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Wolbachia has an obligatory mutualistic relationship with many onchocercid nematodes of the subfamilies Dirofilariinae and Onchocercinae. Till date, no attempts have been made for the in vitro cultivation of this intracellular bacterium from the filarioid host. Hence, the current study attempted cell co-culture method using embryonic Drosophila S2 and the LD cell lines to cultivate Wolbachia from Dirofilaria immitis microfilariae (mfs) harvested from infected dogs. Microfilariae (mfs = 1500) were inoculated in shell vials supplemented with Schneider medium using both cell lines. The establishment and multiplication of the bacterium were observed during the initial inoculation, at day 0 and before every medium change (from days 14 to 115). An aliquot (50 µl) from each time point was tested by quantitative real-time PCR (qPCR). Comparing the average of Ct values, obtained by the tested parameters (i.e., LD/S2 cell lines and mfs with/without treatment), the S2 cell line without mechanical disruption of mfs provided the highest Wolbachia cell count by qPCR. Despite the maintenance of Wolbachia within both S2 and LD-based cell co-culture models for up to 115 days, a definitive conclusion is still far. Further trials using fluorescent microscopy and viable staining will help to demonstrate the cell line infection and viability of Wolbachia. Use of considerable amount of untreated mfs to inoculate the Drosophilia S2 cell lines, as well as the supplementation of the culture media with growth stimulants or pre-treated cells to increase their susceptibility for the infection and development of a filarioid-based cell line system are recommended for the future trials., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Bacteria of the genus Wolbachia are endosymbionts of parasitic filarial nematodes, including Dirofilaria immitis, and are a target for the treatment of canine heartworm disease. In the present study, 53 naturally-infected dogs were divided in three groups, based on their positivity to D. immitis by antigen and Knott tests, to assess the efficacy of doxycycline treatment in eliminating Wolbachia from circulating blood. At T0, dogs that scored positive to both tests (G1) or to antigen only (G2) were submitted to doxycycline (10 mg/kg BID PO) treatment and to 10% Imidacloprid + 2.5% Moxidectin (Advocate®), while those negative to both tests (G3) received only 10% Imidacloprid + 2.5% Moxidectin (Advocate®). All dogs were followed-up for one year, monthly treated with Advocate® and regularly monitored by antigen and Knott tests. During the whole period, all blood samples were screened for Wolbachia-D. immitis DNA load by quantitative real-time PCR (qPCR). At T0, 88.2% of the microfilariemic dogs were positive for Wolbachia DNA, while none of the dogs from G2 or G3 were positive. Wolbachia DNA was no longer detectable in dogs from G1 following 1 month of doxycycline treatment and microfilariae (mfs) were cleared at T2. All dogs from the G1 and G2 were negative for D. immitis antigen at 12 months. Results of this study suggest that successful elimination of mfs by doxycycline is associated with complete clearance of Wolbachia DNA in D. immitis-naturally infected dogs., (Copyright © 2022. Published by Elsevier B.V.)

Dirofilariosis by Dirofilaria immitis and Dirofilaria repens is endemic in dogs from countries of the Mediterranean basin. Both species may infect humans, with most of the infected patients remaining asymptomatic. Based on the recent description of the southernmost hyperendemic European focus of heartworm disease in dogs from the Pelagie archipelagos, we performed a serological and molecular survey in human population of that area. Human blood samples were collected in the islands of Linosa (n=101) and Lampedusa (n=296) and tested by ELISA and molecular test for the detection of D. immitis and D. repens. Samples were also screened for filarioid-associated endosymbionts, Wolbachia sp. The seroprevalence of D. immitis and D. repens was, respectively, 7.9% and 3.96% in Linosa, and 7.77% and 19.93% in Lampedusa. Out of 397 human blood samples tested molecularly, 4 scored positive (1%) for Dirofilaria spp. by qPCR (i.e., three for D. immitis and one for D. repens) and 6 (1.5%) for Wolbachia. Of the qPCR positive for Dirofilaria spp., only D. repens was amplified by cPCR and was positive for Wolbachia. In the phylogenetic analysis, the sequence of Wolbachia detected in D. repens positive samples clustered along with other C supergroup filarioids. Our results overlap with the recent prevalence data collected on dogs from the same area, where D. immitis is prevalent in Linosa and D. repens prevails in Lampedusa. Molecular detection of D. immitis in human blood is quite unusual considering that humans are dead-end hosts for dirofilarial infection and most of the human cases described so far in Europe were ascribed to D. repens. An integrative diagnostic approach using serum analysis and Wolbachia detection is also presented. In endemic areas for canine dirofilarioses humans are exposed to the infection, suggesting the importance of One Health approach in diagnosing, treating and controlling this zoonotic parasitosis., (Copyright © 2021. Published by Elsevier B.V.)

A study on the occurrence of Dirofilaria immitis and its vectors was carried out in order to assess the prevalence of the disease in dogs in previously non-endemic areas of southern Italy. Blood samples (n = 385) and mosquitoes (n = 1540) were collected in two dog shelters and analysed by Knott's test and duplex real-time PCR, respectively. Dirofilaria immitis was the most prevalent filarioid (44.2%), while Culex pipiens was the most prevalent mosquito species (68.8%). This high prevalence of D. immitis infection confirms this location as one of the most hyperendemic foci of dirofilariosis in Europe., (Copyright © 2020 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Background: Dirofilaria immitis is a mosquito-borne pathogen that is spreading worldwide, and the associated infection (i.e. dirofilariosis) is becoming a threat to animals and humans living in endemic areas. Little is known about the occurrence and risk of infection of D. immitis in pinnipeds. Here we report dirofilariosis by D. immitis in several pinniped species kept in captivity in Portugal., Methods: Animals were housed in an oceanographic park located in Algarve, southern Portugal, a geographical area endemic for canine dirofilariosis. To assess the occurrence of D. immitis, blood was collected from the park's resident pinniped population, which consisted of 16 animals (5 common seals Phoca vitulina, 2 grey seals Halichoerus grypus, 3 California sea lions Zalophus californianus and 6 South African fur seals Arctocephalus pusillus pusillus). Dirofilaria immitis nematodes were detected by real-time PCR and by the presence of circulating antigens. In addition, modified Knott's technique was performed to detect circulating microfilariae. Necropsies and histopathological examination of two animals which died during the study were also conducted., Results: Out of the 16 pinnipeds housed at the park, seven (43.8%) were positive for D. immitis by real-time PCR (3 P. vitulina, 2 Z. californianus and 2 A. p. pusillus), two of which (P. vitulina) were also positive for the nematode's antigen. Additionally, D. immitis microfilariae were detected in one A. p. pusillus. Furthermore, several D. immitis specimens were retrieved from the right ventricle and pulmonary arteries at the necropsy of one P. vitulina and one A. p. pusillus., Conclusions: This study provides new epidemiological data on D. immitis infection in pinnipeds diagnosed through clinical, molecular and pathological findings. Additionally, the South African fur seal is herein reported as a new host for this zoonotic filarioid. The situation herein described could also occur in other parks located in areas where canine dirofilariosis is endemic. Active surveillance and preventive measures of dirofilariosis in pinnipeds on a local and global scale are therefore vital to improve the early diagnosis and control of dirofilariosis.

In the past years, canine and human cases of infestation by Dirofilaria repens (Spirurida, Onchocercidae) have been increasingly reported in several European countries. Subcutaneous dirofilariosis by D. repens may either be asymptomatic in dogs or may be characterized by subcutaneous nodules and other symptoms. Information on the periodicity of D. repens microfilariae in naturally infested animals is scant, and this might impair the accurate diagnosis of subcutaneous dirofilariosis and appropriate control plans. In the present study, eight dogs infested with D. repens were sampled twice daily at 12-h intervals for ten consecutive days, and the dog with the highest mean value of microfilariaemia was further sampled every 4 h for four consecutive days. The blood was microscopically and molecularly examined for microfilariae, and, additionally, negative samples were also subjected to a real-time PCR to evaluate the level of circulating DNA. The results demonstrated significant variations in circadian rhythms of D. repens larvae, with higher values of microfilariae per milliliter in the evening samples. A significant variation was also found at the individual level for the dogs with the highest values of microfilariaemia. All samples which were negative at the light microscopy and positive at the real-time PCR displayed levels of circulating parasite DNA <1 microfilaria per milliliter. Biological and clinical implications have been here discussed.

Given the spread of Aedes albopictus from northern to southern Italy, and the lack of updated data on Dirofilaria infections, this study was carried out to assess the infection risk for dogs and cats in Apulia region. During a 2-year study, 175 A. albopictus female specimens and samples of blood from 427 dogs (309 privately owned dogs and 118 shelter dogs) and 12 cats were collected. All blood samples were subjected to a modified Knott method, to a test for the detection of circulating Dirofilaria immitis antigen, and to a Dirofilaria species-specific real-time PCR for the simultaneous detection of D. immitis and Dirofilaria repens, targeting on partial cytochrome oxidase subunit 1 and internal transcribed spacer-2, respectively. Two abdomen and one thorax pools from A. albopictus were positive for D. immitis, with minimum infection rates of 1.14 and 0.51, respectively, and a probability of a single positive specimen to be infected of P = 0.6 % (95 % confidence interval (CI) = 0.12-1.73). Out of 439 examined subjects, 22 (5.0 %) tested positive for Dirofilaria spp. in at least one diagnostic test. A specific D. immitis infestation rate of 3.5 % was found among the privately owned dogs, while shelter dogs tested positive only for D. repens with a prevalence of 3.4 %; one cat tested molecularly positive for D. immitis. There was a significantly higher rate of positivity among guard dogs for D. immitis (odds ratio, 6.24, 95 % CI, 1.26-25.28; P < 0.05). The increasing risk of D. immitis infection in southern Italy is supported by the noteworthy positivity of A. albopictus populations and the cat. Our data highlight the usefulness to include filarioid infestation in routine diagnosis.

This study describes a duplex real-time polymerase chain reaction (PCR) assay for the detection and differentiation between Dirofilaria immitis and Dirofilaria repens in dog blood and mosquitoes. Regions of a cytochrome oxidase 1 (cox1) mitochondrial DNA fragment and the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA were amplified from microfilariae and adult worm samples, using a sensitive SsoFast™ EvaGreen(®) based real-time PCR method coupled with melting-curve analysis. The limit of the real-time PCR in detecting microfilaria and adult worm DNA was also tested both in dog blood and in artificially infected microfilarial. Two peaks at different melting temperatures (T(m)) for D. immitis (mean ± SD=75.7 ± 0.3°C) and D. repens (mean ± SD=70 ± 0.7°C), respectively, were obtained for microfilarial and adult positive controls of both species when examined separately and together. The real-time PCR protocol was also efficient in detecting microfilarial and adult DNA of both species when tested in samples spiked with DNA from Aedes albopictus, in Aedes aegypti experimentally infected by D. repens and in Culex pipiens naturally infected by D. repens and D. immitis. The high sensitivity of real-time PCR confirmed its reliability in detecting small amounts of genomic DNA either in dog blood or mosquitoes (2.5 pg/μl and 3 × 10(-1)pg/μl for D. immitis and D. repens, respectively). This assay is proposed as a tool for the epidemiological surveillance of the two most important Dirofilaria species in areas where they are endemic and sympatric., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Dirofilaria immitis and Dirofilaria repens are transmitted by bloodsucking culicid mosquitoes belonging to Culex, Aedes, Ochlerotatus, Anopheles and Mansonia genera. The detection of filarioids in mosquitoes for assessing distribution of vectors and/or of pathogens in a given area (also known as "xenomonitoring"), when based on individual dissection of wild-caught female mosquitoes is time consuming and hardly applicable in large epidemiological surveys. Our study aimed to evaluate the recently developed duplex real-time PCR for screening large number of culicids and to assess their positivity for D. immitis and D. repens in an area where both species are endemic., Methods: A duplex real-time PCR was used to detect and differentiate D. immitis and D. repens in mosquitoes collected in six provinces of the Veneto region using 43 carbon dioxide-baited traps under the frame of an entomological surveillance program to monitor the vectors of West Nile disease. From early May till October 2010, unfed female mosquitoes (n = 40,892) were captured in 20 selected sites., Results: Mosquitoes identified as Culex pipiens, Ochlerotatus caspius, Aedes vexans and Culex modestus were grouped into 995 pools according to species, day and site of collection (from minimum of 1 to maximum of 57). Out of 955 pools, 23 (2.41 %) scored positive for Dirofilaria spp. of which, 21 (2.2 %) for D. immitis and two (0.21 %) for D. repens. An overall Estimated Rate of Infection (ERI) of 0.06 % was recorded, being higher in Och. caspius and Ae. vexans (i.e., 0.18 % and 0.14 %, respectively). At least one mosquito pool was positive for Dirofilaria spp. in each province with the highest ERI recorded in Vicenza and Padova provinces (i.e., 0.42% and 0.16 %, respectively). Mosquitoes collected in all provinces were positive for D. immitis whereas, only two (i.e., Padova and Rovigo) provinces scored positive for D. repens. All mosquito species, except for Cx. modestus, were positive for D. immitis, whereas D. repens was only found in Cx. pipiens., Conclusions: The results suggest that both Dirofilaria species are endemic and may occur in sympatry in the examined area. The molecular approach herein used represents a powerful tool for surveillance programs of D. immitis and D. repens in the culicid vectors towards a better understanding of the epidemiology of the infections they cause and their seasonal transmission patterns.