Molecular Approach for the Diagnosis of Blood and Skin Canine Filarioids

Abstract

The zoonotic Onchocerca lupi and tick-transmitted filarioids of the genus Cercopithifilaria remain less well known due to the difficulties in accessing to skin samples as target tissues. Here, we proposed a molecular approach reliying on multiplex qPCR assays that allow the rapid identification of filarioids from canine blood, skin, and tick samples. This includes two newly developed duplex qPCR tests, the first one targeting filarial and C. grassii DNA (CanFil-C. grassii). and the second qPCR assay designed for the detection of Cercopithifilaria bainae and Cercopithifilaria sp. II DNAs (C. bainae-C.spII). The third one is a triplex TaqMan cox 1 assay targeting DNA of blood microfilariae (e.g., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum). The novel duplex qPCRs developed were validated in silico and by screening of known DNA collection. The qPCR assays were also used for screening the blood and tick samples of 72 dogs from Algeria. This allowed the identification of canine filariasis infection with 100% of specificity and 89.47% and 100% of sensitivity from naturally infected blood and tick samples, respectively. The prevalences of 26.39% for D. immitis and 5.56% for both D. repens and A. reconditum were reported in blood and tick samples. Cercopithifilaria DNAs were detected only in tick samples, with a prevalence of 4.17% and 5.56% for C. bainae and Cercopithifilaria sp. II, respectively. Co-infections were diagnosed in 6.94% and 13.89% of blood and tick samples, respectively. Whereas all samples were negative for C. grassii DNA. The use of engorged ticks instead of blood and skin samples could be an easier option for the surveillance of all canine filarioids herein investigated. The multiplex qPCR assays herein validated were shown to be useful in the detection of filarial co-infections by overcoming sequencing of positive samples.

Detection of Dirofilaria immitis via integrated serological and molecular analyses in coyotes from Texas, United States

“…However, there are important limitations to consider when interpreting the results from serologic analyses, including the occurrence of false-positive and false-negative results. Molecular approaches have been developed as an alternative and/or complementary diagnostic method for the detection of D. immitis in companion animals ( Laidoudi et al, 2020a , 2020b ). However, the diagnostic potential of molecular tools for the screening of heartworm disease in wild carnivores has not been fully tapped.…”

“…Even though molecular techniques present some limitation, these methods can provide a rapid, sensitive, and accurate diagnoses, and prevent the occurrence of false-positive results due to cross-reactivity. Several studies have reported the use of molecular approaches targeting conserved DNA regions in the diagnosis of numerous filarial nematodes ( Laidoudi et al, 2020a , 2020b ; Panetta et al, 2021 ; Rishniw et al, 2006 ). As expected, with the present study the real-time PCR technique had a higher positive rate than conventional PCR and seems to be an alternative diagnostic method for detecting D .…”

Haematological and biochemical abnormalities in hunting dogs infected with Acanthocheilonema reconditum, associated risk factors, and a European overview

“…Moreover, the routine use of in-clinic rapid tests for the detection of D. immitis antigens in blood samples has facilitated the diagnosis of this major filarial worm in dogs, but has likely contributed to a reduction in the opportunity to diagnose A. reconditum infections through the Knott's test that is a time-consuming procedure (Magnis et al 2013;Evans et al 2019). Recently, the molecular methods that allow to achieve a diagnosis at species level for A. reconditum have increased compared to testing modalities used in the past (Laidoudi et al 2020a(Laidoudi et al , 2020b, but as they are cannot yet be considered a routine diagnostic resource.…”

Identity of Microfilariae Circulating in Dogs from Western and South-Western Romania in the Last Decade

“…Occult infections with D. immitis can represent about 20-30% of all cases. In this situation, it is recommended to perform a method of detection/identification of microfilariae (Knott) as well as an antigen test [6,10,37,43,[55][56][57].…”