda infection in red foxes, dogs and cat in
Bosnia and Herzegovina and Croatia and data presented here suggest that reports of thelaziosis in other Balkan
areas are, as yet, not diagnosed most likely due to the lack of awareness of practitioners. In addition, data regarding
the spread of the infection in Europe over the last ten years suggests that an increasing pattern in the distribution
of this disease in domestic and wild animals should be expected in the future
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