Marco Genchi

Climate and Dirofilaria infection in Europe

Climatic changes, together with an increase in the movement of cats and dogs across Europe, have caused an increase in the geographical range of several vector borne parasites like Dirofilaria, and in the risk of infection for animals and humans. The present paper reviews the effects of climate and other global drivers on Dirofilaria immitis and Dirofilaria repens infections in Europe and the possible implications on the transmission and control of these mosquito-borne nematodes. In the last several years, growing degree day (GDD)-based forecast models, which use wide or local scale temperature data, have been developed to predict the occurrence and seasonality of Dirofilaria in different parts of the world. All these models are based on the fact that: there is a threshold of 14 degrees C below which Dirofilaria development will not proceed; and there is a requirement of 130 GDD for larvae to reach infectivity and a maximum life expectancy of 30 days for a vector mosquito. The output of these models predicts that the summer temperatures (with peaks in July) are sufficient to facilitate extrinsic incubation of Dirofilaria even at high latitudes. 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. These factors not only favour incubation of Dirofilaria, but also impact on mosquito species. Recent findings have also demonstrated that Aedes albopictus is now considered to be an important, competent vector of Dirofilaria infections. This mosquito species could spread from southern to northern European countries in the near future, changing the epidemiological patterns of dirofilariosis both in humans and animals.

A combination of doxycycline and ivermectin is adulticidal in dogs with naturally acquired heartworm disease (Dirofilaria immitis).

Canine heartworm disease is caused by infection with Dirofilaria immitis, a filarial nematode that resides in the pulmonary arteries and occasionally in the right heart chambers of infected dogs. Here the authors evaluated the effect of a combination of doxycycline (10 mg/kg/sid for 30 days) and ivermectin–pyrantel(6μg/kg [DOSAGE ERROR CORRECTED] of ivermectin+5mg/kg of pyrantel every 15 days for 180 days) on microfilariemia, antigenemia and parasite load at echocardiography in naturally infected dogs from an endemic region of Italy. Dogs were examined monthly for 6 months and followed-up 4 months later. One hundred percent of dogs became negative for circulating microfilariae by day 90, while 8/11 (72.7%) of dogs became antigen-negative by day 300. Of the 7 dogs that were positive for visualization of parasites at echocardiography, 6 (85.7%) became negative by day 300. Treatment was well-tolerated by all dogs. These results suggest that a combination of doxycycline and ivermectin is adulticide in dogs with D. immitis.

Th1 response in BALB/c mice immunized with Dirofilaria immitis soluble antigens: a possible role for Wolbachia?

The immune response to filarial infection has been shown to be of both the Th1 and Th2 types. Studies aimed at developing immunization strategies against Dirofilaria immitis infection in dogs have shown that protection against larval challenge is of the Th2 type and that several proteins are recognized by immunized or infected animals. The bacterial endosymbiont Wolbachia, harbored by many filarial species including D. immitis, has recently been shown to interact with the host immune system. Specific antibodies to the Wolbachia recombinant surface protein (WSPr) have been observed in cats infected with D. immitis. In this work the authors have determined cytokine production and antibody response in BALB/c mice inoculated with soluble antigens from third stage larvae or from adult worms of D. immitis. Inoculated mice first produced IFN-gamma followed by a peak in IL-4. Specific antibodies to the Wolbachia protein WSPr were exclusively IgG2a, while antibodies against peptides derived from antigens of D. immitis were in the IgG1 and IgE subclasses. The cytokine response is thus similar to that reported for other filarial infection, where Th1 response shifts towards Th2. Antibody response indicates that Wolbachia may induce preferentially a Th1 response during filarial infection, while nematode antigens may be involved in Th2 response. There is thus an overall agreement with current opinions on the role of bacterial versus nematode molecules in driving the response towards the different directions.

A duplex real-time polymerase chain reaction assay for the detection of and differentiation between Dirofilaria immitis and Dirofilaria repens in dogs and mosquitoes.

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.

Evaluation of lung pathology in Dirofilaria immitis-experimentally infected dogs treated with doxycycline or a combination of doxycycline and ivermectin before administration of melarsomine dihydrochloride.

Adulticide therapy in heartworm (Dirofilaria immitis)-infected dogs can lead to thromboembolism, which can seriously compromise post-treatment health status. Lung pathology following adulticide therapy was evaluated in three groups of experimentally infected dogs. Group 1 was treated with doxycycline at 20 mg/kg per os once daily for 30 days post infection followed by an intramuscular injection of melarsomine dihydrochloride (2.5 mg/kg) at Week 12, followed 1 month later by two injections 24 h apart. Group 2 was treated as described for Group 1, with the addition of ivermectin at 6 mcg/kg given monthly per os for 24 weeks post-infection. Group 3 received melarsomine alone, as described above. All dogs were necropsied at Week 24 and lung pathology was evaluated. Lesion criteria included perivascular inflammation and endothelial proliferation. Lesions were scored by two independent pathologists who were blinded as to treatment. Results indicate that doxycycline treatment alone or combined with ivermectin had lower lesion scores than lungs from dogs who had received melarsomine alone. Dogs that received the combined doxycycline/ivermectin protocol and treated with adulticide showed less severe arterial lesions and the virtual absence of thrombi.

Efficacy of moxidectin microsphere sustained release formulation for the prevention of subcutaneous filarial (Dirofilaria repens) infection in dogs.

A study was carried out to assess the efficacy of a moxidectin microsphere sustained release (SR) injectable formulation (Guardian SR Iniettabile, Fort Dodge) for the prevention of Dirofilaria repens infection in experimentally infected dogs. On day 0, 18 Beagle dogs, 9 male dogs and 9 female dogs, weighing 12-16 kg were ranked in ascending order of body weight (b.w.) and blocked into pairs. Within each pair, dogs were allocated to Group 1 or Group 2 at random. On the same day, dogs in Group 1 were injected with 0.05 ml/kg b.w. of saline solution and dogs in Group 2 were injected with moxidectin SR at the label dose 0.17 mg/kg b.w. (0.05 ml/kg b.w.). Six months after moxidectin SR or saline injection, on day 180, each dog in the two groups was challenged with 50 infective larvae of D. repens collected from laboratory-reared, experimentally infected Aedes aegypti. Dogs were humanely euthanized on day 380 of the study, approximately 7 months from D. repens challenge. At necropsy, no worms were found in dogs treated with moxidectin SR (Group 2) while adult worms were found in saline-treated dogs (total 90; 38 males and 52 females; arithmetic mean 10, standard error 0.96, median 9, range 7-15) (Group 1 vs Group 2 P<0.001). In this experimental study, moxidectin SR injectable showed full efficacy (100%) lasting at least 6 months, and is able to prevent subcutaneous D. repens patent infection throughout the entire transmission season in Europe.

Can heartworm prevalence in dogs be used as provisional data for assessing the prevalence of the infection in cats

Cats are considered a susceptible host for Dirofilaria immitis; however, increased host resistance is reflected by relatively low adult worm burdens in natural and experimental infections; the prolonged prepatent period (8 months); the low level and short duration of microfilaremia; and the short life span of adult worms (2-3 years). From April to September 2006, 212 cats and 608 dogs, all exposed for at least one transmission season, were screened for D. immitis infection in a multi-center study in the Po River Valley in northern Italy. Cats were initially evaluated by antibody testing; positive subjects were followed up by antigen testing and echocardiography (and necropsy if death occurred). The prevalence in dogs was 29% by a modified Knott test and antigen testing compared with a prevalence of 4.7% in cats by an antibody test; six of these infections (2.8%) were confirmed by the follow-up evaluations. This field study demonstrated that the prevalence of heartworm infection in cats in this area is within the expected limits of 9-18% of the prevalence in dogs. Antibody testing likely underestimates the real prevalence of D. immitis infection in cats. These results also emphasize the importance of preventive treatment in cats.

Evaluation of the Efficacy of Imidacloprid 10 % / Moxidectin 2.5 % (Advocate®, Advantage® Multi, Bayer) for the Prevention of Dirofilaria repens Infection in Dogs

The efficacy of imidacloprid 10 %/moxidectin 2.5 % (Advocate®, Advantage® Multi, Bayer) against experimental Dirofilaria (D.) repens infection in dogs was evaluated in a blinded, negative controlled randomised laboratory efficacy study. On SD (study day) 0, eight dogs received a spot-on treatment at a dose of 10 mg imidacloprid and 2.5 mg moxidectin per kg body weight. Another 8 dogs were left untreated. On SD 28 each dog was infected with approximately 75 infective D. repens larvae. Blood samples were collected every 4 weeks after treatment. A modified Knott test was conducted to detect mf (microfilaria). PCR analysis was performed with mf-positive blood samples. On SDs 245 and 246, all dogs were euthanised for detection of D. repens worms. Blood samples of all treated dogs were negative for mf at all sampling days. Blood samples of control dogs were positive for mf in 5 out of 8 control dogs. Individual mf counts ranged from 7 to 2800 mf/ml. In mf-positive blood samples, only D. repens was identified by PCR analysis. During necropsy D. repens worms could be detected in eight untreated control dogs (range: 3–21 worms per dog), whereas no worm could be detected in any of the treated dogs. These results indicate a 100 % preventive efficiency of a single spot-on treatment of imidacloprid 10 %/moxidectin 2.5 % in dogs against experimental infection with D. repens (L3 larvae). The product was well tolerated in all study animals, no treatment related adverse reactions were observed throughout the study.

Rapid differentiation of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood by real-time PCR coupled to high resolution melting analysis.

Dirofilaria immitis and D. repens are the principal causative agents of canine filariosis and, although the number of dogs subjected to specific prevention is increasing, the prevalence of these parasites remains high in many areas of the world. The discrimination between the two Dirofilaria species using the classical diagnostic methods can be difficult and may lead to misdiagnosis especially on samples from areas where both Dirofilaria are present. Over the last years, several molecular methods with higher sensitivity and specificity compared to classical microscopy and ELISA assays were designed. Nevertheless, a need for simple, rapid, and cost-effective molecular protocols to accurately discriminate between D. immitis and D. repens still remains. High resolution melting analysis coupled to real-time PCR (real-time PCR-HRMA) is a widely used technique to target sequence polymorphisms of the same gene in different species without the need to perform DNA sequencing or to use species-specific probes. In this work, a fast and cost-effective real-time PCR-HRMA protocol to detect and differentiate simultaneously and unequivocally D. immitis and D. repens microfilarial DNA extracted from peripheral dog blood samples is described. The present method is simpler to use than most other DNA-based methods and provides comparable discrimination between the two sibling species.

Comparative evaluation of two ivermectin injectable formulations against psoroptic mange in feedlot cattle

A study was carried out to compare the efficacy of two injectable formulations of ivermectin, Ivomec,(1) Merial (IVM reference) and Ivogell,(2) Intervet (IVM generic) in the treatment of psoroptic mange (Psoroptes ovis) in Charollais feedlot cattle. A total of 22 animals were ranked in order of the severity of mange and allocated to 11 replicates of 2 animals each. Within each replicate, one animal was randomly allocated to IVM reference product treatment (Group 1) and one to IVM generic (Group 2). Animals were treated on Day 0 and on Day 8 at the recommended dosage of 200 microg ivermectin/kg bodyweight. The pharmacokinetics profiles (pK) of both IVM formulations were evaluated in plasma samples taken from 6 cattle randomly chosen per group on Day 0, before treatment, and then at 6, 12, 24 hours and daily from Day 2 to Day 7 after the treatment on Day 0. Additionally, the severity of mange lesions was assessed and mites were counted in skin scrapings on Days 0, 8, 15 and 25. Animals were weighed on Day 0 and 25 and body weight and average daily gains (ADG) were evaluated. No statistical differences were found between the cattle of the two groups in any pK parameters, although the mean IVM plasma concentrations in cattle treated with the IVM reference product were consistently higher than those found in cattle treated with the generic compound. By Day 25, all animals in Group 1 had recovered clinically and parasitologically from psoroptic mange while cattle from Group 2 still had mange lesions and, in two animals, living mites were found in the skin scrapings; these differences were significant (P<0.001). The mean body weight of the two groups was significantly different on Day 25 (P<0.01) when animals in Group 1 weighed 20 kg more than those in Group 2. In conclusion, despite similarities in their pharmacokinetic profiles and formulations, the clinical efficacy of the two injectable formulations of IVM differed significantly in their therapeutic efficacy against psoroptic mange in feedlot cattle up to 25 days after treatment: this difference in response was reflected in an incomplete clinical and parasitological response in Group 2 and a slower growth rate.