Rapid differentiation of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood by real-time PCR coupled to high resolution melting analysis

Albonico, F.; Loiacono, Monica; Gioia, G.; Genchi, C.; Genchi, Marco; Mortarino, Michele

doi:10.1016/j.vetpar.2013.11.027

Cited by 31 publications

(22 citation statements)

References 17 publications

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“…Here, the ability of the MALDI-TOF MS to detect filariae in mosquitoes was evaluated using qPCR as a “gold standard”/reference. The reliability of nucleic acid amplification techniques for filariae detection in vectors has been addressed in a number of studies [ 25 , 26 , 29 ]. These studies showed that these PCR assays had high sensitivity and specificity toward the detection of the filariae in mosquitoes.…”

Section: Discussionmentioning

confidence: 99%

Assessment of MALDI-TOF mass spectrometry for filariae detection in Aedes aegypti mosquitoes

et al. 2017

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Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is an emerging tool for routine identification of bacteria, archaea and fungi. It has also been recently applied as an accurate approach for arthropod identification. Preliminary studies have shown that the MALDI-TOF MS was able to differentiate whether ticks and mosquitoes were infected or not with some bacteria and Plasmodium parasites, respectively. The aim of the present study was to test the efficiency of MALDI-TOF MS tool in distinguishing protein profiles between uninfected mosquitoes from specimens infected by filarioid helminths. Aedes aegypti mosquitoes were engorged on microfilaremic blood infected with Dirofilaria immitis, Brugia malayi or Brugia pahangi. Fifteen days post-infective blood feeding, a total of 534 mosquitoes were killed by freezing. To assess mass spectra (MS) profile changes following filariae infections, one compartment (legs, thorax, head or thorax and head) per mosquito was submitted for MALDI-TOF MS analysis; the remaining body parts were used to establish filariae infectious status by real-time qPCR. A database of reference MS, based on the mass profiles of at least two individual mosquitoes per compartment, was created. Subsequently, the remaining compartment spectra (N = 350) from Ae. aegypti infected or not infected by filariae were blind tested against the spectral database. In total, 37 discriminating peak masses ranging from 2062 to 14869 daltons were identified, of which 17, 11, 12 and 7 peak masses were for legs, thorax, thorax-head and head respectively. Two peak masses (4073 and 8847 Da) were specific to spectra from Ae. aegypti infected with filariae, regardless of nematode species or mosquito compartment. The thorax-head part provided better classification with a specificity of 94.1% and sensitivity of 86. Author summaryFilariosis is a disease group affecting humans and animals, caused by nematode parasites of the family Onchocercidae, superfamily Filarioidea. These parasites can be transmitted, essentially, by mosquitoes during blood meals of infected female specimens. Screening vectors for these filariae currently relies on time-and resource-consuming methods such as dissection and polymerase chain reaction-based methods. Here, we applied matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to assess whether this tool can detect changes in the protein profiles of Aedes aegypti infected with filarioid helminths compared to those uninfected by testing different parts of mosquitoes. First a reference mass spectra database from Ae. aegypti infected or not infected by filariae was created using MS from 47 specimen compartments. Then we tested the remaining mass spectra (350 x 4) in a blind validation test. Regardless of filariae species, the best correct classification rate was obtained from the thorax-head part with a specificity of 94.1% and sensitivity of 86.6, 71.4 and 68.7% for non-infected and D. immitis, B. malayi and B. pahang...

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Section: Discussionmentioning

confidence: 99%

Assessment of MALDI-TOF mass spectrometry for filariae detection in Aedes aegypti mosquitoes

et al. 2017

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“…PCR testing was carried out on 48 randomly selected whole blood samples using previously reported primers for Diro laria mitochondrial cytochrome oxidase gene, subunit I 21 . After optimisation of the nucleic acid extraction and PCR protocols using whole blood from dogs con rmed to be infected using microscopy, 100 ul of whole blood was diluted with 100 ul PBS, prior to extraction of genomic DNA using a QIAmp blood mini kit (Qiagen, Hildon, Germany) with elution into 200 ul AE buffer.…”

Section: Methodsmentioning

confidence: 99%

Prevalence of Infection With Dirofilaria Immitis in Cats in Townsville, Australia

Adagra

Squires

Busst

et al. 2020

Preprint

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Objective Dirofilaria immitis commonly infects Australian dogs. Studies on the prevalence of infection by this parasitic helminth in Australia cats are rare and relatively old. Data obtained from other countries would suggest a likely prevalence of 4.7–16%. The current study aimed to determine the prevalence of D. immitis in an endemic region of Australia by antigen, antibody and PCR testing. Methods 172 healthy cats over 6 months of age from the Townsville region of Australia were tested for D. immitis specific antibodies and antigen using a commercially available kit. 50 samples were subsequently retested using a second commercially available antibody kit. 48 of these samples were checked for D. immitis DNA using PCR. Results No cat tested positive on any test. The Ausvet Epitools epidemiology calculator was used to calculate prevalences. Maximum antigen (1.27%), antibody (2.1%) and PCR (2.1%) prevalences were calculated. Conclusion Our results suggest that the prevalence of heartworm infection in pet cats in this region of Australia is lower than expected based on data from other areas around the world.

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“…A real-time PCR targeting the mitochondrial COX 1 gene [ 41 ] of D. immitis and D. repens was designed using GenScript ( www.genscript.com/ssl-bin/app/primer ). Forward and reverse primers and the Taqman® probe were as follows: Diro-f: 5′-GGT GTT TGG GAT TGT TAG TGA A-3′; Diro-r: 5′-CAG CAA TCC AAA TAG AAG CAA-3′; Diro-p: 5′-FAM-TCT GGC CAA ACA AAC GAT CCT TAT CA-TAMRA-3′.…”

Section: Methodsmentioning

confidence: 99%

Development of Dirofilaria immitis and Dirofilaria repens in Aedes japonicus and Aedes geniculatus

et al. 2017

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BackgroundThe mosquito-borne filarial nematodes Dirofilaria immitis and Dirofilaria repens primarily affect dogs but also cats, causing heartworm disease or subcutaneous dirofilariosis, respectively, and both may also cause zoonotic diseases in humans. Several mosquito species have been reported as competent vectors for these nematodes, but no data are available for the invasive mosquito species Aedes japonicus (Theobald, 1901). The objective of this study was to describe the development of both D. immitis and D. repens under standardised experimental laboratory conditions in mosquitoes.MethodsFor this purpose, both a laboratory strain and field-collected individuals of the invasive mosquito species Ae. japonicus and, for comparative purposes, a laboratory strain of Aedes geniculatus, a rare indigenous species sharing habitats with Ae. japonicus, and of the tropical species Aedes aegypti were used. Anticoagulated microfilariaemic blood was fed at a density of 3000 mf/ml to mosquitoes with a hemotek system. Blood-fed mosquitoes were incubated at 27 °C and 85% relative humidity, and specimens were dissected under the microscope at pre-set time points to observe developmental stages of both Dirofilaria species. Additionally, real-time PCRs were carried out in some microscopically negative samples to determine the infection rates.ResultsIn field-collected Ae. japonicus infectious L3 larvae of both D. immitis and D. repens developed, rendering this mosquito species an efficient vector for both filarial species. Additionally, Ae. geniculatus was shown to be an equally efficient vector for both filarial species. Aedes japonicus mosquitoes from a laboratory colony were refractory to D. immitis but susceptible to D. repens, whereas Ae. aegypti was refractory to both filarial species.ConclusionsTo our knowledge, Aedes japonicus was for the first time shown to be an efficient vector for both D. immitis and D. repens, indicating that this invasive and locally highly abundant species may contribute to a transmission of filarial worms. The data emphasize the necessity to perform vector competence studies with local mosquito populations as basis for risk assessments. We further demonstrated that detection of filarial DNA in a mosquito species alone does not allow to draw reliable conclusions with regard to its vector competence.

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Rapid differentiation of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood by real-time PCR coupled to high resolution melting analysis

Cited by 31 publications

References 17 publications

Assessment of MALDI-TOF mass spectrometry for filariae detection in Aedes aegypti mosquitoes

Assessment of MALDI-TOF mass spectrometry for filariae detection in Aedes aegypti mosquitoes

Prevalence of Infection With Dirofilaria Immitis in Cats in Townsville, Australia

Development of Dirofilaria immitis and Dirofilaria repens in Aedes japonicus and Aedes geniculatus

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