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A simple molecular method for discriminating common filarial nematodes of dogs (Canis familiaris)
Abstract
Accurate diagnosis of canine filariosis is essential for choosing correct therapeutic approach. Therefore, reliable methods for discriminating among the different filarial infections in dogs are needed. The authors report simple and highly specific molecular methods that identify the three most common filarial nematodes of European dogs: Dirofilaria immitis, D. repens and Acanthocheilonema (syn. Dipetalonema) reconditum, based on (1) PCR amplifications of mitochondrial DNA (12S rDNA and coxI) with general filarial primers followed by digestion with restriction enzymes that generates band polymorphisms clearly discriminating the three species and (2) PCR amplifications with species-specific primers to support the restriction analysis, in particular in the case of multiple infections.
... Each reaction was prepared in a final volume of 50 ll ( The following primers were used for the COI gene target: forward COIfilF (5 0 -TDTCTWTRDTDATTCGTT-3 0 ) and reverse D.imm-COIR (5 0 -GCACTGACAATACCAAT-3 0 ), and those for the 12S target: forward D.imm-12SF (5 0 -ATTTGTTGTAA T A T T A C G A -3 0 ) and reverse 12SR (5 0 -A T T G A C GGATGRTTTGTACC-3 0 ). These primers amplify 500 bp and 470 bp fragments, respectively, for mitochondrial genes COI and 12S rDNA when the reference sequence of complete mitochondrial genome of D. immitis is used (GenBank accession number AJ537512) (Casiraghi et al., 2001(Casiraghi et al., , 2004(Casiraghi et al., , 2006. PCR thermal cycles were conducted as follows: denaturation step for 5 min at 94 C and a set of 45 cycles at 94 C for 45 sec, 50 C for 45 sec, and 72 C for 60 sec, followed by a final 7 min extension step. ...
... All sequences showed 100% identity for 12S rDNA and COI with sequences from D. immitis specimens isolated from different hosts worldwide (Tables I, II). According to Casiraghi et al. (2006) filarial nematodes have very low intraspecific variation of mDNA. Likewise, Belanger and Perkins (2010) showed that nematodes that have Wolbachia as symbionts present less genetic variability compared to those species that are not hosts of this bacterium. ...
... Likewise, Belanger and Perkins (2010) showed that nematodes that have Wolbachia as symbionts present less genetic variability compared to those species that are not hosts of this bacterium. Perhaps the findings of Casiraghi et al. (2006) and Belanger and Perkins (2010) partly justify the similarity observed between D. immitis sequences from different geographical areas. Belanger et al. (2011) studied D. immitis isolates from different parts of the United States and Mexico, and the authors verified a population structuration explained in part by geographic barriers and corridors of contiguous habitats that will influence the gene flow between species, and they drew attention to a possible spread of drug resistance alleles. ...
Canine dirofilariasis is common in Brazil, but molecular diagnosis is rare even though molecular studies increase our knowledge about molecular epidemiology and circulating genotypes from helminths worldwide. This study aims to estimate the prevalence of infection with a modified Knott's test and to perform molecular characterization of Dirofilaria immitis (Leidy, 1856) Railliet and Henry, 1911, in dogs from endemic areas of Maricá and Niterói municipalities, Rio de Janeiro State, Brazil. Molecular characterization was performed in 33 blood samples from dogs positive for microfilariae and 4 adult worms obtained from 2 other dogs. DNA extraction followed by PCR for mitochondrial target 12S rDNA and cytochrome oxidase subunit 1 (COI) of D. immitis were performed, and the amplified products were sequenced. All sequences were identical for both gene targets and showed 100% identity with D. immitis sequences from different animal species from various countries. The study concluded that this genotype of D. immitis might be dispersed worldwide.
... The specific diagnosis of these infections in domestic dogs in the Amazon is important because it can be used as a basis to direct health public policies to the proper treatment and control of this infection in both the canine and human populations [15]. ...
... However, circulatory larval forms (microfilariae) may be confused when observed by light microscopy in routine laboratory diagnosis, even though morphological and biochemical differences have been described among them [32][33][34]. Although the advantages of the molecular diagnosis of canine filariasis have already been highlighted in other regions around the world [15,17,[35][36], morphological differentiation is still necessary for taxonomic identification and the detection of A. reconditum in the Amazon Region opens up new possibilities for research on morphological and molecular differentiation of this filarid species. ...
Dirofilaria immitis and Acanthocheilonema reconditum are common parasites in dogs but have also been reported parasitizing humans. The differential diagnosis and epidemiological evaluation of these zoonoses are important to the development of efficient public health policies and control strategies. The purpose of this study was to carry out an epidemiological survey by using molecular methods for the specific identification of filarid parasites of domestic dogs in the Marajó mesoregion, State of Pará (PA), Brazil. A total of 418 canine blood samples from Marajó mesoregion (Northern Brazil) were collected, submitted to DNA extraction, polymerase chain reaction (PCR) with “pan filarial” primer, subsequent sequencing and sequence analysis using BLASTn software comparison with previously deposited sequences in GenBank. After that, a phylogenetic analysis by Maximum Parsimony was performed to aid the specific diagnosis. The obtained sequences showed the occurrence of 9 (2.15%) dogs infected with D. immitis and 30 (7.18%) by A. reconditum, with a confidence interval of 95%, there were no cases of co-infection. We observed that male dogs were more likely to D. immits and A. reconditum infection. However, age was not significant to both infections. This study reports for the first time the occurrence of A. reconditum in the northern region of Brazil and confirmed the presence of D. immitis in the Marajó mesoregion.
... The analytical specificity and sensitivity of the cPCR for the specific amplification of cox1 gene fragment (*689bp; [32]) was assessed by testing genomic DNA of: i) skin samples with different parasitic load of O. lupi (Table 3), ii) serial dilution of O. lupi mfs DNA (i.e., from 3.6 ×10 1 pg/2μl to 3.6 ×10 −3 pg/2μl) and iii) DNA of adult specimens (i.e., from 8 ×10 1 ng/2μl to 8 x 10 −3 fg/2μl). ...
... Though few Onchocerca species DNA were herein tested, which may represent a limitation of the qPCR assay, this new tool provides an alternative to the labor intensive microscopic examination of skin snip samples and to cPCR for the diagnosis of O. lupi [38]. The qPCR assay was highly specific in revealing O. lupi DNA both in coinfected samples from dogs as well as in potential vector species, avoiding the sequencing confirmation needed using cPCR with filarioid generic primers [32]. Overall, the positive fluorescent signal from samples of O. lupi, from different geographical areas (i.e., Europe and USA), which displayed genetic intraspecific variability [18], indicates the usefulness of the qPCR also for the surveillance of O. lupi where the parasite has been reported [13,14,16,17,19,[39][40][41]. ...
The ocular onchocercosis is caused is by the zoonotic parasite Onchocerca lupi (Spirurida: Onchocercidae). A major hindrance to scientific progress is the absence of a reliable diagnostic test in affected individuals. Microscopic examination of skin snip sediments and the identification of adults embedded in ocular nodules are seldom performed and labour-intensive. A quantitative real-time PCR (qPCR) assay was herein standardized for the detection of O. lupi DNA and the results compared with microscopic examination and conventional PCR (cPCR). The specificity of qPCR and cPCR was assessed by processing the most common filarial nematodes infecting dogs, skin samples from O. lupi infected (n = 35 dogs) or uninfected animals (n = 21 dogs; n = 152 cats) and specimens of potential insect vector (n = 93 blackflies; n = 59 mosquitoes/midges). The analytical sensitivity of both assays was assessed using 10-fold serial dilutions of DNA from adult specimen and from a pool of microfilariae. The qPCR on skin samples revealed an analytical specificity of 100% and a sensitivity up to 8 x 10-1 fg/2μl O. lupi adult-DNA and up to 3.6 x 10-1 pg/2μl of mfs-DNA (corresponding to 1 x 10-2 mfs/2μl). Only 9.5% O. lupi-infected skin samples were positive for cPCR with a sensitivity of 8 x 10-1 pg/2μl of DNA. Out of 152 blackflies and mosquitoes/midges, eight specimens experimentally infected (n = 1 S. erythrocephalum; n = 1 S. ornatum; n = 6 Simulium sp.) were positive by qPCR. The qPCR assay herein standardized represents an important step forward in the diagnosis of zoonotic onchocercosis caused by O. lupi, especially for the detection and quantification of low number of mfs. This assay provides a fundamental contribution for the establishment of surveillance strategies aiming at assessing the presence of O. lupi in carnivores and in insect species acting as potential intermediate hosts. The O. lupi qPCR assay will enable disease progress monitoring as well as the diagnosis of apparently clinical healthy dogs and cats.
... These Mf can be diagnosed by microscopy through specific morphological identification or Mf histochemical staining [9,10]. Other diagnostic methods are also available, such as detection of circulating adult female antigens (currently only for D. immitis) and molecular approaches [1,11,12]. Modified Knott's and acid phosphatase histochemical staining tests of blood smears remain the most commonly used parasitological tests for Mf detection, but are labourintensive and require expertise. Thus, the prevalence of Dirofilaria spp. ...
... can be over-estimated if other filarial species are present and misidentified [13,14]. Molecular protocols have been developed for reliable detection and differentiation of filarial species, in particular, a speciesspecific PCR assay and multiplex PCR and restriction fragment length polymorphism (RFLP) assays for simultaneous detection of different Dirofilaria spp., either in the vector or in blood [12,[14][15][16][17][18][19][20][21]. ...
Background
Dirofilariosis is a potentially zoonotic parasitic disease, mainly transmitted by mosquito vectors in many parts of the world. Data concerning the canine Dirofilaria species currently circulating in Portugal is scarce. Thereby, a large-scale study was conducted to determine the Dirofilaria spp. present in Portugal, based on a molecular approach, and also to optimize a reliable and highly sensitive species-specific polymerase chain reaction (PCR) assay that could be used for the simultaneous detection and differentiation of Dirofilaria immitis, Dirofilaria repens, and other concurrent filarial species in animal reservoirs.
Methods
Blood samples were collected from three districts of Portugal (Coimbra, Santarém and Setúbal) between 2011 and 2013. Samples were tested using rapid immunomigration tests (Witness® Dirofilaria), modified Knott’s technique and acid phosphatase histochemical staining. In addition, molecular analysis was performed by amplification of the internal transcribed spacer (ITS) region using two different PCR protocols, specific for molecular screening of canine filarial species.
Results
Of the 878 dogs sampled, 8.8% (n = 77) were positive for D. immitis circulating antigen and 13.1% (n = 115) positive for microfilariae by the modified Knott’s technique. Of the 134 samples tested by acid phosphatase histochemical staining, 100 (74.6%) were positive for D. immitis. Overall, 13.7% (n = 120) were positive by PCR for D. immitis by ITS2, of which 9.3% (67/720) were also positive by ITS1. ITS2 PCR was the most sensitive and specific method, capable of detecting mixed D. immitis and A. reconditum infections. Heterozygosity, in the form of double peaks, was detected by sequencing of both ITS regions. No D. repens was detected by any of the diagnostic methods.
Conclusions
The present study confirmed D. immitis as the dominant species of the genus Dirofilaria infecting Portuguese dogs, based on sequencing of ITS1 and ITS2 PCR fragments. Additionally, ITS2 PCR was the most adequate method for diagnosis and prevalence estimation.
... A férgek mikrofiláriái alapján azonban nem könnyű a fajok elkülönítése, sőt a taxonómiában jártas szakértők alapvetően óva intenek a fajoknak csupán a mikrofiláriájuk alapján történő azonosításától (1). Sajnos sem a morfológiai, sem az antigénekben meglévő különbségek nem oly megbízhatóak, hogy csupán önmagukban, ezek segítségével, minden esetben feltétlenül azonosítani tudnánk a mikrofiláriákat (2,4). A konzervatív nukleinsavszakaszok felépítésében megnyilvánuló sajátosságok mutatkoztak eddig a legmegbízhatóbb elkülönítési lehetőségnek, de a PCR sikeres kivitelezéséhez is legalább olyan lárvasűrűség szükséges, amikor egy milliliternyi vérben több lárva van (2,8,13). ...
... Sajnos sem a morfológiai, sem az antigénekben meglévő különbségek nem oly megbízhatóak, hogy csupán önmagukban, ezek segítségével, minden esetben feltétlenül azonosítani tudnánk a mikrofiláriákat (2,4). A konzervatív nukleinsavszakaszok felépítésében megnyilvánuló sajátosságok mutatkoztak eddig a legmegbízhatóbb elkülönítési lehetőségnek, de a PCR sikeres kivitelezéséhez is legalább olyan lárvasűrűség szükséges, amikor egy milliliternyi vérben több lárva van (2,8,13). (Ennél kisebb lárvakoncentráció esetén a hemolízisen vagy más eljáráson alapuló lárvakoncentrálást kell igénybe venni az elegendő mennyiségű nukleinsav kinyeréséhez.) Ezért ideális és indokolt esetben több vizsgálati módszert kell együttesen alkalmazni a mikrofiláriák hovatartozásának biztos meghatározására (3,4). ...
In the first part of this article the authors described some ways to detect microfilariae of filarioid worms of dog. The second part of the article deals with the identification of microfilariae. By morphological examination of Giemsa-stained microfilariae we can clearly distinguish the Dirofilaria immitis and Dirofilaria repens species in most cases. Direct examination of the larvae is particularly useful when there is a mixed infection, in which case the serological and molecular tests do not give necessarily appreciable results. D. immitis larvae are much smaller than the microfilariae of the other species and their tapered head and the distribution of cells in their body makes them distinguishable from D. repens larvae. It is useful if one cane compare the freshly found microfilariae to previously defined reference specimens stored on micrscopic slide. In addition, the authors suggest a simple serial dilution method of haemolysed blood to estimate the amount of larvae that does not require any special devices.
... D. immitis diagnosis in dogs has been simplified in recent years following development of serological tests for detection of specific circulating antigens, and several brands of these tests are now commercially available (Klotins et al., 2000;McCall et al., 2008). Of late, molecular PCR and DNA sequencing techniques applied on blood samples have moreover proved valuable as highly sensitive and specific tools for detecting and identifying filarial infections in dogs (Casiraghi et al., 2006;Rishniw et al., 2006;Latrofa et al., 2012). ...
... The ITS2 region was PCR amplified in a total volume of 20 l using the primers DIDR-F1 and DIDR-R1 (Rishniw et al., 2006) under standard PCR conditions using 1 l DNA as template and an annealing temperature of 58 • C. Likewise, a part of the mitochondrial cox1 gene was PCR amplified by primers COIintF and COIintR (Casiraghi et al., 2006) using annealing temperature of 48 • C. PCR products were stained using GelRed TM (Biotium) and visualized under UV light in 1.5% agarose gels. PCR products were cleaned up and sequenced in both directions using the PCR primers by Macrogen Inc. (Seoul, South Korea). ...
Filariae are common parasites of dogs in many parts of the world, but little is known about the status of these infections in sub-Saharan Africa. A study was carried out to determine the occurrence and species of filariae among 272 dogs in Lusaka, Zambia. Giemsa stained blood smear and Knott's concentration methods revealed microfilariae in 16 (5.9%) of the dogs. PCR confirmed that most of these dogs had Acanthocheilonema reconditum infection. Ten (4.0%) of the examined dogs were positive for Dirofilaria immitis circulating antigen (by DiroCHEK(®) test), but D. immitis microfilariae were not identified in any of the dogs and the status of this infection remains unclear. Further studies are needed to explore the occurrence of filariae in Zambian dogs and the zoonotic potential for humans.
Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
... Species identification was also confirmed by 12S rDNAbased PCR in all cases (Casiraghi et al., 2001). DNA was extracted from worms and purified as described (Sréter-Lancz et al., 2007). ...
... DNA was extracted from worms and purified as described (Sréter-Lancz et al., 2007). The fragments of 12S rDNA were amplified (Casiraghi et al., 2001), and amplicons were further characterised by sequence analysis (Sréter-Lancz et al., 2007). Sequencing data were visually inspected for reading errors and combined using Chromas (Technelysium, Tewantin) and MultAlin (Corpet, 1988) programmes. ...
... 14 Mitochondrial DNA (mtDNA) sequencing has been used for identification of filariae and for investigating genetic variability and phylogenetic relationships; however, these studies were limited to mitochondrial DNA gene partial fragment analysis. 15,16 In this study, we determined the complete nucleotide sequences of ND1 and 16S rDNA D. immitis isolates from seven different dogs, and reconstructed phylogenetic trees using maximum parsimony and Bayes methods. This work provides foundation data for diagnosis, prevention, and control of heartworm disease. ...
... This is in accordance with other reports on the phylogeny of the Filarioidea superfamily, based on molecular approaches. 15,16,[22][23][24] The ND1 and 16S rDNA gene complete sequences are therefore suitable markers for phylogenetic analysis of these organisms. The phylogenetic tree constructed with the 16S sequences showed more detail than did the tree based on ND1, in particular regarding the topology of the two samples DiP1 and DiC4. ...
Dirofilaria immitis (heartworm) is the causative agent of an important zoonotic disease that is spread by mosquitoes. In this study, molecular and phylogenetic characterization of D. immitis were performed based on complete ND1 and 16S rDNA gene sequences, which provided the foundation for more advanced molecular diagnosis, prevention, and control of heartworm diseases. The mutation rate and evolutionary divergence in adult heartworm samples from seven dogs in western China were analyzed to obtain information on genetic diversity and variability. Phylogenetic relationships were inferred using both maximum parsimony (MP) and Bayes methods based on the complete gene sequences. The results suggest that D. immitis formed an independent monophyletic group in which the 16S rDNA gene has mutated more rapidly than has ND1.
... Morphological identification of circulating microfilariae requires special fixation protocols and expertise and is less accurate than genotyping (2, 3). The usefulness of PCR methods for the identification of D. immitis microfilaria in dog blood (the definitive host) has been reported in several recent publications (2–4), however there are no reports of genotyping from paraffin-embedded tissues. ...
... D. immitis in dogs can be diagnosed through careful morphological examination of circulating microfilariae, detection of circulating antigens, histochemical or immuno-histochemical staining of circulating microfilariae or, more recently, through molecular approaches. Morphological identification of circulating microfilariae requires special fixation protocols and expertise and is less accurate than genotyping (2, 3). The usefulness of PCR methods for the identification of D. immitis microfilaria in dog blood (the definitive host) has been reported in several recent publications (2–4), however there are no reports of genotyping from paraffin-embedded tissues. ...
The heartworm disease is an infectious disease of dogs with Dirofilaria immitis combined with cardiovascular and circulatory abnormalities. The heartworm disease can become a serious health risk when associated with a severe infection. In this study, a male, 8 year-old dog that died suddenly was necropsied and all tissues were examined grossly.
Major organs including heart, lungs, liver, spleen, kidneys, brain, eyes, and testis were fixed in 10% neutral formalin, embedded in paraffin, sectioned at 5-µm thickness, stained with hematoxylin and eosin, and examined with a light microscope. For each examined organ, paraffin-embedded tissues were cut and placed in eppendorf tubes for genomic DNA extraction. PCR was performed using two sets of primers for amplification of a 302 bp ITS-2 gene fragment and a 203 bp cytochrome oxidase subunit 1 (CO1) gene fragment of D. immitis.
During the necropsy examination, 46 adult D. immitis were found in the portal vein, right ventricle, and atrium of the heart and pulmonary trunk. Microscopically, microfilarias were found throughout the vessels of different organs including lungs, kidneys, liver, heart, brain, and spleen. All tissues examined by PCR were positive for D. immitis ITS-2 and CO1.
PCR technique now represents an effective method for identification of D. immitis from formalin-fixed samples.
... The amplicons HELMINTHOLOGIA, 46, 3: 159 -161, 2009 were then gel purified (QIAquick gel extraction kit, QIAGEN), and directly sequenced using ABI technology. Species-specific PCR reactions were carried out to detect both D. repens and D. immitis microfilariae, during which process 12S general reverse and species-specific forward primers were used as described by Casiraghi et al., 2006. Two days later after admitting the dog was humanely euthanized following the owner's request, due to the rapid worsening of clinical conditions and necropsy performed. ...
... In order to identify the species of microfilariae in the sample several molecular methods were used. However, a species-specific PCR-based exam (Casiraghi et al., 2006) carried out on the blood confirmed the presence of both D. repens and D. immitis microfilariae, these latter probably in a too low number to be evidenced by Knott test. It is likely that heartworm infection was not the primary cause of the dog's condition, considering that other pathological lesions were found (necrotic lesions in the liver, grey-whitish nodules in the kidney cortex and deep mucosal ulcers on the pyloric part of the duodenum). ...
A 4 year-old, male Hungarian Vizsla dog which had never been abroad was referred with poor general condition, decrease in A 4 year-old, male Hungarian Vizsla dog which had never been abroad was referred with poor general condition, decrease in
body weight, haematemesis and jaundice to the Central Clinic of Veterinary Science University, Budapest. After symptomatic body weight, haematemesis and jaundice to the Central Clinic of Veterinary Science University, Budapest. After symptomatic
treatment abdominal ultrasonography and diagnostic laparatomy were carried out. The dog was humanely euthanized two days later treatment abdominal ultrasonography and diagnostic laparatomy were carried out. The dog was humanely euthanized two days later
following owner’s consent because of sudden worsening of clinical conditions. Two adult heartworms (Dirofilaria immitis) were found in the right ventricle partially coiling around the tricuspid valve. PCR on blood was positive for both D. immitis and Dirofilaria repens while only D. repens microfilariae were found by modified Knott’s test and the serological test was negative for D. immitis antigens. This is the first, confirmed report of autochthonous canine heartworm infection in Hungary. following owner’s consent because of sudden worsening of clinical conditions. Two adult heartworms (Dirofilaria immitis) were found in the right ventricle partially coiling around the tricuspid valve. PCR on blood was positive for both D. immitis and Dirofilaria repens while only D. repens microfilariae were found by modified Knott’s test and the serological test was negative for D. immitis antigens. This is the first, confirmed report of autochthonous canine heartworm infection in Hungary.
... Filariae exhibit complex life cycles involving different hosts, as they develop through several larval stages. Considering that different specific vectors (mosquitoes, flies, ticks, fleas, and lice) are involved in their transmission to complete their biological cycle, the varying rates of infection, coinfection, and symptomatology, and the different treatments required to treat the infections caused by them, it is necessary to differentiate the various filariae species present in the dog for an accurate diagnosis of canine filariosis (2,5,43,44). In our study, firstly, dogs with circulating microfilariae in the blood were identified by the Woo test, which was used as a screening test (45). ...
Canine filariosis is caused by filiform nematodes and affects several species of animals as well as humans. The disease produces a wide range of symptoms that can often be confused with other diseases, which increases the complexity of its diagnosis. The search for methodologies to facilitate its diagnosis is a challenge, and specific and differential identification of the parasite species causing the disease holds key to a successful diagnosis. In Colombia, there is a problem of underdiagnosis of filariosis in microfilaremic dogs infected by Dirofilaria immitis and Acanthocheilonema reconditum, and of microfilaremias not related to heartworm disease. The highest prevalences have been reported for D. immitis infections, although new cases of A. reconditum infections are beginning to appear. The aim of this study was to differentiate the microfilariae infections caused by D. immitis and A. reconditum by a morphological and molecular characterization of microfilariae so as to facilitate an accurate diagnosis of canine filariosis in the metropolitan area of Bucaramanga (Colombia). For this purpose, 400 blood samples with anticoagulants were collected from the dogs and analyzed with the help of a commercial immunochromatography kit for the detection of D. immitis circulating antigen. The Woo, Knott, and polymerase chain reaction (PCR) techniques were employed for determining the parasite count, morphological observation, and molecular identification of microfilariae present in the dogs respectively. The prevalence of microfilaremic dogs in Bucaramanga metropolitan area was 18.75% (75/400). The prevalence of dogs that tested positive for D. immitis in the antigen and in PCR tests was 1.25% (5/400) and 1% (4/400), respectively. Furthermore, the PCR test revealed that 17.75% of the microfilaremic dogs tested positive for A. reconditum (71/400) (first report in the metropolitan area of Bucaramanga), with one animal co-infected by both species, and 0% for D. repens (0/400). However, by morphological characterization, 4% of the microfilariae (3/75) corresponded to D. immitis, 20% (15/75) to D. repens, and 76% (57/75) to A. reconditum. The use of molecular diagnostic methods such as PCR aids in the specific identification of the parasite, thus making it a more accurate method than the morphological characterization of microfilariae. The identification of the parasites by PCR helps improve the veterinary diagnosis of canine filariosis in Colombia, which would lead to the establishment of an appropriate treatment protocol for each species of filaria and also to the generation of reliable data to be used at the clinical and epidemiological levels.
... Twenty-five samples presenting microfilaria at buffy coat analysis were randomly chosen randomly and were submitted to a PCR assay using filarioid-generic primers, namely NTF (5′-TGATTGGTGGTTTTGGTAA-3′) and NTR (5′-ATAAGTACGAGTATCAATATC-3′), that amplifies a fragment (689 bp) of the mitochondrial cox1 gene. For further characterization, samples were submitted to PCR assays targeting the 12S rRNA (12SF 5′-CGGGAGTAAAGTTTTGTTTAACGG-3′ and 12SR 5′-CATTGACGGAT GGTTTGTACCAC-3′), 18S rRNA (NC18SF1 5′-AAAGATTAAGCCA TCCA-3′ and NC5BR 5′-GCAGGTTCACCTACAGAT-3′) (Casiraghi et al., 2004(Casiraghi et al., , 2006Ferri et al., 2009), hsp70 (h70ManF 5′-TGAGACAGCTGGAGGTGTTATG-3′ and h70ManR 5′-ATCTTTCT GTGCCTCATCATCTG-3′), and myoHC genes (MyManF 5′-GAAGCTGAG GCTCAAGCAAT-3′ and MyManR 5′-TCTGTTTTGCTCATCGCATT-3′) (Moraes et al., 2022). Ultra-pure sterile water (Life Technologies®, Carlsbad, CA, USA) was used as a negative control in all PCR assays. ...
Coatis (Nasua nasua) are wild carnivorous well adapted to anthropized environments especially important because they act as reservoirs hosts for many arthropod-borne zoonotic pathogens. Information about filarioids from coatis and associated Wolbachia spp. in Brazil is scant. To investigate the diversity of filarial nematodes, blood samples (n = 100 animals) were obtained from two urban areas in midwestern Brazil and analyzed using blood smears and buffy coats and cPCR assays based on the cox1, 12S rRNA, 18S rRNA, hsp70 and myoHC genes for nematodes and 16S rRNA for Wolbachia. When analyzing coati blood smears and buffy coats, 30% and 80% of the samples presented at least one microfilaria, respectively. Twenty-five cox1 sequences were obtained showing 89% nucleotide identity with Mansonella ozzardi. Phylogenetic analyses clustered cox1 sequences herein obtained within the Mansonella spp. clade. Sequences of both myoHC and two hsp70 genes showed 99.8% nucleotide identity with Mansonella sp. and clustered into a clade within Mansonella sp., previously detected in coatis from Brazil. Two blood samples were positive for Wolbachia, with a 99% nucleotide identity with Wolbachia previously found in Mansonella perstans, Mansonella ozzardi and Mansonella atelensis and in ectoparasites of the genus Pseudolynchia, Melophagus and Cimex. The study showed a high prevalence of Mansonella sp. in the coati population examined, suggesting that this animal species play a role as reservoirs of a novel, yet to be described, species within the Onchocercidae family.
... Among filaria, the genera Dipetalonema, Paulianfilaria, Courduriella, and Protofilaria are known to occur in wild lemurs (reviewed in Irwin & Raharison, 2009;Klein, 2019). However, except for the genus Dipetalonema, which includes species infecting dogs, rodents, neotropical primates, and also humans, these filarial genera seem to be unique to lemurs (de Argôlo et al., 2018;Casiraghi et al., 2006). To the authors' knowledge, no human case of zoonotic filarial infection has been reported in Madagascar to date. ...
The relevance of emerging infectious diseases continues to grow worldwide as human activities increasingly extend into formerly remote natural areas. This is particularly noticeable on the island of Madagascar. As closest relatives to humans on the island, lemurs are of particular relevance as a potential origin of zoonotic pathogen spillover. Knowledge of pathogens circulating in lemur populations is, however, very poor. Particularly little is known about lemur hemoparasites. To infer host range, ecological and geographic spread of the recently described hemoparasitic nematode Lemurfilaria lemuris in northwestern Madagascar, a total of 942 individuals of two mouse lemur species (Microcebus murinus [n = 207] and Microcebus ravelobensis [n = 433]) and two rodent species (the endemic Eliurus myoxinus [n = 118] and the invasive Rattus rattus [n = 184]) were captured in two fragmented forest landscapes (Ankarafantsika National Park and Mariarano Classified Forest) in northwestern Madagascar for blood sample examination. No protozoan hemoparasites were detected by microscopic blood smear screening. Microfilaria were present in 1.0% (2/207) of M. murinus and 2.1% (9/433) of M. ravelobensis blood samples but not in rodent samples. Internal transcribed spacer 1 (ITS‐1) sequences were identical to an unnamed Onchocercidae species previously described to infect a larger lemur species, Propithecus verreauxi, about 650 km further south. In contrast to expectations, L. lemuris was not detected. The finding of a pathogen in a distantly related host species, at a considerable geographic distance from the location of its original detection, instead of a microfilaria species previously described for one of the studied host species in the same region, illustrates our low level of knowledge of lemur hemoparasites, their host ranges, distribution, modes of transmission, and their zoonotic potential. Our findings shall stimulate new research that will be of relevance for both conservation medicine and human epidemiology. A microscopic analysis of 942 blood smears from Microcebus murinus, M. ravelobensis and two rodent species from northwestern Madagascar revealed the presence of an unnamed Onchocercidae species with low prevalence (1.0%−2.1%) in both mouse lemur species, but not in the rodents. This microfilaria species was previously described to infect Propithecus verreauxi about 650 km further south. This pathogen therefore shows a wider geographical distribution than its hosts and a rather low host specificity within lemurs. An undescribed Onchocercidae gen.sp. was detected in the blood of two sympatric mouse lemur species, Microcebus murinus and M. ravelobensis (Cheirogaleidae). This microfilaria species was previously found in Propithecus verreauxi (Indriidae), demonstrating its large geographic distribution and low host specificity within lemurs. An undescribed Onchocercidae gen.sp. was detected in the blood of two sympatric mouse lemur species, Microcebus murinus and M. ravelobensis (Cheirogaleidae). This microfilaria species was previously found in Propithecus verreauxi (Indriidae), demonstrating its large geographic distribution and low host specificity within lemurs.
... Dirofilaria immitis in dogs can be diagnosed through careful morphological examination of circulating microfilariae, detection of circulating antigens, histochemical or immuno-histochemical staining of circulating microfilariae or, more recently, through molecular approaches. Morphological identification of circulating microfilariae, however, is not always easy and is potentially misleading (Casiraghi et al., 2006;Rishniw et al., 2006). ...
We estimated the prevalence of Dirofilaria immitis infection in domestic dogs in five Turkish provinces-Sakarya, Kocaeli, Ankara, Elazig and Mersin-using a commercial ELISA kit for detecting circulating antigen and a PCR test for detecting circulating microfilarial DNA.
... However, most of these methods lack the ability to discriminate between the various filarioid worms, hence the need to combine two or more methods before confirmatory diagnosis. To overcome this, highly sensitive and specific molecular approaches have been developed that allow easy discrimination of the main canine filarial nematodes (MAR et al., 2002;CASIRAGHI et al., 2006;RISHNIW et al., 2006;THANCHOMNANG et al., 2010;WONGKAMCHAI et al., 2012;ROJAS et al., 2015). ...
... Although the gold standard method for the diagnosis of microfilariae in a blood smear is microscopic examination on glass slides stained with Giemsa or hematoxylin and eosin [29,30], this technique requires considerable expertise, has low sensitivity, and cannot clearly discriminate among closely related species of filarial nematodes, such as D. immitis, D. repens, and D. reconditum or B. malayi and B. pahangi [31]. Serologic and molecular techniques with high sensitivity and specificity for filariasis diagnosis have been developed as additional methods for the detection of microfilariae [6,[32][33][34]. In this study, microscopic examination gives a low positive result for microfilariae (28.07%) compared to PCR. ...
Canine filariasis is caused by several nematode species, such as Dirofilaria immitis, Dirofilaria repens, Brugia pahangi, Brugia malayi, and Acanthocheilonema reconditum. Zoonotic filariasis is one of the world’s neglected tropical diseases. Since 2000, the World Health Organization (WHO) has promoted a global filarial eradication program to eliminate filariasis by 2020. Apart from vector control strategies, the infection control of reservoir hosts is necessary for more effective filariasis control. In addition, many studies have reported that Wolbachia is necessary for the development, reproduction, and survival of the filarial nematode. Consequently, the use of antibiotics to kill Wolbachia in nematodes has now become an alternative strategy to control filariasis. Previously, a case of subconjunctival dirofilariasis caused by Dirofilaria spp. has been reported in a woman who resides in the center of Bangkok, Thailand. Therefore, our study aimed to principally demonstrate the presence of filarial nematodes and Wolbachia bacteria in blood collected from domestic dogs from the Bangkok Metropolitan Region, Thailand. A total of 57 blood samples from dogs with suspected dirofilariasis who had visited veterinary clinics in Bangkok were collected. The investigations for the presence of microfilaria were carried out by using both microscopic and molecular examinations. PCR was used as the molecular detection method for the filarial nematodes based on the COI and ITS1 regions. The demonstration of Wolbachia was performed using PCR to amplify the FtsZ gene. All positive samples by PCR were then cloned and sequenced. The results showed that the filarial nematodes were detected in 16 samples (28.07%) using microscopic examinations. The molecular detection of filarial species using COI-PCR revealed that 50 samples (87.72%) were positive; these consisted of 33 (57.89%), 13 (22.81%), and 4 (7.02%) samples for D. immitis, B. pahangi, and B. malayi, respectively. While the ITS1-PCR showed that 41 samples (71.93%) were positive—30 samples (52.63%) were identified as containing D. immitis and 11 samples (19.30%) were identified to have B. pahangi, whereas B. malayi was not detected. Forty-seven samples (82.45%) were positive for Wolbachia DNA and the phylogenetic tree of all positive Wolbachia was classified into the supergroup C clade. This study has established fundamental data on filariasis associated with Wolbachia infection in domestic dogs in the Bangkok Metropolitan Region. An extensive survey of dog blood samples would provide valuable epidemiologic data on potential zoonotic filariasis in Thailand. In addition, this information could be used for the future development of more effective prevention and control strategies for canine filariasis in Thailand.
... Differentiating the sheathed microfilariae of B.malayi from the non-sheathed microfilariae of D.(N.) repens by microscopy was relatively easy, but species differentiation of Brugia microfilariae based on morphology is difficult, as documented by others [14,26,27]. PCR with Comparison of the morphometry of the B. malayi microfilariae detected in the current survey with those recorded in scientific literature showed similarity in its body length measurements, but the longer innenkorper was more in keeping with B. pahangi [11]. ...
Human brugian filariasis has re-emerged in Sri Lanka after a quiescent period of four decades. This study investigated the prevalence of canine and feline filarial parasites in three localities with human sub-periodic brugian filariasis, in order to determine their potential reservoir status. All reachable dogs and cats, both stray and domestic, within a 350m radius of an index case of brugian filariasis in three locations (Madampe, Wattala and Weliweriya) were screened for microfilariae using Giemsa stained thick blood smears. A representative sample of canine and feline blood samples positive for Brugia spp. microfilariae by microscopy, from each of the three locations, were further analyzed by PCR with specific primers for internal transcribed spacer region 2 (ITS2) of the ribosomal DNA. A total of 250 dogs and 134 cats were screened. The overall microfilaraemia rates were high among both dogs (68.8%) and cats (47.8%). The prevalence of microfilaraemia was significantly higher among dogs than cats (p<0.05). Two filarial species were identified based on morphology of microfilariae: Dirofilaria (Nochtiella) repens (dogs, 54.4% and cats, 34.3%) and Brugia spp. (dogs, 51.6% and cats, 30.6%). PCR analysis of canine (n = 53) and feline (n = 24) samples elicited bands in the region of 615bp, which confirmed Brugia malayi infection. Co-infection with D.(N.) repens was detected by PCR with an additional band at 484bp, in 36 canine and 17 feline samples. Overall microfilaraemia rates of dogs (81.8%) and cats (75%) in Madampe (rural) were significantly higher than in urbanized Wattala (dogs, 62.4% and cats, 26.0%) (p<0.05). High rates of zoonotic filarial infections strongly implicate dogs and cats as potential reservoirs for human dirofilariasis and brugian filariasis in Sri Lanka.
... The differential diagnosis of the visceral localization of disease frequently (but not only) includes malignancy, requiring a biopsy or surgery for a conclusive histologic diagnosis. More recently, the introduction of molecular methods based on polymerase chain reaction and sequencing has improved diagnostic accuracy [14][15][16][17]. The use of minimally invasive techniques for the diagnostic and therapeutic excision of lung nodules of unclear origin should be strongly encouraged. ...
Background:
Human pulmonary dirofilariasis is a rare zoonosis caused by the dog worm Dirofilaria spp., a parasite transmitted by mosquitos and resulting in peripheral lung nodules. The filarial nematode enters the subcutaneous tissue, travels to the right ventricle and dies causing a small pulmonary infarction that may embolize through the pulmonary vessels and may appear as a solitary nodule. These nodules are usually incidentally identified in asymptomatic patients undergoing chest imaging studies, and are generally interpreted to be malignant.
Case presentation:
We present the case report of a human dirofilariasis in a patient with multiple pulmonary nodules resected using video-assisted thoracic surgery (VATS). According to our literature review, this is the first case with double synchronous lung nodules reported in Italy.
Conclusions:
Minimally invasive resection with histologic examination may be the best approach for the diagnosis and treatment of pulmonary dirofilariasis. Polymerase Chain Reaction testing may provide a more accurate etiological diagnosis in case of an inconclusive pathology result.
... La técnica Panfilaria se caracteriza por la obtención de fragmentos de diferente longitud de acuerdo con la especie de filárido hallado (Rishniw et al., 2006). En el protocolo COI se analiza la presencia de una banda de aproximadamente 650 pares de bases (pb) para diferentes filáridos, diferenciables mediante RFLP (restriction fragment length polymorphism) o secuenciación (Casiraghi et Impresa ISSN 0365514-8 Electrónica ISSN 1514-2590 Analecta Vet 2017; 37 (2): -al., 2001,2006). ...
Canine dirofilariasis in Argentina extends from Buenos Aires province to the North. All documented cases were due to the dog heartworm, Dirofilaria immitis, the unique species recorded in natural infected dogs in the country. In this study, we present morphologic, serologic and molecular evidences of the infection with another Dirofilaria species in 8 dogs from Neuquén city, in the northern extreme ofPatagonia. The observed microfilariae had a mean of 370 mm long and 7.1 mm wide. The sequences obtained showed homologies lower than 95% with other filarid sequences reported in the Genbank
... Bu nedenle filarial parazitlerin spesifik teşhisinde de özellikle nükleik asit tabanlı yöntemler kullanılmaya başlanmıştır (26). Mikrofilaremi görülen köpek ve kedilerde başta PCR, Nested PCR ve Real Time PCR gibi moleküler tanı yöntemleri ile filarial enfeksiyonların kesin teşhisi sağlanmış ve konvansiyonel yöntemlerde ortaya çıkabilecek hatalı identifikasyonların önüne geçilmiştir (13,(27)(28)(29). Ayrıca söz konusu teknikler miks enfeksiyonların saptanmasında da oldukça duyarlıdır. ...
This study was carried out to determine the prevalence of filarial nematode infections in stray dogs in Avanos
and vicinity. For this aim, a total of 138 blood samples were collected from stray dogs in different ages, sexes
and breeds between May-September 2013. The blood samples were firstly examined by wet mount (direct
microscopic examination), modified knott’s and membrane filtration-acid phosphates histochemical staining
techniques in order to detect the circulating microfilaria in peripheral blood. In the next step, genomic DNA extraction was obtained from collected blood samples, and the obtained genomic DNAs were analyzed by PCR with primers amplifying specific gene regions of 5.8S-ITS2-28S and cytochrome oxidase subunit 1 (COI) of Dirofilaria immitis. Three (2.17%) out of 138 blood samples were found to be D. immitis positive whereas no other
filarial nematode species were detected. Two out of three positive samples determined as molecularly positive
were also found positive by peripheral blood examination techniques. The PCR product belonging to one of
positive isolates was gel purified and sequenced in both directions for two gene regions. The identity rates between the obtained D. immitis isolate obtained from Avanos region and the other homologous isolates from
the world GenBank were determined as 99.5%-100% and 94.3%-100% according to the phylogenetic analyses
of 5.8S-ITS2-28S and COI gene regions, respectively. Both microscopic and molecular methods the prevalence
of D. immitis from stray dogs of Avanos region was revealed and the molecular characterization and phylogenetic
analyses of isolate for 5.8S-ITS2-28S and COI gene regions were investigated in this study.
... PCR (polymerase chain reaction) is a sensitive and accurate tool to discriminate microfilariae from the different filarial worms able to infect dogs and cats. Its use is advisable in case of morphological abnormalities of microfilariae, not infrequent in dogs treated incorrectly with preventive drugs or when multiple infections with more than one species of filarial worm makes difficult to differentiate microfilariae Mar et al., 2002;Casiraghi et al., 2006;Rishniw et al., 2006). ...
... Genomic DNA was prepared through proteinase-K treatment according to a previously described method [13]. A 450 bp fragment of the mitochondrial DNA (mtDNA; 12S rDNA) was amplified using specific primers and thermal profiles described by Casiraghi et al. [14][15][16]. In agreement with morphological identification, BLAST (http://blast.ncbi.nlm.nih.gov) ...
This report describes a rare case of ophthalmic dirofilariasis in a 2-year-old boy with redness, irritation, pain and foreign body sensation in the right eye. Slit lamp examination demonstrated a thread-like whitish nematode in the anterior chamber of the right eye that twisted around itself. The nematode worm (35 mm long and 150–200 μm width) was removed surgically. The presence of the smooth cuticular surface without longitudinal ridges and the vulva showed that it could be a female Diroflaria immitis. PCR amplification was done to verify the Diroflaria species. PCR amplification and sequence analysis of mitochondrial 12S rDNA confirmed that recovered worm was D. immitis. Ocular dirofilariosis caused by D. immitis is very rare, but it must be considered in humans living in endemic areas.
... Blood samples were tested by the following methods: modified Knott's method, Dirofilaria (Ag) ELISA tests (Witness ® Dirofilaria Test, SNAP ® 4Dx ® Plus Test). In addition, in 35 cases PCR was used for the specific detection of D. immitis and D. repens as described by Casiraghi et al. (2006). In 11 cases PCR analysis was not performed. ...
Dirofilariosis is an emerging mosquito-borne veterinary and medical problem in the Northern hemisphere. The ecological investigation of 56 canine dirofilariosis cases in new endemic locations was performed in Szeged, Hungary. The aim was to analyse the influence of the spatial patterns of dog abundance and the potential mosquito breeding habitats on the spatial occurrence patterns of dirofilariosis in the city of Szeged. The limnoecological characterisation was based on the fluvial habitat classification of Amoros of natural water bodies; the built environment was evaluated using the UrbanisationScore urbanisation intensity measuring software. Dirofilaria immitis accounted for 51% and D. repens for 34.3% of the dirofilariosis cases, and in 20% of the cases only the Knott's test was positive. It was concluded that most of the cases were related to locations with a medium to high urbanisation index, although the proximity of mosquito-bearing waters also played an important role in the observed spatial infection patterns. We found that the distance from potential mosquito habitats and the urbanisation intensity determine the abundance of dirofilariosis in urban environments.
... An aliquot of ∼100-150 l of blood from the bottom of each tube was collected and the DNA was extracted with the commercial kit NucleoSpin ® Blood (Macherey-Nagel, Duren, Germany) following manufacturing instructions. A species-specific region within the mitochondrial gene encoding for subunit b of the cytochrome oxidase 1 (cox1) was amplified as previously described (Casiraghi et al., 2006). The amplification protocol was also performed on specimens negative to microscopic examinations. ...
The present study evaluated the microfilaricidal efficacy of a single application of the spot-on containing imidacloprid 10%/moxidectin 2.5% (Advocate®, Bayer Animal Health) in dogs naturally infected either by Dirofilaria immitis or Dirofilaria repens. Dogs living in north-eastern and central-southern Italy, endemic for D. immitis and D. repens respectively, were randomly screened. Sixteen animals, eight infected with D. immitis and eight with D. repens, and fulfilling inclusion criteria were enrolled. Dogs infected with D. immitis received an adulticide treatment prior to the study and Advocate® 3 weeks after. The animals were divided in blocks of two (1:1, T1:T2) animals each, where Day 0 (D0) had an interval of 15 days to compare T2 vs. T1 dogs during the first fortnight of examination (i.e. T2 dogs acted as control animals at each examination). At baseline (Days −15 and 0 for T2 and T1 dogs, respectively) the animals had a range of microfilaraemia of 180-99.700 mff/ml (D. immitis) and 60-750 mff/ml (D. repens). All animals received a topical administration of Advocate® at D0 and were examined for microfilariae with microscopic and molecular tests at D15, D30, D60 and D90. All animals scored negative for mff at the first control post-treatment and throughout the study, with the exception of two D. immitis- infected animals that had a 2 mff/ml count at D15, and then become negative from Day 30 onwards. No adverse events were observed. The present study demonstrates the safety and the high microfilaricidal efficacy (99.97% and 100% for D. immitis and D. repens, respectively) of a single dose of moxidectin contained in Advocate® in naturally infected dogs.
... PCR (polymerase chain reaction) is a sensitive and accurate tool to discriminate microfilariae from the different filarial worms able to infect dogs and cats. Its use is advisable in case of morphological abnormalities of microfilariae, not infrequent in dogs treated incorrectly with preventive drugs or when multiple infections with more than one species of filarial worm makes difficult to differentiate microfilariae Mar et al., 2002;Casiraghi et al., 2006;Rishniw et al., 2006). ...
... PCR (polymerase chain reaction) is a sensitive and accurate tool to discriminate microfilariae from the different filarial worms able to infect dogs and cats. Its use is advisable in case of morphological abnormalities of microfilariae, not infrequent in dogs treated incorrectly with preventive drugs or when multiple infections with more than one species of filarial worm makes difficult to differentiate microfilariae Mar et al., 2002;Casiraghi et al., 2006;Rishniw et al., 2006). ...
... PCR (polymerase chain reaction) is a sensitive and accurate tool to discriminate microfilariae from the different filarial worms able to infect dogs and cats. Its use is advisable in case of morphological abnormalities of microfilariae, not infrequent in dogs treated incorrectly with preventive drugs or when multiple infections with more than one species of filarial worm makes difficult to differentiate microfilariae Mar et al., 2002;Casiraghi et al., 2006;Rishniw et al., 2006). ...
... The zoonotic filariae (Phylum: Nematoda, Class: Secrenentia, Subclass: Spiruria, Order: Spirurida, Family: Dirofilaridae), Dirofilaria repens and D. immitis, have become increasingly recognized worldwide as inadvertent human pathogens (1)(2)(3)(4) . These filariae are spread by mosquito species belonging to Anopheles, Culex and Aedes (5,6) . ...
Background: The zoonotic filariae: Dirofilaria repens and Dirofilaria immitis infect man as accidental dead end hosts in whom adult worms do not reach maturity. Human infection presents with either subcutaneous nodules or lung parenchymal disease that may be asymptomatic. Many sporadic cases have been diagnosed in Egypt. Objective: The aim of this paper is to present two cases of dirofilariasis repens. The first case was an adult male who presented to the surgical outpatient clinic of the Medical Research Institute Hospital complaining of a painful nodule in the right arm. The second case was a woman from the same locality (Ezbet Mohsen in Alexandria, Egypt) who presented to the dermatologist with a nodule on the right side of the chest. Methodology: One worm was extracted from each nodule and the identification of the collected worms depended on parasitological, histological and scanning electron microscope (SEM) studies. Criteria for morphological identification were worm length, cuticle thickness and the longitudinal ridges on the cuticle circumference. Results: The two cases were diagnosed as locally acquired dirofilariasis (D. repens) infection. Conclusion: D. repens human infections have become increasingly recognized worldwide as inadvertent human pathogens. This resulted from increased incidence as well as more refined diagnostic techniques. Recommendations: Some subcutaneous and pulmonary lesions of human dirofilariasis may be recognized as malignant tumors, thus requiring refined diagnostic modalities before invasive procedures are taken.
... Da sie zudem den Nachweis von O. lupi nicht ermöglicht, bietet sie keine Alternative zu der hier entwickelten Real-time PCR. Eine auf Restriktionsverdau beruhende Gel-PCR wird auch bei [Casiraghi et al., 2006] beschrieben. Es wird auch von Real-time PCRs berichtet, diese sind aber in der Regel nur in der Lage eine (z. ...
The present study was performed aiming to investigate a large number and a wide variety of mosquitoes
from various regions of Germany for the presence of defined groups of parasites. Accordingly, during
three successive years between 2010 and 2012, more than 140.000 mosquitoes, trapped at various
sites in Germany, were characterized and analysed. The selected trapping sites, most of them located
in southwest and northeast Germany, are known tor their abundant occurrence of mosquitoes, such
as the foodplains of the river Isar. In addition, some of them represent important stepping stones for
migrating birds, such as the Lake Chiemsee or the Lake Constance.
The mosquitoes were characterized on the basis of morphological criteria. The results indicated, that
the main autochthonous species generally present in Germany were trapped, in particular abundant
species such as Aedes vexans and Culex pipiens s. l.. As morphological criteria do not allow the
discrimination of members of the Culex pipiens complex or between females of Culex pipiens and
Culex torrentium, a multiplex real-time PCR was developed for the rapid genetic differentiation of
the various Culex species and biotypes. Using this method, hybrids of Cx. pipiens pipiens biotype
pipiens and biotype molestus were detected in Germany for the first time. These hybrids are known
as important bridge vectors for the transmission of West Nile virus from birds to humans.
To fascilitate the detection of parasites in such a large number of mosquio samples within a reasoonable
period of time, further multiplex real-time PCR assays were developed to allow analyses of
respective microbial DNAs by high-throughput screening. The results indicated that a considerable
number of German mosquitoes are carrying filariae. In this context, a new filarial species was detected,
occuring primarily in southern Germany. The reservoir of this new filarial species is probably birds, as
it was detected mainly in ornithophilic mosquitoes. Studies on Dirofilaria immitis, Dirofilaria repens
as well as Onchocerca lupi, three zoonotic filarial species endemis in southern Europe and pathofenic
for humans, revealed the detection of a focus of stable transmission of D. repens in the federal state
of Brandenburg. This rilarial species was detected in several mosquito catches from 2011 and 2012,
along the Oder Valley in the vincinity of the town of Eberswalde.
As there were reports on cases of avian malaira in the zoo of Heidelberg in 2010 screening of mosquitoes
was extended for the presence of Haemosporidia, such as Haemoproteus sp., Leukocytozoon
sp. or Plasmodium sp.. A multiplex real-time PCR was developed able to detect nematodes as well
as blood protozoans. Depending on the catchment area, more than 70 % of the mosquitoes were
positive for at least one of the two groups of parasites.
... Um outro método que permite fazer a distinção destas espécies é um método histoquímico, baseado na coloração das microfilárias pela fosfatase ácida (Casiraghi et al., 2006;Carvalho & Santos, 2006). Este método permite a diferenciação mais precisa pela utilização de corantes histoquímicos para a actividade da fosfatase ácida. ...
... Wherever human filarial cases are detected, animal reservoirs are present. The present finding supports the previous reports that observed D. repens infection in humans from the study area [6], using PCR as molecular diagnostic tool, which is useful to differentiate D. immitis from other filarial parasites [8,12]. ...
Objective: To access the prevalence of Dirofilaria repens (D. repens) in dogs from Assam,
India.
Methods: A total of 223 blood samples from local dogs were examined with conventional (wet
film and Knott’s concentration technique), serological (ELISA test using Snap4Dx kits) and
molecular techniques (targeting internal transcribed spacer-2 region using panfilarial primers)
in Guwahati, Assam, India.
Results: The study revealed 4 (1.79%) cases of asymptomatic canine dirofilariasis caused by
D. repens. The blood samples were positive for D. repens with microfilaremia on wet blood
film, at Giemsa stained smear and under Knott’s concentration technique, but were negative at
Snap®4Dx test (IDEXX Laboratory, Westbrook, USA) which is specific for Dirofiaria immitis.
D. repens could be detected by molecular test. Further confirmation was obtained on the basis
of DNA sequencing and homology searching by basic local alignment search tool. Sequence
analysis revealed that the species prevalent in Guwahati was genetically distinct from the other
D. repens reported from elsewhere.
Conclusions: Occurrence of D. repens in dogs from this part of India was recorded for the
first time, confirming the presence of a autochthonous canine reservoir for the zoonotic filarial
nematode in Assam, India, where three cases of human subcutaneous and ocular infection with
D. repens (dirofilariasis) have been reported.
... Serological diagnosis of D. immits is based on the detection of a female adult antigen, and has been applied for clinical purposes and in epidemiological studies [11]; however, it restricts detection only to D. immitis, disregarding other canine filarioids. Molecular detection techniques have been designed to detect different genetic loci that identify canine filarioids in general or certain species with high sensitivity and specificity [12][13][14][15]. Nevertheless, none of previous studies have compared the validity of the Knott's test to all the other diagnostic methods included in this study. ...
Canine filarioids are important nematodes transmitted to dogs by arthropods. Diagnosis of canine filariosis is accomplished by the microscopic identification of microfilariae, serology or PCR for filarial-DNA. The aim of this study was to evaluate a molecular assay for the detection of canine filariae in dog blood, to compare its performance to other diagnostic techniques, and to determine the relationship between microfilarial concentration and infection with other vector-borne pathogens.
Blood samples from 146 dogs from Costa Rica were subjected to the detection of canine filarioids by four different methods: the microhematocrit tube test (MCT), Knott's modified test, serology and a high resolution melt and quantitative real-time PCR (HRM-qPCR). Co-infection with other vector-borne pathogens was also evaluated.
Fifteen percent of the dogs were positive to Dirofilaria immitis by at least one of the methods. The HRM-qPCR produced distinctive melting plots for the different filarial worms and revealed that 11.6% of dogs were infected with Acanthocheilonema reconditum. The latter assay had a limit of detection of 2.4x10-4 mf/μl and detected infections with lower microfilarial concentrations in comparison to the microscopic techniques and the serological assay. The MCT and Knott's test only detected dogs with D. immitis microfilaremias above 0.7 mf/μl. Nevertheless, there was a strong correlation between the microfilarial concentration obtained by the Knott's modified test and the HRM-qPCR (r = 0.906, p < 0.0001). Interestingly, one dog was found infected with Cercopithifilaria bainae infection. Moreover, no association was found between microfilaremia and co-infection and there was no significant difference in microfilarial concentration between dogs infected only with D. immitis and dogs co-infected with Ehrlichia canis, Anaplasma platys or Babesia vogeli.
This is the first report of A. reconditum and C. bainae in Costa Rica and Central America. Among the evaluated diagnostic techniques, the HRM-qPCR showed the most sensitive and reliable performance in the detection of blood filaroids in comparison to the Knott's modified test, the MCT test and a serological assay.
... Dipetalonema) reconditum. Species found in the subcutis such as Dirofilaria repens or Dipetalonema reconditum were considered nonpathogenic (3,9). ...
This work deals about some pathological manifestations found in dogs' dirofilariosis. The dogs were examined in the Parasitology and Parasitological diseases and Small Animal Pathology clinics of the Faculty of Veterinary Medicine, Timisoara, Romania. The pathological manifestations were studied on 4 dogs of different sex, breed and age. In chronic evolution of heartworm disease in most infected dogs do not show clinical signs. Serological tests for heartworm disease can be negative in non-endemic areas and that's why additional methods are compulsory.The application of prophylactic measures in dogs throughout the period of mosquito activity can be decreased incidence and dissemination of dirofilariosis. Dirofilariosis is a disease caused by filarial worms of the genus Dirofilaria (12). Filarial nematodes are common parasites of many vertebrates (1, 5). The nematodes of genus Dirofilaria belong to family Onchocercidae and subfamily Dirofilariinae of the order Spirurida (1, 8). This genus consists of 27 valid species, and 15 species of questionable validity (2, 12). The genus Dirofilaria is divided in two subgenera. The subgenus Dirofilaria includes Dirofilaria immitis and the subgenus Nochtiella includes Dirofilaria repens (8). In European dogs, the most common species of filariae are Dirofilaria immitis, D. repens and Acanthocheilonema (syn. Dipetalonema) reconditum. Species found in the subcutis such as Dirofilaria repens or Dipetalonema reconditum were considered nonpathogenic (3, 9). D. immitis and D. repens are transmitted by over 60 common mosquito species. The intermediate hosts of D. reconditum are fleas and lice. Because dirofilariosis is a zoonotic helminthosis and are imprecisely diagnosed we proposed to present some pathological manifestations in dogs' dirofilariosis.
... An aliquot of~150 μl of blood collected from the bottom of each tube was subjected to the DNA extraction using the commercial kit NucleosSpin® Blood (Macherey-Nagel, Duren, Germany). All DNA extracts were undertaken to semi-nested PCR protocols specific for mitochondrial gene encoding for subunit b of the cytochrome oxidase 1 (cox1) of canine filariae as previously described [31,39]. In the first round, the universal primer set for spirurid nematodes COIintF (5′-TGATTGGTG GTTTTGGTAA-3′) and COIintR (5′-ATAAGTACG AGTATCAATATC-3′) were used. ...
Background
Dirofilaria repens is the causative agent of subcutaneous dirofilariosis of dogs, other animals and humans. This nematode is transmitted by mosquitoes of Aedes, Anopheles and Culex genera. In dogs, the parasite may cause subclinical infection or cutaneous signs. Recently, D. repens has emerged and spread in different geographical areas, with an increase of cases in dogs and humans. Chemoprevention in dogs in endemic areas is the most reliable approach for controlling this infection. This paper describes a randomized, blocked and multicentric clinical field study investigating the efficacy of an oral, chewable formulation containing milbemycin oxime/praziquantel (Milbemax®, Novartis Animal Health) in the chemoprevention of subcutaneous dirofilariosis in dogs.
Methods
This study was conducted in endemic areas of Italy. A total of 249 dogs, at two sites, negative for D. repens, were allocated into two groups (i.e. Treated -T1 vs Untreated-T2) with a ratio of 1:1, and subjected to clinical visits and blood sampling once monthly until the end of the study. All blood samples were microscopically and genetically examined. Animals belonging to T1 group received a minimum target dose of 0.5 mg/kg bodyweight of milbemycin oxime and 5 mg/kg of praziquantel in commercial tablets (Milbemax®) according body weight once every 4 weeks. Animals of group T2 were not treated with Milbemax® but received, when necessary, specific parasiticide treatments. The study duration was 336 ± 2 days for each dog.
Results
A total of 219 dogs completed the study (i.e. 111 in T1 and 108 in T2), while 30 dogs (i.e. 13 in T1, 17 in T2) were withdrawn for a variety of reasons unrelated to administration of Milbemax®. The percentages of animals not showing microfilariae of D. repens were 100% (111 animals) in T1 and 94.7% (108 animals out of 114) in group T2. Milbemax® was shown to be safe in treated dogs.
Conclusions
The results of this study confirm that the monthly use of Milbemax® in dogs is effective and safe for the prevention of subcutaneous dirofilariosis in endemic areas.
... However, mf may be absent from dog blood for several reasons (drug treatment, age of adult worms, etc.), and imperfect sensitivity is a concern with antigen detection methods [3]. Polymerase chain reaction (PCR)based methods are the most accurate diagnostic approach for dirofilariosis to date [9,10], but are not routinely employed. A major issue with currently available filarial diagnostic tests is their inability to predict adult worm burden and viability. ...
Filarial nematodes cause chronic and profoundly debilitating diseases in both humans and animals. Applications of novel technology are providing unprecedented opportunities to improve diagnosis and our understanding of the molecular basis for host-parasite interactions. As a first step, we investigated the presence of circulating miRNAs released by filarial nematodes into the host bloodstream. miRNA deep-sequencing combined with bioinformatics revealed over 200 mature miRNA sequences of potential nematode origin in Dirofilaria immitis-infected dog plasma in two independent analyses, and 21 in Onchocerca volvulus-infected human serum. Total RNA obtained from D. immitis-infected dog plasma was subjected to stem-loop RT-qPCR assays targeting two detected miRNA candidates, miR-71 and miR-34. Additionally, Brugia pahangi-infected dog samples were included in the analysis, as these miRNAs were previously detected in extracts prepared from this species. The presence of miR-71 and miR-34 discriminated infected samples (both species) from uninfected samples, in which no specific miRNA amplification occurred. However, absolute miRNA copy numbers were not significantly correlated with microfilaraemia for either parasite. This may be due to the imprecision of mf counts to estimate infection intensity or to miRNA contributions from the unknown number of adult worms present. Nonetheless, parasite-derived circulating miRNAs are found in plasma or serum even for those species that do not live in the bloodstream.
... Proper identification of circulating microfilariae based on morphology requires the involvement of a well-trained parasitologist. Filarial infection with multiple species and morphological alterations of microfilariae due to incorrect preventive treatment of dogs are not easily differentiated morphologically even by trained persons [31]. ...
A very high prevalence of microfilaremia of 42.68 per cent out of 164 canine blood samples examined was observed in Cherthala (of Alappuzha district of Kerala state), a known human Brugia malayi endemic area of south India. The species of canine microfilariae were identified as Dirofilaria repens, Brugia malayi, and Acanthocheilonema reconditum. D. repens was the most commonly detected species followed by B. pahangi. D. immitis was not detected in any of the samples examined. Based on molecular techniques, microfilariae with histochemical staining pattern of “local staining at anal pore and diffuse staining at central body” was identified as D. repens in addition to those showing acid phosphatase activity only at the anal pore. Even though B. malayi like acid phosphatase activity was observed in few dogs examined, they were identified as genetically closer to B. pahangi. Hence, the possibility of dogs acting as reservoirs of human B. malayi in this area was ruled out.
... However, the identification of the parasite species using the traditional approaches sometimes can be problematic, as in cases where the morphological recognition of microfilariae proves to be difficult, or in case of mixed infections (Genchi et al., 2007). For these reasons, several different molecular protocols have been developed during the last years including single species identification using species-specific PCR assays, or simultaneous detection of the different Dirofilaria species using multiplex PCR and PCR-RFLP assays (Favia et al., 1996;Mar et al., 2002;Nuchprayoon et al., 2005;Casiraghi et al., 2006;Rishniw et al., 2006;Gioia et al., 2010;Latrofa et al., 2012). Highresolution melting analysis (HRMA) is a modern molecular technique with increasing use in diagnostic microbiology and parasitology for species identification and genotyping. ...
... However, following the increasing demand for accurate, fast, sensitive and efficient detection methods in all hosts, molecular tools have been steadily improved in recent years (Nuchprayoon et al. 2005;Duscher et al. 2009a;Gioia et al. 2010;Latrofa et al. 2012a, b). Molecular-based assays can identify and discriminate different filarioid species (Nuchprayoon et al. 2005;Casiraghi et al. 2006;Rishniw et al. 2006) and have been tested on blood samples (Gioia et al. 2010), skin samples (Latrofa et al. 2012b) and mosquitoes (Latrofa et al. 2012a). So far, all these methods employ DNA preparation prior to PCR detection which increases both the sample turnover time and the costs, prohibiting their large-scale application in epidemiological screening. ...
A novel direct PCR assay for the detection of Dirofilaria spp. from EDTA blood, Knott test, FTA cards, adult filarial worms, skin nodules and Dirofilaria spp.-infected mosquitoes was tested. Larval and adult DNA of Dirofilaria spp. from FTA cards, from the mosquito vector and from worm fragments without prior DNA extraction was successfully obtained. As little as 3.11 larvae/100 μl blood on FTA cards could be detected. Thus, direct PCR is capable of directly detecting first larval stages in the blood, third larval stages in the mosquito vector and pieces of mature stages of Dirofilaria spp. The assay is a rapid, sensitive and cost-effective alternative to standard PCR.
... Recent molecular-based assays have been reported for the unequivocal identification of filarioids, irrespective of their life cycle stage. Ribosomal or mitochondrial DNA sequence fragments of D. immitis, D. repens and A. reconditum may be amplified and analysed with a restriction fragment length polymorphism, specific PCR amplifications or with primers yielding species-specific amplicons [126,127] and their usefulness has been demonstrated in epidemiological and clinical studies [112,128,129]. Recently, a duplex real-time PCR has been assessed for the discrimination between D. immitis and D. repens and their quantification in blood samples and mosquitoes [130]. ...
Presently, 45% of the total human population of Europe, as well as their domestic and companion animals are exposed to the risk of vector-borne helminths (VBH) causing diseases. A plethora of intrinsic biological and extrinsic environmental factors affect the relationship among helminths, vectors and animal hosts, in a constantly changing environment. Although canine dirofilarioses by Dirofilaria immitis and Dirofilaria repens are key examples of the success of VBH spreading into non-endemic areas, another example is represented by Thelazia callipaeda eyeworm, an emergent pathogen of dogs, cats and humans in several regions of Europe. The recent finding of Onchocerca lupi causing canine and human infestation in Europe and overseas renders the picture of VBH even more complicated. Similarly, tick-transmitted filarioids of the genus Cercopithifilaria infesting the skin of dogs were recently shown to be widespread in Europe. Although for many of the VBH above there is an increasing accumulation of research data on their distribution at national level, the overall impact of the diseases they cause in dogs and humans is not fully recognised in many aspects. This review investigates the reasons underlying the increasing trend in distribution of VBH in Europe and discusses the diagnostic and control strategies currently available. In addition, this article provides the authors' opinion on some topics related to VBH that would deserve further scientific investigation.
... Therefore, modified Knott's technique to identify mf on the basis of differential morphometric and morphological features of canine filariae (Lindsey 1965;Hahn 1994) was used, and a microfilaremia by D. repens of 490 mf/ml was assessed. A blood sample and a single paraffin-embedded skin biopsy were subjected to PCRs specific for the mitochondrial gene cox1 of canine filariae (Casiraghi et al. 2006). The sequence from amplicons produced for both the blood and the skin showed 100% homology with each other and 99.8% with the D. repens sequence registered under the GenBank TM accession number DQ358814.1. ...
Adult stages of Dirofilaria repens (Nematoda, Filarioidea) reside in the subcutaneous tissues of the definitive or occasional host as dogs, other animals, and humans, and it is transmitted by mosquitoes. Canine infections with adults and circulating larvae of D. repens are often considered asymptomatic, although in some cases, the parasite causes subcutaneous nodules, diffused dermatitis, skin lesions, and itching. This report provides a complete clinical description of an unusual case of allergic diffused dermatitis caused by D. repens in a naturally infected dog and its successful treatment with the use of a spot-on solution containing imidacloprid 10%/moxidectin 2.5%. The dog presented multiple pustules and alopecic areas with lichenification, hyperpigmentation, and erythematous scaling margins without pruritus. Histological examination was compatible with allergic dermatitis. After being unsuccessfully managed for suspected food hypersensitivity, with a significantly worsening of the lesions, a Knott’s analysis detected nematode larvae in the blood. Morphological and molecular identification showed them to be D. repens. The dog was then treated with a single administration of a spot-on formulation containing imidacloprid 10%/moxidectin 2.5%, and the dermatological signs completely resolved within 2 months after treatment. The dog showed no recurrence of the lesions, and no circulating microfilariae were found upon microscopic and molecular examination for six consecutive months after treatment. This report indicates the apparent primary role of D. repens in causing hypersensitivity-like skin disease without pruritus in a dog. It also confirms, as recently shown elsewhere, the efficacy of imidacloprid 10%/moxidectin 2.5% in the treatment of dermatitis caused by D. repens.
Filarioid nematodes, which are vector-borne parasites of cosmopolitan distribution, of dogs are medically important. They are represented by species in which microfilariae were found to be circulating in the bloodstream (e.g., Dirofilaria sp., Acanthocheilonema sp., and Brugia sp.) or skin-dwelling (e.g., Cercopithifilaria sp. and Onchocerca sp.). Those species whose microfilariae are detected in blood have been extensively studied, especially Dirofilaria immitis, due to their clinical importance. In recent decades, there has been an increased interest by the scientific community in filarioid nematodes whose microfilariae are detected in the skin because of the zoonotic aspect of Onchocerca lupi. In the United States (US), although D. immitis has been considered the main filarioid infecting dogs, the intense animal movement and global canine filarioid diversity may indicate that the likely presence of cutaneous filarioid nematodes is more common than previously expected. Hence, a question remains: Are these canine filarioid nematodes emerging, neglected, or simply underdiagnosed in the US? In this review, we provide an overview of pertinent information that briefly summarizes the biology of the different canine filarioid nematode species, clinical signs associated with infections, and currently available diagnostic tools using molecular and microscopy-based methods and highlight knowledge gaps where research and surveillance efforts remain necessary. The data herein presented serve as an alert to the scientific community about the importance of filarioid nematodes infecting dogs other than D. immitis. Additionally, the zoonotic potential of several filarioid species reinforces the necessity of a proper diagnosis and the need for broader surveillance to understand their diversity and distribution, to highlight the potential introduction of certain species, and mitigate their establishment in the country and new animal and human cases.
Chile is a large country with a marked range of climate conditions that make it an ideal scenario for the study of vector-borne parasites; however, knowledge about their distribution is limited to a few confined areas of this country. The presence of Hepatozoon spp., piroplasmids, Leishmania spp. and filarioids was investigated through molecular and serological methods in blood samples of 764 free-ranging rural dogs, 154 Andean foxes (Lycalopex culpaeus), and 91 South American grey foxes (Lycalopex griseus) from six bioclimatic regions across Chile. Hepato-zoon spp. DNA was exclusively detected in foxes (43% prevalence), including sequences closely related to Hepa-tozoon felis (24.1%; only Andean foxes), Hepatozoon americanum (16.2%; only grey foxes), and Hepatozoon canis (1.25%; in one grey fox). Risk factor assessment identified a higher probability of Hepatozoon infection in juvenile foxes. DNA of piroplasmids was detected in 0.7% of dogs (Babesia vogeli) but in no fox, whilst antibodies against Babesia sp. were detected in 24% of the dogs and 25% of the foxes, suggesting a wider circulation of canine piroplasmids than previously believed. A positive association between the presence of antibodies against Babesia and high Rhipicephalus sanguineus sensu lato burden was observed in dogs. Leishmania spp. DNA and anti-bodies were detected in 0.8% and 4.4% of the dogs, respectively. Acanthocheilonema reconditum was the only blood nematode detected (1.5% of the dogs and no fox). Differences in prevalence among bioregions were observed for some of the VBP. These results expand our knowledge about the occurrence of vector-borne parasites in Chile, some of which are firstly reported herein. This information will facilitate the diagnosis of vector-borne diseases in domestic dogs and improve the control measures for both domestic and wild canids.
To estimate the incidence of Dirofilaria immitis in Austrian shelter dogs and mosquitoes trapped in their proximity, 115 shelter dogs from fourteen animal shelters located in five different Austrian states were examined. Blood samples were screened for D. immitis using ELISA antigen-testing, PCR and microscopical examination for microfilariae. In total, 91% of the dogs originated from countries endemic for dirofilariosis. Eleven dogs (9.6%), all originating from Hungary, tested positive for D. immitis. None of the dogs examined showed microfilaremia. Eight dogs showed no or only mild clinical signs (e.g., infrequent coughing), and three dogs showed frequent coughing, dyspnea, exercise intolerance, blunt fur or weight loss. In total, 205 Mosquitoes of ten different species were caught at five different shelter sites in four different Austrian states, using CO2-baited mosquito traps set once a month (June–September 2019) for 24 h. All 205 mosquitoes tested negative for Dirofilaria spp. via PCR. The risk of endemisation of D. immitis in Austria (and other non-endemic countries in a similar situation) is very serious and its zoonotic potential should be communicated more strongly. To monitor a possible transmission of microfilariae from untreated or even untested positive dogs, e.g., in animal shelters, to mosquitoes in the near surroundings, frequent screening for Dirofilaria in mosquitoes should be used more intensively. Current knowledge on D. immitis should be integrated into daily veterinary practice and dog owners should be proactively educated, especially before traveling to endemic areas or adopting dogs from endemic countries. Animal shelters and animal welfare organizations should be provided with appropriate education and veterinary guidance regarding the testing and treatment of dogs imported from high-risk areas.
Otita la câine este o afectiune cu o incidență ridicată, care uneori întâmpină dificultăți sau erori de diagnostic şi de tratament. Aceste erori pot fi asociate cu o mare diversitate de manifestări clinice, diferite de la un animal la altul. Pseudomonas aeruginosa este frecvent implicată în cazul otitelor medii și externe însoțite de eroziuni, ulcerații și de cantități apreciabile de exsudat galben-deschis, de obicei cu evotuție cronică și recidivantă.
Lotul de studiu a inclus 26 de tulpini Pseudomonas aeruginosa, izolate în perioada 2013-2014 în laboratorul de microbiologie al Centrului Universitar de Cercetări Medical- Veterinare Iași din cadrul FMV –IAȘI.
Am folosit recomandările recente (Magiorakos et al., 2012) pentru a defini tulpinile multidrog rezistente, MDR.
Cele 7 clase de antibiotice cercetate au fost: cefalosporine, combinații penicilină anti-Pseudomonas+inhibitor de β-lactamază, cefalosporine cu spectru extins, monobactame, carbapeneme, fluorochinolone, aminoglicozide și polimixine.
Prezentul studiu a evidențiat 16 tulpini MDR, în consecință în Medicina Veterinară din cauza paletei restrânse de antibiotice de elecție din sfera ORL, concomitent cu capacitatea de rezistență multidrog a tulpinilor de Pseudomonas aeruginosa se impune un protocol de diagnostic etiologic diferit.
In bee families untreated with acaricide substances during the active season population trends of infestation with Varroa destructor mites by determining indicators, daily natural mortality, level of infestation of adult bees and level of infestation of the brood, was monitored. In results interpretation was established that, in climatic conditions of western Romania, V. destructor mite populations in bee families under active season may reach, in the fall at the end of the active beekeeping season, damaging levels in the absence of specific treatment.
Introducción: A la fecha no hay datos concluyentes en Chile respecto a la presencia de dirofilariasis zoonótica en perros. Objetivos: Identificar la presencia de dirofilarias en sangre de perros de una comuna semi-rural cercana a Santiago y comparar su frecuencia en animales con y sin manifestaciones dermatológicas. Materialy Métodos. Se examinó un frotis sanguíneo de 100 perros en busca de microfilarias mediante observación microscópica (técnica de Knott modificada). Cincuenta perros presentaban síntomas o signos dermatológicos que se han asociado a esta parasitosis y 50 eran asintomáticos. Se amplificaron los genes ITS-2 y 12s ADNr de filarías en las muestras con microfilarias al frotis, secuenciando los fragmentos amplificados. Resultados: Se observaron microfilarias en 22 perros (22%), 16/50 (32%) sintomáticos y 6/50 (12%) asintomáticos (p = 0,02). Morfológicamente, la mayoría de las microfilarias observadas fueron similares a D. repens; sin embargo, una gran proporción mostró un tamaño mayor al descrito para esta especie. Las secuencias nucleotídicas de los genes amplificados mostraron una homología no mayor al 95% con las secuencias de D. repens disponibles para comparación. Se identificaron además dos especies poco patógenas, D. reconditum por morfología y secuenciación genética y D. dracunculoides por morfología. Conclusiones: Los resultados indican la existencia de una nueva especie de Dirofilaria cercanamente relacionada a D. repens o de una variante de esta especie.
Introduction:
To date, there has been no definitive confirmation of the presence of zoonotic dirofilariasis in dogs in Chile.
Objectives:
To study the presence of dirofilarias in blood samples from dogs collected in a semi-rural district near Santiago and to compare their frequency in dogs with and without dermatological manifestations.
Methods:
We examined 100 blood samples for dog filariae infections using microscopic methods (modified Knott technique). 50 dogs presented dermatological symptoms or signs compatible with filarial infections and 50 were asymptomatic. ITS-2 and 12s rDNA gene amplification by PCR and sequencing were performed in samples microscopically positive for microfilariae. Results. We observed microfilariae in 22 dogs (22%). Of these, 16/50 (32%) were symptomatic and 6/50 (12%) were asymptomatic (p = 0.02). Morphologically, the majority of micro-filariae were similar to Dirofilaria repens, although many had a bigger size than previously described. Nucleotide sequencing of the amplified genes showed no more than 95% homology with the D. repens sequences available for comparison. D. reconditum and D. dracunculoides infections were also identified.
Conclusions:
These features might indicate the presence of new species of Dirofilaria or a D. repens close related variant in Chile.
Animal cells are the sole habitat for a variety of bacteria. Molecular sequence data have been used to position a number of these intracellular microorganisms in the overall scheme of eubacterial evolution. Most of them have been classified as proteobacteria or chlamydiae. Here we present molecular evidence placing an intracellular symbiont among the flavobacteria-bacteroides. This microorganism inhabits specialized cells in the cockroach fat body and has been described as a mutualistic endosymbiont of uncertain phylogenetic position. The small subunit ribosomal DNA of these bacteria was analysed after polymerase chain reaction amplification to investigate their phylogeny. The endosymbionts of five species of cockroaches were found to make up a coherent group with no close relatives within the eubacterial phylum defined by the flavobacteria. In addition, the relationships among the endosymbionts, as revealed by DNA sequence data, appeared to be congruent with the host taxonomic relationships. Based on the host fossil record, a tentative calibration of the nucleotide substitution rate for the cockroach flavobacteria gave results congruent with those obtained for the aphid endosymbiotic proteobacteria.
CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new
system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing
the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows
the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional
multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order
of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to
be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments
or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with
a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled
on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for
x86 PCs, and Macintosh PowerMac.
Infection with the endosymbiotic bacteria Wolbachia is widespread in filarial nematodes. Previous studies have suggested concordance between the phylogeny of Wolbachia with that of their nematode hosts. However, there is only one published molecular phylogenetic study of filarial species, based on the 5S rRNA gene spacer. The phylogeny proposed by this study is partially incongruent with previous classifications of filarial nematodes, based on morphological characters. Furthermore, both traditional classifications and molecular phylogenies are, in part, inconsistent with the phylogeny of Wolbachia. Here we report mitochondrial cytochrome oxidase I (COI) gene sequences for 11 species of filaria and for another spirurid nematode which was included as an outgroup. In addition, 16S rRNA, wsp and ftsZ gene sequences were generated for the Wolbachia of several filarial species, in order to complete the available data sets and further resolve the phylogeny of Wolbachia in nematodes. We used these data to evaluate whether nematode and Wolbachia phylogenies are concordant. Some of the possible phylogenetic reconstructions based on COI gene were congruent with the phylogeny of Wolbachia and supported the grouping of the rodent filaria Litomosoides sigmodontis with the lymphatic filariae (i.e. Brugia spp. and Wuchereria spp.) and the sister group relationship of Dirofilaria spp. and Onchocerca spp. However, the placement of the Wolbachia-free filaria Acanthocheilonema viteae is ambiguous and dependent on the phylogenetic methods used.
Both Dirofilaria immiti and Dipetalonema reconditum may be found in blood of infected dogs but it is not easy to distinguish D. immitis from D. reconditum in morphology. We cloned and sequenced the contiguous internal transcribed spacer (ITS) region, ITS1-5.8S-ITS2, of these two different parasites and published on GenBank as AF217800 for D. immiti and AF217801 for D. reconditum in this study. We designed two pairs of specific primers derived from ITS2 being used for polymerase chain reaction (PCR). The amplicons of ITS2 from D. immiti and D. reconditum are 302 and 348bp, respectively. Moreover, the limitation for amplifying ITS2 gene using this PCR demonstrated that 1 x 10(-2) microfilaria of each species of parasite smashed or even with mixed samples could be detected and the PCR products were predicted as the same as that described above. Thus, D. immiti and D. reconditum could be differentially diagnosed by this specific PCR. Seventeen clinical cases were evaluated and all of them were correctly identified. In this study, ITS1-5.8S-ITS2 of D. immiti or D. reconditum were the first time sequenced and analyzed. No significant similarity of ITS1 and ITS2 between D. immiti and D. reconditum could be observed.
Wolbachia pipientis is a bacterial endosymbiont associated with arthropods and filarial nematodes. In filarial nematodes, W. pipientis has been shown to play an important role in the biology of the host and in the immuno-pathology of filariasis. Several species of filariae, including the most important parasites of humans and animals (e.g. Onchocerca volvulus, Wuchereria bancrofti and Dirofilaria immitis) have been shown to harbour these bacteria. Other filarial species, including an important rodent species (Acanthocheilonema viteae), which has been used as a model for the study of filariasis, do not appear to harbour these symbionts. There are still several open questions about the distribution of W. pipientis in filarial nematodes. Firstly the number of species examined is still limited. Secondly, it is not clear whether the absence of W. pipientis in negative species could represent an ancestral characteristic or the result of a secondary loss. Thirdly, several aspects of the phylogeny of filarial nematodes are still unclear and it is thus difficult to overlay the presence/absence of W. pipientis on a tree representing filarial evolution. Here we present the results of a PCR screening for W. pipientis in 16 species of filariae and related nematodes, representing different families/subfamilies. Evidence for the presence of W. pipientis is reported for five species examined for the first time (representing the genera Litomosoides, Litomosa and Dipetalonema); original results on the absence of this bacterium are reported for nine species; for the remaining two species, we have confirmed the absence of W. pipientis recently reported by other authors. In the positive species, the infecting W. pipientis bacteria have been identified through 16S rDNA gene sequence analysis. In addition to the screening for W. pipientis in 16 species, we have generated phylogenetic reconstructions based on mitochondrial gene sequences (12S rDNA; COI), including a total of 28 filarial species and related spirurid nematodes. The mapping of the presence/absence of W. pipientis on the trees generated indicates that these bacteria have possibly been lost during evolution along some lineages of filarial nematodes.
This book is a republication in one volume of the 10-part Commonwealth Institute of Helminthology's "Keys to the Nematode Parasites of Vertebrates", first published between 1974 and 1983. The first chapter provides a glossary of morphological terms and taxonomic keys to subclasses (Adenophorea and Secernentea) of nematode parasites of vertebrates. The next 13 chapters describe the taxonomy and provide keys to families, subfamilies, genera and subgenera of 27 superfamilies based on morphological and biological characteristics. These superfamilies include Dioctophymatoidea, Trichinelloidea, Muspiceoidea, Rhabditoidea, Diaphanocephaloidea, Ancylostomatoidea, Strongyloidea, Trichostrongyloidea, Metastrongyloidea, Oxyuroidea, Cosmocercoidea, Seuratoidea, Heterakoidea, Ascaridoidea, Subuluroidea, Camallanoidea, Dracunculoidea, Gnathostomatoidea, Physalopteroidea, Rictularoidea, Thelazioidea, Spiruroidea, Habronematoidea, Acuarioidea, Diplotriaenoidea, Aproctoidea and Filarioidea. It is hoped that, in addition to meeting the need for a means of identification of animal parasitic nematodes, the re-issuing of the keys will also facilitate investigations into both the evolution of nematodes and parasitism within the phylum by helping to link recent molecular insights with those provided to date in numerous morphological studies.
Based on recently published surveys and newly acquired data, a study was conducted to verify the distribution of filarial worm (Filarioidea) infections in Europe, with particular emphasis on canine heartworm infection (Dirofilaria immitis). A Geographic Information System based on thermal regimen was constructed as a means to identify areas potentially suitable for heartworm transmission, taking into account that the development of D. immitis larvae in the mosquito does not occur below the threshold temperature of approximately 14 °C. Furthermore, a bionomic model of D. immitis in its mosquito vectors, which calculates the moving cumulative heartworm development unit parameter, was applied using the available temperature data to assess the theoretic transmission timing of heartworm in Europe. The results show that the earliest infection risk occurs in Spain on March 21 and the latest risk occurs in Spain on September 11. The longest risk period occurs in Spain (Murcia station: March 21–November 11), and the shortest risk period occurs in northeastern Europe. The study also provides the first risk assessment maps for Europe and suggests that if the actual climatic trend continues, filarial infection should spread into previously infection-free areas.
The first edition of this book was published in 1992 (see Helminthological Abstracts (1993) 62 , abstract 1457). This new enlarged edition includes additional relevant information from some 450 articles published between 1989 and 1998 (with a few from 1999), and some articles overlooked or unavailable for the first edition. The number of species covered has been increased by 34 (total now 595). As before, the book is in 2 parts, the Secernentea and Adenophorea, which are now regarded as classes rather than subclasses. The Secernentea covers the orders Rhabditida, Strongylida, Oxyurida, Ascaridida and Spirurida (suborders Camallanina and Spirurina), and the Adenophorea covers the order Enoplida, with the Dioctophymina and Trichinellina now treated as separate suborders. The aim of the book remains "to summarize and synthesize knowledge of the basic features of the development and transmission of parasitic nematodes of vertebrates, and to place this information in the context of the modern classification as found in the CIH Keys to the Nematode Parasites of Vertebrates " [but see the 2 departures from these keys as noted above]. Nematode parasites of humans, domestic animals and wildlife (including fish) are covered. Each chapter or part begins with an overview of the mode of feeding, habitat and life cycles of the group. This is followed by descriptions and illustrations of larval stages of named specific examples. The number of illustrations has been increased from 33 to 43. Comprehensive bibliographies appear at the end of the sections on each order or suborder.
On the basis of known DNA sequences of Dirofilaria repens and D. immitis we designed specific primers for the amplification by Polymerase Chain Reaction (PCR) of the DNA from the two species. The PCR-based identification was found to be unambiguous and allowed specific diagnosis of microfilariae in blood samples, of developing larvae in the mosquito vector and of immature adults in bioptic material, overcoming the serious constraints of the morphological separation of these filarial parasites at the pre-adult stages. The technique was found to be very sensitive and applicable to samples stored either dry or in various preservation media, with the exception of formalin. The reliable identification of D. repens and D. immitis from bioptic material is expected to greatly enhance the chances of detecting human infections and to further clarify the role of the two parasites as pathogens of man. The possibility of routine identification of developing larvae in the vector will substantially improve the perspectives for epidemiological investigations, particularly in Southern European regions, such as Italy, where the two nematode species are largely sympatric.
The diagnosis of canine heartworm infection is based upon the presence of circulating Dirofilaria immitis microfilariae or on techniques for the detection of serum antibodies or antigens. In the first of these, discrimination between D. immitis, D. repens and Acanthocheilonema dracunculoides microfilariae is based upon the acid phosphatase histochemical stain. In this paper, we propose an alternative technique for histochemical staining using a commercial kit test of naphthol-AS-OL (Leucognost-SP). This offers the advantages of speed and simplicity as compared to the standard Barka procedure.
Canine dirofilariasis caused by Dirofilaria immitis is usually diagnosed by specific antigen testing and/or identification of microfilariae. However, D. immitis and at least six other filariae can produce canine microfilaremias with negative heartworm antigen tests. Discriminating these can be of clinical importance. To resolve discordant diagnoses by two diagnostic laboratories in an antigen-negative, microfilaremic dog recently imported into the US from Europe we developed a simple molecular method of identifying different microfilariae, and subsequently validated our method against six different filariae known to infect dogs by amplifying ribosomal DNA spacer sequences by polymerase chain reaction using common and species-specific primers, and sequencing the products to confirm the genotype of the filariae. We identified the filaria in this dog as D. repens. This is the first case of D. repens infection in the United States. Additionally, we examined microfilariae from five additional antigen-negative, microfilaremic dogs and successfully identified the infecting parasite in each case. Our diagnoses differed from the initial morphological diagnosis in three of these cases, demonstrating the inaccuracy of morphological diagnosis. In each case, microfilariae identified morphologically as A. reconditum were identified as D. immitis by molecular methods. Finally, we demonstrated that our PCR method should amplify DNA from at least two additional filariae (Onchocerca and Mansonella), suggesting that this method may be suitable for genotyping all members of the family Onchocercidae.
Species-specific PCR amplifications of D. immitis and D. repens 12S rDNA and of A. reconditum coxI. (a) PCR amplification using D
- Fig
Fig. 2. Species-specific PCR amplifications of D. immitis and D.
repens 12S rDNA and of A. reconditum coxI. (a) PCR amplification
using D. immitis 12S rDNA specific primers on D. immitis DNA (lanes
- M Casiraghi
M. Casiraghi et al. / Veterinary Parasitology 141 (2006) 368–372
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Histochemical differentiation of Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema dracunculoides microfilariae by staining with a commercial kit
- M A Peribáñez
- J Lucientes
- S Arce
- M Morales
- J A Castello
- M J Grazia
Peribáñez, M.A., Lucientes, J., Arce, S., Morales, M., Castello, J.A.,
Grazia, M.J., 2001. Histochemical differentiation of Dirofilaria
immitis, Dirofilaria repens and Acanthocheilonema dracunculoides microfilariae by staining with a commercial kit, Leucognost-SP 1. Vet. Parasitol. 102, 173-175.