Major antigen and paramyosin proteins as candidate biomarkers for serodiagnosis of canine infection by zoonotic Onchocerca lupi

Onchocerca lupi (Spirurida: Onchocercidae) is a filarial worm parasitizing domestic carnivores and humans. Adult nematodes usually localize beneath in the sclera or in the ocular retrobulbar of infected animals, whilst microfilariae are found in the skin. Therefore, diagnosis of O. lupi is achieved by microscopic and/or molecular detection of microfilariae from skin biopsy and/or surgical removal of adults from ocular tissues of infected hosts. An urgent non-invasive diagnostic tool for the diagnosis of O. lupi in dog is mandatory. In this study, an immunoproteomic analyses was performed using a combination of immunoblotting and mass spectrometry techniques. Onchocerca lupi major antigen (Ol-MJA) and paramyosin (Ol-PARA) proteins were identified as potential biomarkers for serodiagnosis. Linear epitopes were herein scanned for both proteins using high-density peptide microarray. Sera collected from dog infected with O. lupi and healthy animal controls led to the identification of 11 immunodominant antigenic peptides (n = 7 for Ol-MJA; n = 4 for Ol-PARA). These peptides were validated using sera of dogs uniquely infected with the most important filarioids infesting dogs either zoonotic (Dirofilaria repens, Dirofilaria immitis) or not (Acanthocheilonema reconditum and Cercopithifilaria bainae). Overall, six antigenic peptides, three for Ol-MJA and for Ol-PARA, respectively, were selected as potential antigens for the serological detection of canine O. lupi infection. The molecular and proteomic dataset herein reported should provide a useful resource for studies on O. lupi toward supporting the development of new interventions (drugs, vaccines and diagnostics) against canine onchocercosis.

Molecular Approach for the Diagnosis of Blood and Skin Canine Filarioids

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.

Ταυτοποίηση, φυλογένεση και καθορισμός των παραγόντων επικινδυνότητας των ειδών του γένους Cercopithifilaria, που προσβάλουν το σκύλο και τους κρότωνες στην Ελλάδα

Τα είδη του γένους Cercopithifilaria, που προσβάλλουν τον σκύλο, δεν έχουν μελετηθεί εκτενώς μέχρι σήμερα, αν και έχουν διαπιστωθεί σποραδικά σε αρκετές χώρες. Η φιλαρίωση αυτή παρέμεινε για αρκετά χρόνια άγνωστη, λόγω της υποδόριας εντόπισης των ενηλίκων παρασίτων και της πρακτικής δυσκολίας ανίχνευσης των μικροφιλαριών (Otranto et al., 2013a). Ο βιολογικός κύκλος είναι έμμεσος, με ενδιάμεσο ξενιστή τους κρότωνες της οικογένειας Ixodidae (Winkhardt, 1980, Petit et al., 1988, Bain et al., 1986, Spratt and Haycock, 1988). Στο σκύλο έχουν ταυτοποιηθεί 3 είδη, τα Cercopithifilaria bainae, Cercopithifilaria grassi και Cercopithifilaria sp. II. Η παρουσία των ενηλίκων παρασίτων στον σκύλο και η εντόπιση των μικροφιλαριών στην επιδερμίδα, έχουν συσχετισθεί με διάφορες παθολογικές καταστάσεις όπως η δερματοπάθεια, υποδόρια κύστη και η αρθροπάθεια (Otranto et al., 2011a, Otranto et al., 2012a, Boyd et al., 2019, Soares et al., 2020a). Σήμερα, τα δεδομένα σχετικά με την Cercopithifilaria spp., που προσβάλλει τον σκύλο, είναι περιορισμένα παγκοσμίως. Στη χώρα μας το είδος Cercopithifilaria bainae έχει βρεθεί σε σκύλους σε μια πιλοτική έρευνα, που πραγματοποιήθηκε μόνο σε μια περιοχή της Θράκης (Otranto et al., 2012b), καθώς και σε κρότωνες, που συλλέχθηκαν από σκύλους και εξετάστηκαν, μόνο με τη χρήση μοριακών μεθόδων (Latrofa et al., 2017b). Ωστόσο, υπάρχει ένδεια επιστημονικών δεδομένων, σχετικά με τη γεωγραφική κατανομή, την ταυτοποίηση των ειδών και τη γενετική ποικιλομορφία του παρασίτου αυτού στην Ελλάδα. Στην παρούσα μελέτη με σκοπό τη διερεύνηση της γεωγραφικής κατανομής του παρασίτου, συλλέχθηκαν δείγματα ιστοτεμαχίων δέρματος και κρότωνες από σκύλους. Ταυτόχρονα με τη συλλογή των δειγμάτων, πραγματοποιήθηκε η καταγραφή όλων των ατομικών στοιχείων του κάθε σκύλου με σκοπό τη περαιτέρω στατιστική ανάλυση. Η μελέτη περιελάμβανε τη μικροσκοπική εξέταση των ιστοτεμαχίων δέρματος, με σκοπό την ανεύρεση των δερματικών μιρκοφιλαριών και τη μορφολογική ταυτοποίηση τους. Συνολικά από τους 500 σκύλους οι 70 (14%, CI 95%, 11 to 17%) βρέθηκαν να είναι μολυσμένοι με δερματικές φιλάριες του γένους Cercopithifilariα. Συγκεκριμένα, ταυτοποιήθηκαν 2 είδη, το είδος Cercopithifilaria bainae και Cercopithifilaria sp. II. Επίσης, ταυτοποιήθηκε μορφολογικά ένα επιπλεόν αταυτοποίητο είδος Cercopithifilaria. Ακόμα, πραγματοποιήθηκε η μοριακή ανάλυση των ιστοτεμαχίων δέρματος, καθώς και ενός αντιπροσωπευτικού δείγματος από τους συλλεχθέντες κρότωνες, με τη μέθοδο της αλυσιδωτής αντίδρασης της πολυμεράσης (PCR), με σκοπό την ενίσχυση του μιτοχονδριακού γονιδίου cox1 (~304 ζεύγη βάσεων). Τα δείγματα, τόσο των ιστοτεμαχίων δέρματος, όσο και των κροτώνων, που βρέθηκαν θετικά στην παρουσία Cercopithifilaria, με τη μέθοδο της PCR, επεξεργάσθηκαν περαιτέρω με τη μέθοδο Sanger, κατά την οποία προσδιορίστηκε η νουκλεοδιτική αλληλουχία του γονιδίου cox1. Στη συνέχεια, επεξεργάστηκαν περαιτέρω, με σκοπό την πολλαπλή στοίχιση τους, χρησιμοποιώντας το ειδικό αυτοματοποιημένο λογισμικό βιοπληροφορικής BLAST (Basic Logic Alignment Serach Tool). Τα αποτελέσματα, που προέκυψαν από την ανάλυση, έδειξαν πως υπήρχε υψηλή νουκλεοτιδική συγγένεια με τις αλληλουχίες cox1 (99% με 100%) τού είδους C. bainae. Από τη μέθοδο BLAST προσδιορίστηκαν 7 συνολικά διαφορετικοί απλότυποι. Ο πιο συχνός απλότυπος ήταν ο απλότυπος I, ο απλότυπος XVIII, και εν συνεχεία ο απλότυπος II και IX. Επιπροσθέτως, ταυτοποιήθηκαν τρεις νέοι απλότυποι, XIX, XX και XXI με υψηλή νουκλεοτιδική συγγένεια (99,58%) με τις αλληλουχίες cox1 του είδους C. bainae. Οι αλληλουχίες, που προέκυψαν από τη μέθοδο Sanger, επεξεργάστηκαν με σκοπό τη φυλογενετική ανάλυση. Το φυλογενετικό δένδρο που προέκυψε, ομαδοποίησε όλους τους απλοτύπους, που βρέθηκαν στην παρούσα εργασία, σε ένα μονοφυλετικό κλάδο του είδους C. bainae, γεγονός που υποστηρίζεται επιπλέον, από τη σχετικά υψηλή τιμή του παράγοντα bootstrap (99%). Από τη στατιστική ανάλυση για τη διερεύνηση των διαφόρων παραγόντων επικινδυνότητας, προέκυψε πως διάφοροι ατομικοί παράγοντες των σκύλων σχετίζονται και προδιαθέτουν για μόλυνση, από τα είδη Cercopithifilaria. Ανάμεσα στους διαφόρους παράγοντες επικινδυνότητας συμπεριλαμβάνονται, ο χώρος διαβίωσης, το βάρος, το ιστορικό προσβολής από κρότωνες, καθώς και η χρήση εξω-παρασιτοκότνων σκευασμάτων. Η περαιτέρω μελέτη της επιδημιολογίας και γεωγραφικής κατανομής των παρασίτων Cercopithifilaria, καθώς και η διερεύνηση των υποκείμενων μηχανισμών παθογένειας, του βιολογικού κύκλου και της μεγάλης γενετικής ποικιλομορφίας είναι αναγκαία, με σκοπό να απαντηθούν αρκετά ερευνητικά ερωτηματικά. Τέλος, τονίζεται η ανάγκη για ενημέρωση των κτηνίατρων της χώρας, σχετικά με την παρουσία των νηματωδών παρασίτων στους σκύλους και τις διαθέσιμες μεθόδους διάγνωσης και πρόληψης.

Detection of Canine Vector-Borne Filariasis and Their Wolbachia Endosymbionts in French Guiana

In French Guiana, canine heartworm disease is well known, but the diversity of filarial parasites of dogs remains largely unknown. A total of 98 canine blood samples from Cayenne and Kourou were assessed by a blood wet mount preparation, heartworm antigen test and molecular exploration of filarioid and Wolbachia DNAs, followed by a multiplex species-specific qPCR’s identification and a subsequent sequencing analysis. Thereafter, a phylogeny based on maximum likelihood was carried out to facilitate specific identification. Five dogs were microfilaremic. Heartworm antigens were detected in 15 (15.3%) dogs. Of these, six (6.1%) were considered as occult infections as neither microfilariae nor Dirofilaria immitis DNA were detected. The 11 (11.2%) D. immitis isolates corresponded to a low virulent strain. Six of the D. immitis isolates were positive for Wolbachia endosymbionts of D. immitis belonging to the clade C DNA. Acanthocheilonema reconditum DNA was detected in 3 (3.1%) samples. Of these latter, one was found co-infected with the Brugia sp. genotype and the DNA of the clade D of the Wolbachia endosymbiont of Brugia species. This latter was also detected in two filarioid DNA-free samples. Finally, two samples were positive for Cercopithifilaria bainae genotype, which is distinct from those identified in Europe. The present study highlights the urgent need to implement chemoprophylaxis associated with anti-Wolbachia drugs to control these potential zoonoses.

Microfilaremia by Cercopithifilaria bainae in a dog from the central western region of Brazil: case report

ABSTRACT Cercopithifilaria bainae is a nematode belonging to the family Onchocercidae that parasitizes the subcutaneous tissue of dogs. Its transmission occurs through the tick Rhipicephalus sanguineus and its geographical distribution overlaps that of this vector. The present study reports the detection of microfilaremia by C. bainae in an eight-year-old male dog that presented anorexia, hyperthermia, motor incoordination, mydriasis, a nodule in the left testicle and concomitant infection by Ehrlichia sp. Blood samples were analyzed using microscopy, PCR and DNA sequencing. Microfilariae measuring 150±5.5μm in length and 7±1.8μm in width were retrieved. The DNA sequence exhibited 98% identity with C. bainae sequences available in Genbank. This is the first report of microfilaremia by C. bainae in a dog in the central western region of Brazil.

Detection of Cercopithifilaria bainae infection in shelter dogs and ticks in Oklahoma, USA

Background Cercopithifilaria bainae is a filarioid nematode of dogs. Infection with the parasite was not reported in the USA until 2017, when a dog with skin lesions in Florida was diagnosed. Brown dog ticks, Rhipicephalus sanguineus sensu lato, are the purported tick vectors, and are widespread in the USA. Therefore, C. bainae is likely present in additional states. Here, we tested dogs and ticks in Oklahoma for evidence of C. bainae infection. Methods Dermal punch biopsies were opportunistically collected from municipal shelter and client-owned dogs. Multiple skin samples collected from interscapular and head regions were tested by saline sedimentation to recover live microfilariae for morphometric identification and by PCR to amplify a 330 bp region of the filarioid 12S rRNA gene. Also, ticks observed on surveyed dogs were collected, identified to species level, and tested for filarioid DNA. Results A total of 496 saline sedimentations were performed on 230 shelter and 20 client-owned dogs. Cercopithifilaria bainae infections were identified in 2.6% (6/230) of shelter dogs by morphometry of microfilariae in sedimentations and/or amplification of DNA from skin. DNA sequences amplified from PCR positive skin samples were 99–100% identical to C. bainae reported in Italy. All skin samples from client-owned dogs were negative for filarioid infection by saline sedimentation and PCR. A total of 112 ticks, comprised of four species, were collected. Two of 72 R. sanguineus s.l., both engorged females found attached to a C. bainae infected dog, harbored C. bainae DNA (99–100% identity). One attached R. sanguineus s.l. male on the same dog harbored filarioid DNA sequence which was difficult to interpret at numerous base-pair locations, but was closest in identity (~80%) to C. bainae . Conclusions The distribution of C. bainae is more widespread than previously known. Here, we document C. bainae infections in dogs and DNA in brown dog ticks in Oklahoma for the first time. As brown dog ticks are commonly found throughout the USA, veterinarians in this region should consider C. bainae infection as a differential diagnosis in canine patients with dermatitis or polyarthritis.

Detection of Cercopithifilaria bainae infection in shelter dogs and ticks in Oklahoma, USA

Background Cercopithifilaria bainae is a filarioid nematode of dogs. Infection with the parasite was not reported in the USA until 2017, when a dog with skin lesions in Florida was diagnosed. Brown dog ticks, Rhipicephalus sanguineus sensu lato, are the purported tick vectors, and are widespread in the USA. Therefore, C. bainae is likely present in additional states. Here, we tested dogs and ticks in Oklahoma for evidence of C. bainae infection. Methods Dermal punch biopsies were opportunistically collected from municipal shelter and client-owned dogs. Multiple skin samples collected from interscapular and head regions were tested by saline sedimentation to recover live microfilariae for morphometric identification and by PCR to amplify a 330 bp region of the filarioid 12S rRNA gene. Also, ticks observed on surveyed dogs were collected, identified to species level, and tested for filarioid DNA. Results A total of 496 saline sedimentations were performed on 230 shelter and 20 client-owned dogs. Cercopithifilaria bainae infections were identified in 2.6% (6/230) of shelter dogs by morphometry of microfilariae in sedimentations and/or amplification of DNA from skin. DNA sequences amplified from PCR positive skin samples were 99–100% identical to C. bainae reported in Italy. All skin samples from client-owned dogs were negative for filarioid infection by saline sedimentation and PCR. A total of 112 ticks, comprised of four species, were collected. Two of 72 R. sanguineus s.l., both engorged females found attached to a C. bainae infected dog, harbored C. bainae DNA (99–100% identity). One attached R. sanguineus s.l. male on the same dog harbored filarioid DNA sequence which was difficult to interpret at numerous base-pair locations, but was closest in identity (~80%) to C. bainae . Conclusions The distribution of C. bainae is more widespread than previously known. Here, we document C. bainae infections in dogs and DNA in brown dog ticks in Oklahoma for the first time. As brown dog ticks are commonly found throughout the USA, veterinarians in this region should consider C. bainae infection as a differential diagnosis in canine patients with dermatitis or polyarthritis.