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Domenico Otranto - One of the best experts on this subject based on the ideXlab platform.
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major antigen and paramyosin proteins as candidate biomarkers for serodiagnosis of canine infection by zoonotic onchocerca lupi
PLOS Neglected Tropical Diseases, 2021Co-Authors: Maria Stefania Latrofa, Giada Annoscia, Domenico Otranto, Giuseppe Palmisano, Ciro Leonardo Pierri, Ramaswamy ChandrashekarAbstract:Onchocerca lupi (Spirurida: Onchocercidae) is a filarial worm parasitizing domestic carnivores and humans. Adult nematodes usually localize beneath in the sclera or in the ocular retrobulbar of infected animals, whilst microfilariae are found in the skin. Therefore, diagnosis of O. lupi is achieved by microscopic and/or molecular detection of microfilariae from skin biopsy and/or surgical removal of adults from ocular tissues of infected hosts. An urgent non-invasive diagnostic tool for the diagnosis of O. lupi in dog is mandatory. In this study, an immunoproteomic analyses was performed using a combination of immunoblotting and mass spectrometry techniques. Onchocerca lupi major antigen (Ol-MJA) and paramyosin (Ol-PARA) proteins were identified as potential biomarkers for serodiagnosis. Linear epitopes were herein scanned for both proteins using high-density peptide microarray. Sera collected from dog infected with O. lupi and healthy animal controls led to the identification of 11 immunodominant antigenic peptides (n = 7 for Ol-MJA; n = 4 for Ol-PARA). These peptides were validated using sera of dogs uniquely infected with the most important filarioids infesting dogs either zoonotic (Dirofilaria repens, Dirofilaria immitis) or not (Acanthocheilonema reconditum and Cercopithifilaria bainae). Overall, six antigenic peptides, three for Ol-MJA and for Ol-PARA, respectively, were selected as potential antigens for the serological detection of canine O. lupi infection. The molecular and proteomic dataset herein reported should provide a useful resource for studies on O. lupi toward supporting the development of new interventions (drugs, vaccines and diagnostics) against canine onchocercosis.
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overview on dirofilaria immitis in the americas with notes on other filarial worms infecting dogs
Veterinary Parasitology, 2020Co-Authors: Filipe Dantastorres, Domenico OtrantoAbstract:Canine dirofilariosis in the Americas is primarily caused by Dirofilaria immitis, a mosquito-borne filarial worm infecting dogs, which may also infect and cause disease in other mammalian hosts, including humans. Morphological and molecular evidence gathered in the past decades suggest that other Dirofilaria spp. may infect dogs in the American continent, though the actual taxonomic identity of them still needs to be elucidated. In addition, filarial worms belonging to distinct genera have also been reported in dogs in this part of the world, including Acanthocheilonema reconditum, Cercopithifilaria bainae, and Onchocerca lupi. Among these, O. lupi is a species of major clinical significance, which has been reported not only in dogs, but also in cats and humans in North America. The present article provides a current overview on canine dirofilariosis in the Americas, with notes on other filarial worms infecting dogs. It also provides some insights into outstanding research questions that should be responded to fill some of the several knowledge gaps related to this group of parasites in this continent.
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Molecular Approach for the Diagnosis of Blood and Skin Canine Filarioids
Microorganisms, 2020Co-Authors: Younes Laidoudi, Domenico Otranto, Bernard Davoust, Maria Stefania Latrofa, Samia Bedjaoui, Hacene Medkour, Abdeslam Mekroud, Idir Bitam, Oleg MediannikovAbstract:The zoonotic Onchocerca lupi and tick-transmitted filarioids of the genus Cercopithifilaria remain less well known due to the difficulties in accessing to skin samples as target tissues. Here, we proposed a molecular approach reliying on multiplex qPCR assays that allow the rapid identification of filarioids from canine blood, skin, and tick samples. This includes two newly developed duplex qPCR tests, the first one targeting filarial and C. grassii DNA (CanFil-C. grassii). and the second qPCR assay designed for the detection of Cercopithifilaria bainae and Cercopithifilaria sp. II DNAs (C. bainae-C.spII). The third one is a triplex TaqMan cox 1 assay targeting DNA of blood microfilariae (e.g., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum). The novel duplex qPCRs developed were validated in silico and by screening of known DNA collection. The qPCR assays were also used for screening the blood and tick samples of 72 dogs from Algeria. This allowed the identification of canine filariasis infection with 100% of specificity and 89.47% and 100% of sensitivity from naturally infected blood and tick samples, respectively. The prevalences of 26.39% for D. immitis and 5.56% for both D. repens and A. reconditum were reported in blood and tick samples. Cercopithifilaria DNAs were detected only in tick samples, with a prevalence of 4.17% and 5.56% for C. bainae and Cercopithifilaria sp. II, respectively. Co-infections were diagnosed in 6.94% and 13.89% of blood and tick samples, respectively. Whereas all samples were negative for C. grassii DNA. The use of engorged ticks instead of blood and skin samples could be an easier option for the surveillance of all canine filarioids herein investigated. The multiplex qPCR assays herein validated were shown to be useful in the detection of filarial co-infections by overcoming sequencing of positive samples.
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current surveys on the prevalence and distribution of dirofilaria spp and Acanthocheilonema reconditum infections in dogs in romania
Parasitology Research, 2015Co-Authors: Angela Monica Ionică, Mirabela Oana Dumitrache, Adriana Győrke, Gianluca Damico, Ioana Adriana Matei, Giada Annoscia, Nikola Pantchev, Katka Albrechtova, Viorica Mircean, Domenico OtrantoAbstract:During the last decades, Dirofilaria spp. infection in European dogs has rapidly spread from historically endemic areas towards eastern and northeastern countries, but little or no information is available from these geographical regions. The present study provides a picture of filarial infections in dogs from Romania and compares two tests for the diagnosis of Dirofilaria immitis. From July 2010 to March 2011, blood samples were collected from 390 dogs from nine counties of Romania and serological SNAP tests were performed for the detection of D. immitis antigen. The remaining blood clots were subsequently used for DNA extraction followed by multiplex PCR for assessing filarioid species diversity (i.e. D. immitis, Dirofilaria repens and Acanthocheilonema reconditum). Based on molecular detection, an overall prevalence of 6.92 % (n = 27; 95 % confidence interval (CI) 4.70–10.03 %) for D. repens, 6.15 % (n = 24; 95 % CI 4.07–9.14 %) for D. immitis and 2.05 % (n = 8; 95 % CI 0.96–4.16 %) for A. reconditum was recorded, with significant variations according to sampling areas. Coinfections of D. immitis and D. repens were recorded in 23.91 % (n = 11) positive dogs. A slightly higher prevalence for D. immitis was detected at the SNAP test (n = 28, 7.17 %; 95 % CI 4.91–10.33 %), but this difference was not statistically significant (p = 0.66). However, only 53.57 % (n = 15) of antigen-positive dogs were confirmed by PCR, while other dogs (n = 9) PCR positive for D. immitis were negative at the serology. The present study shows that Dirofilaria species are endemic in the southern and southeastern areas of Romania, This article also provides, for the first time, an epidemiological picture of the distribution of A. reconditum in Romania.
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RESEARCH On a Cercopithifilaria sp. transmitted by Rhipicephalus sanguineus: a neglected, but widespread filarioid of dogs Open Access
2013Co-Authors: Domenico Otranto, Emanuele Brianti, Giada Annoscia, Salvatore Giannetto, Gabriella Gaglio, Maria Stefania Latrofa, Stefania Weigl, Ettore Napoli, Riccardo Paolo Lia, E PapadopoulosAbstract:Background: This study was aimed at investigating the distribution of a Cercopithifilaria sp. sensu Otranto et al., 2011 with dermal microfilariae recently identified in a dog from Sicily (Italy). A large epidemiological survey was conducted by examining skin samples (n = 917) and ticks (n = 890) collected from dogs at different time points in Italy, central Spain and eastern Greece. Results: The overall prevalence of Cercopithifilaria sp. in the sampled animal populations was 13.9 % and 10.5 % by microscopy of skin sediments and by PCR on skin samples, respectively. Up to 21.6 % and 45.5 % of dogs in Spain were positive by microscopical examination and by PCR. Cumulative incidence rates ranging from 7.7 % to 13.9% were estimated in dogs from two sites in Italy. A low level of agreement between the two diagnostic tests (microscopical examination and PCR) was recorded in sites where samples were processed in parallel. Infestation rate as determined by tick dissection (from 5.2 % to 16.7%) was higher than that detected by PCR (from 0 % to 3.9%); tick infestation was significantly associated with Cercopithifilaria sp. infestation in dogs from two out of four sites. Developing larvae found in ticks were morphometrically studied and as many as 1469 larvae were found in a single tick. Conclusions: Our data suggest that, in addition to the most common species of filarioids known to infest dogs (i. e., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum), Cercopithifilaria sp. with dermal microfilariae should be considered due to its widespread distribution in southern Europe and high frequency in tick-exposed dogs
Giada Annoscia - One of the best experts on this subject based on the ideXlab platform.
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major antigen and paramyosin proteins as candidate biomarkers for serodiagnosis of canine infection by zoonotic onchocerca lupi
PLOS Neglected Tropical Diseases, 2021Co-Authors: Maria Stefania Latrofa, Giada Annoscia, Domenico Otranto, Giuseppe Palmisano, Ciro Leonardo Pierri, Ramaswamy ChandrashekarAbstract:Onchocerca lupi (Spirurida: Onchocercidae) is a filarial worm parasitizing domestic carnivores and humans. Adult nematodes usually localize beneath in the sclera or in the ocular retrobulbar of infected animals, whilst microfilariae are found in the skin. Therefore, diagnosis of O. lupi is achieved by microscopic and/or molecular detection of microfilariae from skin biopsy and/or surgical removal of adults from ocular tissues of infected hosts. An urgent non-invasive diagnostic tool for the diagnosis of O. lupi in dog is mandatory. In this study, an immunoproteomic analyses was performed using a combination of immunoblotting and mass spectrometry techniques. Onchocerca lupi major antigen (Ol-MJA) and paramyosin (Ol-PARA) proteins were identified as potential biomarkers for serodiagnosis. Linear epitopes were herein scanned for both proteins using high-density peptide microarray. Sera collected from dog infected with O. lupi and healthy animal controls led to the identification of 11 immunodominant antigenic peptides (n = 7 for Ol-MJA; n = 4 for Ol-PARA). These peptides were validated using sera of dogs uniquely infected with the most important filarioids infesting dogs either zoonotic (Dirofilaria repens, Dirofilaria immitis) or not (Acanthocheilonema reconditum and Cercopithifilaria bainae). Overall, six antigenic peptides, three for Ol-MJA and for Ol-PARA, respectively, were selected as potential antigens for the serological detection of canine O. lupi infection. The molecular and proteomic dataset herein reported should provide a useful resource for studies on O. lupi toward supporting the development of new interventions (drugs, vaccines and diagnostics) against canine onchocercosis.
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current surveys on the prevalence and distribution of dirofilaria spp and Acanthocheilonema reconditum infections in dogs in romania
Parasitology Research, 2015Co-Authors: Angela Monica Ionică, Mirabela Oana Dumitrache, Adriana Győrke, Gianluca Damico, Ioana Adriana Matei, Giada Annoscia, Nikola Pantchev, Katka Albrechtova, Viorica Mircean, Domenico OtrantoAbstract:During the last decades, Dirofilaria spp. infection in European dogs has rapidly spread from historically endemic areas towards eastern and northeastern countries, but little or no information is available from these geographical regions. The present study provides a picture of filarial infections in dogs from Romania and compares two tests for the diagnosis of Dirofilaria immitis. From July 2010 to March 2011, blood samples were collected from 390 dogs from nine counties of Romania and serological SNAP tests were performed for the detection of D. immitis antigen. The remaining blood clots were subsequently used for DNA extraction followed by multiplex PCR for assessing filarioid species diversity (i.e. D. immitis, Dirofilaria repens and Acanthocheilonema reconditum). Based on molecular detection, an overall prevalence of 6.92 % (n = 27; 95 % confidence interval (CI) 4.70–10.03 %) for D. repens, 6.15 % (n = 24; 95 % CI 4.07–9.14 %) for D. immitis and 2.05 % (n = 8; 95 % CI 0.96–4.16 %) for A. reconditum was recorded, with significant variations according to sampling areas. Coinfections of D. immitis and D. repens were recorded in 23.91 % (n = 11) positive dogs. A slightly higher prevalence for D. immitis was detected at the SNAP test (n = 28, 7.17 %; 95 % CI 4.91–10.33 %), but this difference was not statistically significant (p = 0.66). However, only 53.57 % (n = 15) of antigen-positive dogs were confirmed by PCR, while other dogs (n = 9) PCR positive for D. immitis were negative at the serology. The present study shows that Dirofilaria species are endemic in the southern and southeastern areas of Romania, This article also provides, for the first time, an epidemiological picture of the distribution of A. reconditum in Romania.
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Development of Acanthocheilonema reconditum (Spirurida, Onchocercidae) in the cat flea Ctenocephalides felis (Siphonaptera, Pulicidae).
Parasitology, 2014Co-Authors: Ettore Napoli, Emanuele Brianti, Giada Annoscia, Gabriella Gaglio, Filipe Dantas-torres, Luigi Falsone, Sabrina Foit, Francesca Abramo, Salvatore GiannettoAbstract:To investigate larval development of Acanthocheilonema reconditum in the cat flea Ctenocephalides felis, fleas were fed through an artificial feeding system with dog blood containing different concentrations of microfilariae (i.e. low, group L = 250; medium, group M = 500; high, group H = 1500 microfilariae per mL) or no microfilariae (group C). Fleas were sampled at 12 different time-points throughout the study period (D1-D28) and A. reconditum was detected by dissection, PCR and histology. Of 2105 fleas fed with infected dog blood, 891 (38·7%) died during the study before being sampled whilst the remaining (n = 1214) were examined for A. reconditum. Upon dissection, first-stage larvae (L1) were identified after 2 days post infection (D2), second-stage (L2) at D13 and infective third-stage larvae (L3) at D15. Eighteen (30%) of 60 pools of fleas molecularly examined tested positive. Histologically, L2 were detected at D13 in the sub-cuticle region embedded in the back muscle of one female flea. This study provides original data on larval development of A. reconditum in C. felis and reports on the usefulness of the artificial feeding system.
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RESEARCH On a Cercopithifilaria sp. transmitted by Rhipicephalus sanguineus: a neglected, but widespread filarioid of dogs Open Access
2013Co-Authors: Domenico Otranto, Emanuele Brianti, Giada Annoscia, Salvatore Giannetto, Gabriella Gaglio, Maria Stefania Latrofa, Stefania Weigl, Ettore Napoli, Riccardo Paolo Lia, E PapadopoulosAbstract:Background: This study was aimed at investigating the distribution of a Cercopithifilaria sp. sensu Otranto et al., 2011 with dermal microfilariae recently identified in a dog from Sicily (Italy). A large epidemiological survey was conducted by examining skin samples (n = 917) and ticks (n = 890) collected from dogs at different time points in Italy, central Spain and eastern Greece. Results: The overall prevalence of Cercopithifilaria sp. in the sampled animal populations was 13.9 % and 10.5 % by microscopy of skin sediments and by PCR on skin samples, respectively. Up to 21.6 % and 45.5 % of dogs in Spain were positive by microscopical examination and by PCR. Cumulative incidence rates ranging from 7.7 % to 13.9% were estimated in dogs from two sites in Italy. A low level of agreement between the two diagnostic tests (microscopical examination and PCR) was recorded in sites where samples were processed in parallel. Infestation rate as determined by tick dissection (from 5.2 % to 16.7%) was higher than that detected by PCR (from 0 % to 3.9%); tick infestation was significantly associated with Cercopithifilaria sp. infestation in dogs from two out of four sites. Developing larvae found in ticks were morphometrically studied and as many as 1469 larvae were found in a single tick. Conclusions: Our data suggest that, in addition to the most common species of filarioids known to infest dogs (i. e., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum), Cercopithifilaria sp. with dermal microfilariae should be considered due to its widespread distribution in southern Europe and high frequency in tick-exposed dogs
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A multiplex PCR for the simultaneous detection of species of filarioids infesting dogs.
Acta tropica, 2012Co-Authors: Maria Stefania Latrofa, Emanuele Brianti, Giada Annoscia, Filipe Dantas-torres, Stefania Weigl, Donato Traversa, Domenico OtrantoAbstract:The 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.
Oleg Mediannikov - One of the best experts on this subject based on the ideXlab platform.
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multiple vector borne pathogens of domestic animals in egypt
PLOS Neglected Tropical Diseases, 2021Co-Authors: Hend H A M Abdullah, Meriem Louni, Didier Raoult, Nadia Amanzougaghene, Handi Dahmana, Oleg MediannikovAbstract:Vector Borne Diseases (VBDs) are considered emerging and re-emerging diseases that represent a global burden. The aim of this study was to explore and characterize vector-borne pathogens in different domestic animal hosts in Egypt. A total of 557 blood samples were collected from different animals using a convenience sampling strategy (203 dogs, 149 camels, 88 cattle, 26 buffaloes, 58 sheep and 33 goats). All samples were tested for multiple pathogens using quantitative PCR and standard PCR coupled with sequencing. We identified Theileria annulata and Babesia bigemina in cattle (15.9 and 1.1%, respectively), T. ovis in sheep and buffaloes (8.6 and 7.7%, respectively) and Ba. canis in dogs (0.5%) as well as Anaplasma marginale in cattle, sheep and camels (20.4, 3.4 and 0.7%, respectively) and Coxiella burnetii in sheep and goats (1.7 and 3%; respectively). New genotypes of An. centrale, An. ovis, An. platys-like and Borrelia theileri were found in cattle (1.1,3.4, 3.4 and 3.4%, respectively), An. platys-like in buffaloes (7.7%), An. marginale, An. ovis, An. platys-like and Bo. theileri in sheep (3.4, 1.7, 1.7 and 3.4%, respectively), An. platys, An. platys-like and Setaria digitata in camels (0.7, 5.4 and 0.7%, respectively) and Rickettsia africae-like, An. platys, Dirofilaria repens and Acanthocheilonema reconditum in dogs (1.5, 3.4, 1 and 0.5%, respectively). Co-infections were found in cattle, sheep and dogs (5.7, 1.7, 0.5%, respectively). For the first time, we have demonstrated the presence of several vector-borne zoonoses in the blood of domestic animals in Egypt. Dogs and ruminants seem to play a significant role in the epidemiological cycle of VBDs.
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Detection of Canine Vector-Borne Filariasis and Their Wolbachia Endosymbionts in French Guiana
Microorganisms, 2020Co-Authors: Younes Laidoudi, Oleg Mediannikov, Djamel Tahir, Jean-lou Marie, Stephanie Watier-grillot, Bernard DavoustAbstract:In French Guiana, canine heartworm disease is well known, but the diversity of filarial parasites of dogs remains largely unknown. A total of 98 canine blood samples from Cayenne and Kourou were assessed by a blood wet mount preparation, heartworm antigen test and molecular exploration of filarioid and Wolbachia DNAs, followed by a multiplex species-specific qPCR's identification and a subsequent sequencing analysis. Thereafter, a phylogeny based on maximum likelihood was carried out to facilitate specific identification. Five dogs were microfilaremic. Heartworm antigens were detected in 15 (15.3%) dogs. Of these, six (6.1%) were considered as occult infections as neither microfilariae nor Dirofilaria immitis DNA were detected. The 11 (11.2%) D. immitis isolates corresponded to a low virulent strain. Six of the D. immitis isolates were positive for Wolbachia endosymbionts of D. immitis belonging to the clade C DNA. Acanthocheilonema reconditum DNA was detected in 3 (3.1%) samples. Of these latter, one was found co-infected with the Brugia sp. genotype and the DNA of the clade D of the Wolbachia endosymbiont of Brugia species. This latter was also detected in two filarioid DNA-free samples. Finally, two samples were positive for Cercopithifilaria bainae genotype, which is distinct from those identified in Europe. The present study highlights the urgent need to implement chemoprophylaxis associated with anti-Wolbachia drugs to control these potential zoonoses.
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Molecular Approach for the Diagnosis of Blood and Skin Canine Filarioids
Microorganisms, 2020Co-Authors: Younes Laidoudi, Domenico Otranto, Bernard Davoust, Maria Stefania Latrofa, Samia Bedjaoui, Hacene Medkour, Abdeslam Mekroud, Idir Bitam, Oleg MediannikovAbstract:The zoonotic Onchocerca lupi and tick-transmitted filarioids of the genus Cercopithifilaria remain less well known due to the difficulties in accessing to skin samples as target tissues. Here, we proposed a molecular approach reliying on multiplex qPCR assays that allow the rapid identification of filarioids from canine blood, skin, and tick samples. This includes two newly developed duplex qPCR tests, the first one targeting filarial and C. grassii DNA (CanFil-C. grassii). and the second qPCR assay designed for the detection of Cercopithifilaria bainae and Cercopithifilaria sp. II DNAs (C. bainae-C.spII). The third one is a triplex TaqMan cox 1 assay targeting DNA of blood microfilariae (e.g., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum). The novel duplex qPCRs developed were validated in silico and by screening of known DNA collection. The qPCR assays were also used for screening the blood and tick samples of 72 dogs from Algeria. This allowed the identification of canine filariasis infection with 100% of specificity and 89.47% and 100% of sensitivity from naturally infected blood and tick samples, respectively. The prevalences of 26.39% for D. immitis and 5.56% for both D. repens and A. reconditum were reported in blood and tick samples. Cercopithifilaria DNAs were detected only in tick samples, with a prevalence of 4.17% and 5.56% for C. bainae and Cercopithifilaria sp. II, respectively. Co-infections were diagnosed in 6.94% and 13.89% of blood and tick samples, respectively. Whereas all samples were negative for C. grassii DNA. The use of engorged ticks instead of blood and skin samples could be an easier option for the surveillance of all canine filarioids herein investigated. The multiplex qPCR assays herein validated were shown to be useful in the detection of filarial co-infections by overcoming sequencing of positive samples.
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Development of a multiplexed qPCRs-based approach for the diagnosis of Dirofilaria immitis, D. repens, Acanthocheilonema reconditum and the others filariosis
2019Co-Authors: Younes Laidoud, Marie Varloud, Florence Fenollar, Bernard Davoust, Amadou Niang El Hadj, Oleg MediannikovAbstract:Background: The frequent canine filariosis are caused by zoonotic filarial nematodes called Dirofilaria immitis, D. repens and Acanthocheilonema reconditum (Spirurida: Onchocercidae ). The absence of reliable diagnostic tools to diagnose and discriminate between these infections as well as their different forms constitutes a major obstacle to their control. The serological diagnosis of heartworm disease has recently shown both sensitivity and specificity problems. Findings: Herein, we developed and set up a novel molecular approach for an improved detection of the occult and non-occult filarioses, especially those caused by A. reconditum, D. immitis and D. repens as well as their differential diagnosis based on qPCRs assays. This approach designated the (Combined multiplex approach), proceeds as follows: Filaria and wolbachia identification using the newly customized 28S-based pan-filarial and 16S-based pan wolbachia qPCRs, respectively, followed by the fast typing method of positive samples using the triplex qPCR targeting A. reconditum, D. immitis and D. repens , and a duplex qPCR targeting wolbachiae of D. immitis and that of D. repens. The analytical sensitivity of the newly qPCR systems was confirmed by the detection limit of wolbachia and filaria DNA ranged from 5 10 -1 to 1.5 10 -4 mf/ml of blood with an R2 higher than 0.99, Cohen s Kappa agreement ranged from 0.98 to 1. The approach was complemented by a pan-filarial COI and pan- Wolbachia fts Z PCR for the identification of other filarial parasites and their wolbachiae, respectively. When tested on clinical samples, the results are as follows: 29.2 % (49/168) tested positive to at least filariae or wolbachiae DNA. 19 samples of them tested positive for filarial DNA, 9 for wolbachia DNA and 21 for both. Filarial species and Wolbachia genotype were also identified by the combined multiplex approach from all the positive samples. The single DNA of D. immitis was identified in 12 samples, D. repens in 7, and A. reconditum in 15 samples, the co-infection was observed in 5 samples, 4 for both Dirofilaria and one harbored the three species. Therefore, 22 samples were positive for wolbachia endosymbiont of D. immitis, 3 for that of D. repens and 5 for both genotypes. A newly duplex qPCR developed for the differential diagnosis of heartworm and French heartworm (Angiostrongylus vasorum ) was successfully validated in vitro. However, no DNA of this latter was detected in canine blood samples used in this study. The immunochromatographic test for dirofilariasis antigen during evaluation before and after thermal pretreatment of sera showed substantial agreement (K=0.6) and weak agreement (K=0.15), respectively. Conclusion: The proposed molecular tool targeting filarial genes and associated wolbachia genes is a reliable tool for the exploration and diagnosis of occult and nonoccult canine filariasis. We believe that the current diagnosis of heartworm based on antigen detection should be always confirmed by qPCR-based essays. The heat pretreatment of sera is useless and strongly discouraged.
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Prevalence of Anaplasmataceae and Filariidae species in unowned and military dogs in New Caledonia
Veterinary Medicine and Science, 2018Co-Authors: Mustapha Dahmani, Djamel Tahir, Florence Fenollar, Olivier Cabre, Oleg MediannikovAbstract:Dogs are competent reservoir hosts of several zoonotic agents, including Filariidae nematodes and Anaplasmataceae family bacteria. The latter family unites human and veterinary pathogens (Anaplasma, Ehrlichia and Neorickettsia bacteria) with Wolbachia, some of which are obligatory endosymbionts of pathogenic filarial nematodes. The epidemiology of Anaplasmataceae and Filariidae species infecting dogs living in kennels in New Caledonia was studied. 64 EDTA blood samples were screened for the presence of Anaplasmataceae and filarial nematodes. Molecular study was conducted using primers and probe targeting the of 23S rRNA long fragment of Anaplasmataceae species. Next, all blood sample was screened for the presence of Filariidae species targeting the primers and probe targeting the COI gene, as well as primers targeting the COI and 5S rRNA genes of all filarial worms. Anaplasma platys was identified in 8/64 (12.5, 95% confidence interval [CI]: 4.4-20.6%) and Wolbachia endosymbiont of Dirofilaria immitis in 8/64 (12.5%, CI: 4.4-20.6%). Filariidae species investigation was performed and showed that 11/64 (17.2%, CI: 7.9-26.4%) dogs were infected with D. immitis, whereas, 2/64 (3.1%, CI: 0.0-7.3%) were infected with Acanthocheilonema reconditum. Finally, we checked the occurrence of co-infection between Anaplasmataceae and Filariidae species. Co-occurrence with Wolbachia endosymbiont of D. immitis was observed in seven dogs, one dog was co-infected with A. platys and A. reconditum and another was co-infected with Wolbachia endosymbiont of D. immitis and A. reconditum. These results are the first report of Anaplasmataceae and Filariidae occurring in dogs in New Caledonia.
Maria Stefania Latrofa - One of the best experts on this subject based on the ideXlab platform.
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major antigen and paramyosin proteins as candidate biomarkers for serodiagnosis of canine infection by zoonotic onchocerca lupi
PLOS Neglected Tropical Diseases, 2021Co-Authors: Maria Stefania Latrofa, Giada Annoscia, Domenico Otranto, Giuseppe Palmisano, Ciro Leonardo Pierri, Ramaswamy ChandrashekarAbstract:Onchocerca lupi (Spirurida: Onchocercidae) is a filarial worm parasitizing domestic carnivores and humans. Adult nematodes usually localize beneath in the sclera or in the ocular retrobulbar of infected animals, whilst microfilariae are found in the skin. Therefore, diagnosis of O. lupi is achieved by microscopic and/or molecular detection of microfilariae from skin biopsy and/or surgical removal of adults from ocular tissues of infected hosts. An urgent non-invasive diagnostic tool for the diagnosis of O. lupi in dog is mandatory. In this study, an immunoproteomic analyses was performed using a combination of immunoblotting and mass spectrometry techniques. Onchocerca lupi major antigen (Ol-MJA) and paramyosin (Ol-PARA) proteins were identified as potential biomarkers for serodiagnosis. Linear epitopes were herein scanned for both proteins using high-density peptide microarray. Sera collected from dog infected with O. lupi and healthy animal controls led to the identification of 11 immunodominant antigenic peptides (n = 7 for Ol-MJA; n = 4 for Ol-PARA). These peptides were validated using sera of dogs uniquely infected with the most important filarioids infesting dogs either zoonotic (Dirofilaria repens, Dirofilaria immitis) or not (Acanthocheilonema reconditum and Cercopithifilaria bainae). Overall, six antigenic peptides, three for Ol-MJA and for Ol-PARA, respectively, were selected as potential antigens for the serological detection of canine O. lupi infection. The molecular and proteomic dataset herein reported should provide a useful resource for studies on O. lupi toward supporting the development of new interventions (drugs, vaccines and diagnostics) against canine onchocercosis.
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Molecular Approach for the Diagnosis of Blood and Skin Canine Filarioids
Microorganisms, 2020Co-Authors: Younes Laidoudi, Domenico Otranto, Bernard Davoust, Maria Stefania Latrofa, Samia Bedjaoui, Hacene Medkour, Abdeslam Mekroud, Idir Bitam, Oleg MediannikovAbstract:The zoonotic Onchocerca lupi and tick-transmitted filarioids of the genus Cercopithifilaria remain less well known due to the difficulties in accessing to skin samples as target tissues. Here, we proposed a molecular approach reliying on multiplex qPCR assays that allow the rapid identification of filarioids from canine blood, skin, and tick samples. This includes two newly developed duplex qPCR tests, the first one targeting filarial and C. grassii DNA (CanFil-C. grassii). and the second qPCR assay designed for the detection of Cercopithifilaria bainae and Cercopithifilaria sp. II DNAs (C. bainae-C.spII). The third one is a triplex TaqMan cox 1 assay targeting DNA of blood microfilariae (e.g., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum). The novel duplex qPCRs developed were validated in silico and by screening of known DNA collection. The qPCR assays were also used for screening the blood and tick samples of 72 dogs from Algeria. This allowed the identification of canine filariasis infection with 100% of specificity and 89.47% and 100% of sensitivity from naturally infected blood and tick samples, respectively. The prevalences of 26.39% for D. immitis and 5.56% for both D. repens and A. reconditum were reported in blood and tick samples. Cercopithifilaria DNAs were detected only in tick samples, with a prevalence of 4.17% and 5.56% for C. bainae and Cercopithifilaria sp. II, respectively. Co-infections were diagnosed in 6.94% and 13.89% of blood and tick samples, respectively. Whereas all samples were negative for C. grassii DNA. The use of engorged ticks instead of blood and skin samples could be an easier option for the surveillance of all canine filarioids herein investigated. The multiplex qPCR assays herein validated were shown to be useful in the detection of filarial co-infections by overcoming sequencing of positive samples.
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RESEARCH On a Cercopithifilaria sp. transmitted by Rhipicephalus sanguineus: a neglected, but widespread filarioid of dogs Open Access
2013Co-Authors: Domenico Otranto, Emanuele Brianti, Giada Annoscia, Salvatore Giannetto, Gabriella Gaglio, Maria Stefania Latrofa, Stefania Weigl, Ettore Napoli, Riccardo Paolo Lia, E PapadopoulosAbstract:Background: This study was aimed at investigating the distribution of a Cercopithifilaria sp. sensu Otranto et al., 2011 with dermal microfilariae recently identified in a dog from Sicily (Italy). A large epidemiological survey was conducted by examining skin samples (n = 917) and ticks (n = 890) collected from dogs at different time points in Italy, central Spain and eastern Greece. Results: The overall prevalence of Cercopithifilaria sp. in the sampled animal populations was 13.9 % and 10.5 % by microscopy of skin sediments and by PCR on skin samples, respectively. Up to 21.6 % and 45.5 % of dogs in Spain were positive by microscopical examination and by PCR. Cumulative incidence rates ranging from 7.7 % to 13.9% were estimated in dogs from two sites in Italy. A low level of agreement between the two diagnostic tests (microscopical examination and PCR) was recorded in sites where samples were processed in parallel. Infestation rate as determined by tick dissection (from 5.2 % to 16.7%) was higher than that detected by PCR (from 0 % to 3.9%); tick infestation was significantly associated with Cercopithifilaria sp. infestation in dogs from two out of four sites. Developing larvae found in ticks were morphometrically studied and as many as 1469 larvae were found in a single tick. Conclusions: Our data suggest that, in addition to the most common species of filarioids known to infest dogs (i. e., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum), Cercopithifilaria sp. with dermal microfilariae should be considered due to its widespread distribution in southern Europe and high frequency in tick-exposed dogs
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A multiplex PCR for the simultaneous detection of species of filarioids infesting dogs.
Acta tropica, 2012Co-Authors: Maria Stefania Latrofa, Emanuele Brianti, Giada Annoscia, Filipe Dantas-torres, Stefania Weigl, Donato Traversa, Domenico OtrantoAbstract:The 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.
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on a cercopithifilaria sp transmitted by rhipicephalus sanguineus a neglected but widespread filarioid of dogs
Parasites & Vectors, 2012Co-Authors: Domenico Otranto, Emanuele Brianti, Giada Annoscia, Salvatore Giannetto, Gabriella Gaglio, Maria Stefania Latrofa, Stefania Weigl, Ettore Napoli, Riccardo Paolo Lia, E PapadopoulosAbstract:This study was aimed at investigating the distribution of a Cercopithifilaria sp. sensu Otranto et al., 2011 with dermal microfilariae recently identified in a dog from Sicily (Italy). A large epidemiological survey was conducted by examining skin samples (n = 917) and ticks (n = 890) collected from dogs at different time points in Italy, central Spain and eastern Greece. The overall prevalence of Cercopithifilaria sp. in the sampled animal populations was 13.9% and 10.5% by microscopy of skin sediments and by PCR on skin samples, respectively. Up to 21.6% and 45.5% of dogs in Spain were positive by microscopical examination and by PCR. Cumulative incidence rates ranging from 7.7% to 13.9% were estimated in dogs from two sites in Italy. A low level of agreement between the two diagnostic tests (microscopical examination and PCR) was recorded in sites where samples were processed in parallel. Infestation rate as determined by tick dissection (from 5.2% to 16.7%) was higher than that detected by PCR (from 0% to 3.9%); tick infestation was significantly associated with Cercopithifilaria sp. infestation in dogs from two out of four sites. Developing larvae found in ticks were morphometrically studied and as many as 1469 larvae were found in a single tick. Our data suggest that, in addition to the most common species of filarioids known to infest dogs (i.e., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum), Cercopithifilaria sp. with dermal microfilariae should be considered due to its widespread distribution in southern Europe and high frequency in tick-exposed dogs.
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Development of Acanthocheilonema reconditum (Spirurida, Onchocercidae) in the cat flea Ctenocephalides felis (Siphonaptera, Pulicidae).
Parasitology, 2014Co-Authors: Ettore Napoli, Emanuele Brianti, Giada Annoscia, Gabriella Gaglio, Filipe Dantas-torres, Luigi Falsone, Sabrina Foit, Francesca Abramo, Salvatore GiannettoAbstract:To investigate larval development of Acanthocheilonema reconditum in the cat flea Ctenocephalides felis, fleas were fed through an artificial feeding system with dog blood containing different concentrations of microfilariae (i.e. low, group L = 250; medium, group M = 500; high, group H = 1500 microfilariae per mL) or no microfilariae (group C). Fleas were sampled at 12 different time-points throughout the study period (D1-D28) and A. reconditum was detected by dissection, PCR and histology. Of 2105 fleas fed with infected dog blood, 891 (38·7%) died during the study before being sampled whilst the remaining (n = 1214) were examined for A. reconditum. Upon dissection, first-stage larvae (L1) were identified after 2 days post infection (D2), second-stage (L2) at D13 and infective third-stage larvae (L3) at D15. Eighteen (30%) of 60 pools of fleas molecularly examined tested positive. Histologically, L2 were detected at D13 in the sub-cuticle region embedded in the back muscle of one female flea. This study provides original data on larval development of A. reconditum in C. felis and reports on the usefulness of the artificial feeding system.
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RESEARCH On a Cercopithifilaria sp. transmitted by Rhipicephalus sanguineus: a neglected, but widespread filarioid of dogs Open Access
2013Co-Authors: Domenico Otranto, Emanuele Brianti, Giada Annoscia, Salvatore Giannetto, Gabriella Gaglio, Maria Stefania Latrofa, Stefania Weigl, Ettore Napoli, Riccardo Paolo Lia, E PapadopoulosAbstract:Background: This study was aimed at investigating the distribution of a Cercopithifilaria sp. sensu Otranto et al., 2011 with dermal microfilariae recently identified in a dog from Sicily (Italy). A large epidemiological survey was conducted by examining skin samples (n = 917) and ticks (n = 890) collected from dogs at different time points in Italy, central Spain and eastern Greece. Results: The overall prevalence of Cercopithifilaria sp. in the sampled animal populations was 13.9 % and 10.5 % by microscopy of skin sediments and by PCR on skin samples, respectively. Up to 21.6 % and 45.5 % of dogs in Spain were positive by microscopical examination and by PCR. Cumulative incidence rates ranging from 7.7 % to 13.9% were estimated in dogs from two sites in Italy. A low level of agreement between the two diagnostic tests (microscopical examination and PCR) was recorded in sites where samples were processed in parallel. Infestation rate as determined by tick dissection (from 5.2 % to 16.7%) was higher than that detected by PCR (from 0 % to 3.9%); tick infestation was significantly associated with Cercopithifilaria sp. infestation in dogs from two out of four sites. Developing larvae found in ticks were morphometrically studied and as many as 1469 larvae were found in a single tick. Conclusions: Our data suggest that, in addition to the most common species of filarioids known to infest dogs (i. e., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum), Cercopithifilaria sp. with dermal microfilariae should be considered due to its widespread distribution in southern Europe and high frequency in tick-exposed dogs
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A multiplex PCR for the simultaneous detection of species of filarioids infesting dogs.
Acta tropica, 2012Co-Authors: Maria Stefania Latrofa, Emanuele Brianti, Giada Annoscia, Filipe Dantas-torres, Stefania Weigl, Donato Traversa, Domenico OtrantoAbstract:The 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.
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on a cercopithifilaria sp transmitted by rhipicephalus sanguineus a neglected but widespread filarioid of dogs
Parasites & Vectors, 2012Co-Authors: Domenico Otranto, Emanuele Brianti, Giada Annoscia, Salvatore Giannetto, Gabriella Gaglio, Maria Stefania Latrofa, Stefania Weigl, Ettore Napoli, Riccardo Paolo Lia, E PapadopoulosAbstract:This study was aimed at investigating the distribution of a Cercopithifilaria sp. sensu Otranto et al., 2011 with dermal microfilariae recently identified in a dog from Sicily (Italy). A large epidemiological survey was conducted by examining skin samples (n = 917) and ticks (n = 890) collected from dogs at different time points in Italy, central Spain and eastern Greece. The overall prevalence of Cercopithifilaria sp. in the sampled animal populations was 13.9% and 10.5% by microscopy of skin sediments and by PCR on skin samples, respectively. Up to 21.6% and 45.5% of dogs in Spain were positive by microscopical examination and by PCR. Cumulative incidence rates ranging from 7.7% to 13.9% were estimated in dogs from two sites in Italy. A low level of agreement between the two diagnostic tests (microscopical examination and PCR) was recorded in sites where samples were processed in parallel. Infestation rate as determined by tick dissection (from 5.2% to 16.7%) was higher than that detected by PCR (from 0% to 3.9%); tick infestation was significantly associated with Cercopithifilaria sp. infestation in dogs from two out of four sites. Developing larvae found in ticks were morphometrically studied and as many as 1469 larvae were found in a single tick. Our data suggest that, in addition to the most common species of filarioids known to infest dogs (i.e., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum), Cercopithifilaria sp. with dermal microfilariae should be considered due to its widespread distribution in southern Europe and high frequency in tick-exposed dogs.
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On a Cercopithifilaria sp. transmitted by Rhipicephalus sanguineus: a neglected, but widespread filarioid of dogs
Parasites & Vectors, 2012Co-Authors: Domenico Otranto, Emanuele Brianti, Giada Annoscia, Salvatore Giannetto, Gabriella Gaglio, Maria Stefania Latrofa, Stefania Weigl, Ettore Napoli, Riccardo Paolo Lia, E PapadopoulosAbstract:Background This study was aimed at investigating the distribution of a Cercopithifilaria sp. sensu Otranto et al., 2011 with dermal microfilariae recently identified in a dog from Sicily (Italy). A large epidemiological survey was conducted by examining skin samples (n = 917) and ticks (n = 890) collected from dogs at different time points in Italy, central Spain and eastern Greece. Results The overall prevalence of Cercopithifilaria sp. in the sampled animal populations was 13.9% and 10.5% by microscopy of skin sediments and by PCR on skin samples, respectively. Up to 21.6% and 45.5% of dogs in Spain were positive by microscopical examination and by PCR. Cumulative incidence rates ranging from 7.7% to 13.9% were estimated in dogs from two sites in Italy. A low level of agreement between the two diagnostic tests (microscopical examination and PCR) was recorded in sites where samples were processed in parallel. Infestation rate as determined by tick dissection (from 5.2% to 16.7%) was higher than that detected by PCR (from 0% to 3.9%); tick infestation was significantly associated with Cercopithifilaria sp. infestation in dogs from two out of four sites. Developing larvae found in ticks were morphometrically studied and as many as 1469 larvae were found in a single tick. Conclusions Our data suggest that, in addition to the most common species of filarioids known to infest dogs (i.e., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum ), Cercopithifilaria sp. with dermal microfilariae should be considered due to its widespread distribution in southern Europe and high frequency in tick-exposed dogs.