Optimized protocol for DNA/RNA co-extraction from adults of Dirofilaria immitis.

Dirofilaria immitis, the etiologic agent of canine heartworm disease, like several other filarial nematodes, harbors the bacterial endosymbiont Wolbachia. To investigate metabolic and functional pathways of D. immitis and Wolbachia individually, along with their interactions, the use of both transcriptomic and genome analysis has becoming increasingly popular. Although several commercial kits are available for the single extraction of either DNA or RNA, no specific protocol has been described for simultaneous extraction of DNA and RNA from such a large organism like an adult D. immitis, where female worms generally reach ∼25 cm in length. More importantly, adult worms of D. immitis can only be obtained either through necropsy of experimentally infected dogs or by minimally-invasive surgical heartworm removal of naturally infected dogs. This makes each individual worm sample extremely important. Thus, in the context of a project aimed at the evaluation of both gene expression analysis and Wolbachia population assessment following different treatments, an optimized protocol for co-extraction of DNA and RNA from a single sample of adult D. immitis has been developed. •An optimized method for DNA/RNA co-extraction from large size nematodes using TRIzol® reagent.•Allows maximum exploitation of unique samples as adults of D. immitis.

Novel Activity of a Synthetic Decapeptide Against Toxoplasma gondii Tachyzoites.

The killer peptide KP is a synthetic decapeptide derived from the sequence of the variable region of a recombinant yeast killer toxin-like microbicidal single-chain antibody. KP proved to exert significant activities against diverse microbial and viral pathogens through different mechanisms of action, but little is known of its effect on apicomplexan protozoa. The aim of the present study was to evaluate the in vitro activity of KP against Toxoplasma gondii, a globally widespread protozoan parasite of great medical interest. The effect of KP treatment and its potential mechanism of action on T. gondii were evaluated by various methods, including light microscopy, quantitative PCR, flow cytometry, confocal microscopy, and transmission electron microscopy. In the presence of KP, the number of T. gondii tachyzoites able to invade Vero cells and the parasite intracellular proliferation were significantly reduced. Morphological observation and analysis of apoptotic markers suggested that KP is able to trigger an apoptosis-like cell death in T. gondii. Overall, our results indicate that KP could be a promising candidate for the development of new anti-Toxoplasma drugs with a novel mechanism of action.

Heat treatment and false-positive heartworm antigen testing in ex vivo parasites and dogs naturally infected by Dirofilaria repens and Angiostrongylus vasorum.

BACKGROUND: Heartworm antigen testing is considered sensitive and specific. Currently available tests are reported as detecting a glycoprotein found predominantly in the reproductive tract of the female worm and can reach specificity close to 100%. Main concerns regard sensitivity in the case of light infections, the presence of immature females or cases of all-male infections. Research and development have been aimed at increasing sensitivity. Recently, heat treatment of serum prior to antigen testing has been shown to result in an increase in positive antigen test results, presumably due to disruption of natural antigen-antibody complexes. Cross-reactions in dogs with both natural and experimental infections with Angiostrongylus vasorum and Spirocerca lupi have been reported, but cross-reactions with other helminths have not been extensively studied. In order to evaluate potential cross-reactivity with other canine and feline parasites, two studies were performed. Study 1: Live adults of Dirofilaria immitis, Dirofilaria repens, Toxocara canis, Toxocara cati, Dipylidium caninum, Taenia taeniaeformis and Mesocestoides spp. larvae were washed and incubated in tubes with saline solution. All worms were alive at the time of removal from the saline. Saline solutions containing excretory/secretory antigens were then tested for heartworm with six different, commercially available antigen tests. All results were evaluated blind by three of the authors. Study 2: Sera from dogs with natural infections by A. vasorum or D. repens, living in areas free of heartworm disease, were tested with the same tests before and after heat treatment (103 °C for 10 min). RESULTS: Results suggest that antigens detected by currently available tests are not specific for D. immitis. They may give positive results through detection of different parasites' antigens that are normally not released into the bloodstream or released in a low amount and/or bound to antibodies. Tests may even detect antigens released by male D. immitis adult worms. D. repens appears to release more detectable antigens than the other worms studied. CONCLUSIONS: Cross-reaction with A. vasorum and D. repens does occur in the field and could potentially occur with other helminths. Heat treatment decreases specificity by enhancing cross-reactivity.

Clinical assessment of post-adulticide complications in Dirofilaria immitis-naturally infected dogs treated with doxycycline and ivermectin.

This study shows that a combination of doxycycline (10mg/kg/sid for 30 days) and ivermectin (6 µg/kg/every 15 days for 6 months) is well tolerated for the treatment of canine heartworm disease (HWD). Monthly echocardiography showed that 84% of treated dogs either progressively improved parameters indicative of pulmonary hypertension or, following slight worsening, resolved all signs. Thoracic radiography showed the persistence of interstitial inflammation, even though also in this case, approximately 70% of the dogs steadily improved or worsened but then improved by the end of the study.

Wolbachia and its implications for the immunopathology of filariasis.

Filarial infections are characterized by immunopathological phenomena, that are responsible for the onset of often dramatic pathological outcomes, such as blindness (Onchocerca volvulus) and elephantiasis (W. bancrofti). In addition, the long-term survival (as long as 10 years) of these parasites in otherwise immunocompetent hosts indicates that these nematodes are capable of manipulating the host immune response. The ground-breaking discovery of the bacterial endosymbiont Wolbachia, which resides in most filarial nematodes causing disease, has led to increasing interest in the role it may play in immuno-modulation, pro-inflammatory pathology and other aspects of filarial infection. Indeed, Wolbachia has been shown to be responsible for exacerbating inflammation (as in river blindness), while at the same time blocking efficient elimination of parasites through the host immune response (Onchocerca ochengi). While studies aimed at identifying Wolbachia as a potential target for anti-filarial therapy are at the forefront of current research, understanding its role in the immunology of filarial infection is a fascinating field that has yet to uncover many secrets.

Anti-Wolbachia Surface Protein antibodies are present in the urine of dogs naturally infected with Dirofilaria immitis with circulating microfilariae but not in dogs with occult infections.

Heartworm infection (Dirofilaria immitis) can cause kidney damage due to the presence of circulating microfilariae (mf) that contribute to the production and deposit of immune complexes. It has been shown that mf are a major source of Wolbachia antigen during active infection. Here the authors compared urine samples from 19 naturally infected dogs with (mf+) and 12 without (mf-) microfilariae for the presence of proteinuria and anti-Wolbachia Surface Protein (-WSP) IgG in ELISA. Kidneys from 6 mf+ and 3 mf- dogs were also examined by anti-WSP immuno-histochemistry. All infected dogs showed proteinuria, but mf+ dogs had significantly higher values compared to mf-dogs. Mf+ dogs had optical density values for anti-WSP IgG consistently higher than established cut-off values and were significantly higher than values for mf- dogs. Kidneys from mf+ dogs showed Wolbachia+ mf in glomerular capillaries. Results strongly suggest that Wolbachia associated with circulating mf may contribute to immune-mediated kidney disease in dogs with heartworm infection.

Dirofilarial infections in Europe.

Nematodes of the genus Dirofilaria are currently considered emerging agents of parasitic zoonoses in Europe. Climatic changes and an increase in the movement of reservoirs (mostly infected dogs) have caused an increase in the geographical range of these parasites from the traditionally endemic/hyperendemic southern regions, and the risk for human infection has increased. In the last several years, forecast models have predicted that current summer temperatures are sufficient to facilitate extrinsic incubation of Dirofilaria in many areas of Europe. The global warming projected by the Intergovernmental Panel on Climate Change suggests that warm summers suitable for Dirofilaria transmission in Europe will be the rule in the future decades, and if the actual trend of temperature increase continues, filarial infection should spread into previously infection-free areas. Dirofilaria repens is currently the filarial species that is most commonly reported as spreading from southern to northern areas. This article reviews the zoonotic aspects, effects of climate, and other global drivers on Dirofilaria infections in Europe and the possible implications on the transmission and control of these mosquito-borne nematodes.

Prevalence of Theileria equi and Babesia caballi infection in horses from northern Italy.

Babesia caballi and Theileria equi are the causative agents of equine piroplasmosis. In this epidemiological study, 294 horses reared in a rural area of northern Italy were studied. During January 2008-January 2009, blood samples were taken for serology (indirect fluorescent antibody test) and for polymerase chain reaction (PCR). Data on the geographical area, sex, and age were collected for statistical analysis of risk factors associated with infection. A seroprevalence of 8.5% was found: 8.2% of the animals were positive for anti-T. equi antibodies and 0.3% for anti-B. caballi antibodies. No dual infections were observed. Of those horses with positive serology to T. equi, 33% were also positive in PCR, whereas none of the seropositive horses for B. caballi was positive in PCR. No significant correlation between sex or age was found for infection status.

A case of human conjunctival dirofilariosis by Dirofilaria immitis in Italy.

This short communication describes a case of human conjunctival dirofilariosis by Dirofilaria immitis. A 51-year-old man from the Emilia-Romagna region of northeastern Italy referred for ocular foreign body sensation in his right eye. A slight swelling of the bulbar conjunctiva was observed. A live, whitish, 10-cm-long nematode was extracted from the subconjunctival space. Histology revealed typical features of a filarioid nematode belonging to the genus Dirofilaria sp. Molecular analysis with polymerase chain reaction confirmed that the extracted nematode was a male of D. immitis. The removal of the parasite leads to complete resolution of symptoms. Ocular human dirofilariosis caused by infection with the filarioid nematode D. immitis is extremely rare, but should be considered in humans living in Italian endemic areas.

Association of Wolbachia with heartworm disease in cats and dogs.

Although the presence of adult Dirofilaria immitis in the pulmonary arteries and its associated arteritis and thromboembolic disease can explain some of the manifestations of canine and feline heartworm disease, the cause of other findings remains unclear. Cats with D. immitis antibodies but lacking adult parasites in the pulmonary arteries frequently develop histological lesions of the airways, resulting in a condition termed Heartworm-Associated Respiratory Disease. All D. immitis parasites harbor Wolbachia pipientis bacteria and D. immitis-infected animals can have circulating Wolbachia antibodies and pro-inflammatory Wolbachia antigens (WSP) deposited in tissues. Little is known about the role that Wolbachia plays in lung disease of animals naturally infected with D. immitis. The purpose of this study was to determine the contribution of Wolbachia to the pathogenesis of natural heartworm disease in cats and dogs. We hypothesized that animals having sufficient Wolbachia burden to be detected in lung tissue by immunohistochemistry and/or polymerase chain reaction (PCR) would have more severe pulmonary disease than those with bacteria below the limits of detection. We further hypothesized that animals that were immunoreactive to pro-inflammatory WSP would have more severe pulmonary lesions than those that were seronegative for WSP antibodies. Blood and lung tissue samples were collected from cats and dogs representing three different D. immitis infection statuses: heartworm-free, heartworm-exposed, heartworm-infected. There was a positive but weak correlation between the magnitude of D. immitis antibody titers and WSP titers in cats (r=0.57, p<0.001) and in dogs (r=0.39, p<0.001). Pulmonary lesions were more common in HW-infected animals than in HW-free animals. Pulmonary arteriolar occlusion was more common in HW-infected cats (57%; p=0.003) than in HW-infected dogs (17%). Although pulmonary lesions were most common in HW-infected animals, there was no clear additive effect when either Wolbachia DNA/WSP was detected in lung tissue or when circulating Wolbachia antibodies were detected. There were no significant differences in the magnitude of pulmonary lesion scores within each HW-infection status group regardless of whether Wolbachia DNA/WSP or antibodies were detected. The relationship between Wolbachia and lung pathology in heartworm-infected animals remains to be determined. The lack of clear evidence for a role of Wolbachia in heartworm disease creates a dilemma for veterinarians treating animals in D. immitis-endemic areas. Although the indiscriminant use of antibiotics should be avoided, many clinicians prescribe doxycycline based on the favorable responses observed in human filarial diseases and promising results from the first published studies of doxycycline use in D. immitis-infected dogs.

Heartworm disease in animals and humans.

Heartworm disease due to Dirofilaria immitis continues to cause severe disease and even death in dogs and other animals in many parts of the world, even though safe, highly effective and convenient preventatives have been available for the past two decades. Moreover, the parasite and vector mosquitoes continue to spread into areas where they have not been reported previously. Heartworm societies have been established in the USA and Japan and the First European Dirofilaria Days (FEDD) Conference was held in Zagreb, Croatia, in February of 2007. These organizations promote awareness, encourage research and provide updated guidelines for the diagnosis, treatment and prevention of heartworm disease. The chapter begins with a review of the biology and life cycle of the parasite. It continues with the prevalence and distribution of the disease in domestic and wild animals, with emphasis on more recent data on the spreading of the disease and the use of molecular biology techniques in vector studies. The section on pathogenesis and immunology also includes a discussion of the current knowledge of the potential role of the Wolbachia endosymbiont in inflammatory and immune responses to D. immitis infection, diagnostic use of specific immune responses to the bacteria, immunomodulatory activity and antibiotic treatment of infected animals. Canine, feline and ferret heartworm disease are updated with regard to the clinical presentation, diagnosis, prevention, therapy and management of the disease, with special emphasis on the recently described Heartworm Associated Respiratory Disease (HARD) Syndrome in cats. The section devoted to heartworm infection in humans also includes notes on other epizootic filariae, particularly D. repens in humans in Europe. The chapter concludes with a discussion on emerging strategies in heartworm treatment and control, highlighting the potential role of tetracycline antibiotics in adulticidal therapy.

Human subcutaneous dirofilariasis, Russia.

We report 14 cases of human subcutaneous dirofilariasis caused by Dirofilaria repens, diagnosed from February 2003 through July 2004, in patients from Rostovon-Don, Russia. Serologic analysis showed evidence of high risk of exposure to D. repens. Surveillance studies on prevalence and prevention effectiveness of canine infection are needed to control this emerging zoonosis.

Full season efficacy of moxidectin microsphere sustained release formulation for the prevention of heartworm (Dirofilaria immitis) infection in dogs.

The authors report the efficacy of an injectable, moxidectin sustained release (SR) formulation for the prevention of canine heartworm infection in endemic areas in northern and central Italy. Three field trials were carried out on a total of 324 dogs. Two hundred forty-three dogs were treated with moxidectin SR 6 months apart and 81 dogs (positive controls) with moxidectin tablets given monthly for 5 consecutive months during the risk season each year throughout the study. Results of testing for microfilariae and circulating adult female antigens were negative for all the experimentally treated dogs at the 6, 7, 11 and 19 months after the last injection. No adverse reactions to moxidectin SR were observed but a moderate pain at palpation and swelling (5-6 cm) at the injection site after the first treatment. In the study areas, prevalence of Dirofilaria immitis infection calculated by testing dogs which had no preventive treatment in the previous transmission season ranged from 33 to 63%. This study confirms the efficacy and safety of injectable, moxidectin SR formulation in the prevention of heartworm infection in dogs and demonstrates that the prophylactic efficacy lasts for the full season and strongly suggests that the product gives 1-year protection.