André Pereira

Multiple Phlebovirus (Bunyaviridae) genetic groups detected in Rhipicephalus, Hyalomma and Dermacentor ticks from southern Portugal

In the last decade, various research groups have reported a large diversity of new tick-borne phleboviruses, mostly prompted by the discovery of important human pathogens such as the Heartland and severe fever with thrombocytopenia syndrome viruses. Since these analyses have rarely been conducted using ticks collected from Southern Europe, this study was carried out so as to bring new insights into the diversity of phleboviruses circulating in Southern Portugal. Tick specimens were collected from the vegetation (questing ticks) or directly from animals (feeding ticks), and the majority analysed in pools using a detection strategy targeting the large (L) viral genomic segment. A high number of pools revealed the presence of phebovirus sequences, regardless of gender (male/female), origin (questing/feeding) or even species of the tick analysed. These sequences apparently formed three different groups in phylogenetic trees, and encoded L proteins characterized by group-specific amino acid residues. Furthermore, under the conditions used, these viruses failed to replicate in both Vero and DH82 cells. The impact these viruses may have on human/animal health will be addressed in the future.

Tick-borne bacteria and protozoa detected in ticks collected from domestic animals and wildlife in central and southern Portugal

Ticks are vectors of many human and animal pathogens. The aim of this study was to screen bacteria and protozoa from ticks infesting domestic animals and wildlife collected in central and southern Portugal. A total of 593 ticks, comprising 465 (78.4%) adults, 122 (20.6%) nymphs, and six (1.0%) larvae, were collected from 283 hosts of 25 different species (4 domestic and 21 wild). Overall, the analysis of DNA extracts prepared from ticks collected from hosts of 11 different species in the districts of Castelo Branco, Portalegre, Lisboa, Setúbal, Beja and Faro, revealed the presence of genomic sequences from Anaplasma sp., A. ovis, Babesia sp., relapsing fever-like Borrelia sp., Ehrlichia spp., Rickettsia aeschlimannii, Ri. helvetica, Ri. massiliae, Ri. raoultii, Ri. slovaca, Candidatus Ri. barbariae, Theileria annulata and T. ovis, in specimens of Dermacentor marginatus, Hyalomma lusitanicum, Hy. marginatum, Rhipicephalus bursa and Rh. sanguineus sensu lato. The obtained results suggest the circulation of a wide variety of infectious agents, some of zoonotic concern, in hard ticks from Portugal. Further studies should be conducted to better characterize (both genetically and phenotypically) the putative novel microorganisms detected, both in what regards their potential pathogenity towards vertebrates, and to assist the implementation of effective control strategies for the management of ticks and human and animal tick-borne pathogens.

Evaluation of oxfendazole in the treatment of zoonotic Onchocerca lupi infection in dogs

The genus Onchocerca encompasses parasitic nematodes including Onchocerca volvulus, causative agent of river blindness in humans, and the zoonotic Onchocerca lupi infecting dogs and cats. In dogs, O. lupi adult worms cause ocular lesions of various degrees while humans may bear the brunt of zoonotic onchocercosis with patients requiring neurosurgical intervention because of central nervous system localization of nematodes. Though the zoonotic potential of O. lupi has been well recognized from human cases in Europe, the United States and the Middle East, a proper therapy for curing this parasitic infection in dogs is lacking. To evaluate the efficacy of oxfendazole, 11 out of the 21 client-owned dogs (21/123; 17.1%) positive for skin-dwelling O. lupi microfilariae (mfs), were enrolled in the efficacy study and were treated with oxfendazole (50 mg/kg) per OS once a day for 5 (G2) or 10 (G3) consecutive days or were left untreated (G1). The efficacy of oxfendazole in the reduction of O. lupi mfs was evaluated by microfilarial count and by assessing the percentage of mfs reduction and mean microfilaricidal efficacy, whereas the efficacy in the reduction of ocular lesions was evaluated by ultrasound imaging. All dogs where subjected to follow-ups at 30 (D30), 90 (D90) and 180 (D180) days post-treatment. The percentage of reduction of mfs was 78% for G2 and 12.5% for G3 at D180. The mean microfilaricidal efficacy of oxfendazole in the treatment of canine onchocercosis by O. lupi at D30, D90 and D180 was 41%, 81% and 90%, in G2 and 40%, 65% and 70%, in G3, respectively. Retrobulbar lesions did not reduce from D0 to D180 in control group (dogs in G1), whereas all treated dogs (in G2 and G3) had slightly decreased ocular lesions. Percentage of reduction of ocular lesions by ultrasound examination was 50% and 47.5% in G2 and G3 at D180, respectively. Despite the decrease in ocular lesions in all treated dogs (G2 and G3), oxfendazole was ineffective in reducing ocular lesions and skin-dwelling O. lupi mfs in treated dogs (G2 and G3) in a six-month follow-up period. Here we discuss the need for more reliable diagnostic techniques and efficient treatment protocols to better plan future intervention strategies.

Cercopithifilaria sp. II in Vulpes vulpes: new host affiliation for an enigmatic canine filarioid

Cercopithifilaria bainae and Cercopithifilaria grassii (Spirurida, Onchocercidae) are filarioids inhabiting the skin of dogs worldwide. The microfilariae of a third species namely, Cercopithifilaria sp. II sensu Otranto et al. 2013, have been morphologically and molecularly characterized but scientific knowledge of this parasite is minimal. The first case of infection of a red fox (Vulpes vulpes) with the filarioid Cercopithifilaria sp. II is herein described in Castro Marim, Portugal. Microfilariae from skin sediment of the fox’s ear were morphological characterized, and the identification was confirmed molecularly in samples from skin sediment, skin samples, and from Rhipicephalus sanguineus group ticks collected from the animal (99% homology with Cercopithifilaria sp. II). Studies should evaluate if red foxes might play a role in the maintenance and distribution of Cercopithifilaria sp. II infection in dog populations.

Evaluation of Different Methods for the Estimation of the Bitumen Fatigue Life with DSR Testing

Asphalt fatigue cracking is one of the phenomena that contribute most to degradation of road pavements and it may initiate within the bitumen or at the bitumen-aggregate interface. The cohesive cracking resistance can be evaluated with bitumen testing. Commonly, the Dynamic Shear Rheometer (DSR) is used for bitumen testing. This paper presents an evaluation of different methods proposed in literature for the estimation of the bitumen fatigue life. A neat and a polymer modified bitumen (PMB) were tested with time sweep tests (continuous and discontinuous loading) and with incremental load amplitude (linear amplitude sweep test). The results are analysed with the traditional approach (Nf,50 corresponding to 50 % initial modulus reduction) and other methodologies, namely the Ratio of Dissipated Energy Change (RDEC) and the Viscoelastic Continuum Damage (VECD) approach. The results obtained showed, as expected, that the PMB has a higher resistance to fatigue than the neat bitumen. The test conditions and the method used to evaluate the fatigue resistance lead to significant differences in the estimated bitumen fatigue life. The plateau value (RDEC) shows very good correlation with Nf,50 obtained from constant strain amplitude tests, regardless of the type of bitumen or test conditions. The fatigue life parameters obtained from the linear amplitude sweep test is very sensitive to the analysis method. Healing during non-loading periods has a large effect on the PMB fatigue life while no effect in the neat bitumen fatigue life for small to intermediate rest periods.