Dirofilaria immitis in Italian Cats and the Risk of Exposure by Aedes albopictus.

Background: Dirofilaria immitis, the agent of canine and feline heartworm disease (HWD), is a widespread mosquito-borne helminth. The true prevalence of HW infection in cats is likely underestimated due to the difficulty in establishing a definitive diagnosis. Aedes albopictus, a recognized competent vector for D. immitis, is currently considered the most invasive mosquito species worldwide and Italy presents the highest abundance of Ae. albopictus in Europe. Materials and Methods: The present study was aimed at evaluating the current seroprevalence of D. immitis antibodies in cats in Italy. Further, the ecological niche model (ENM) was applied to evaluate the potential future risk of feline HWD associated with the presence of Ae. albopictus. Results: Sera of 812 Italian cats were analyzed, and the average prevalence of D. immitis antibodies was 12%. Seropositivity was significantly associated with age (<6 years), whereas there was no association with sex or geographical area. Data obtained with the risk map showed that the highest risk of infection was found in northern inland areas and along coastal areas, whereas the lowest risk was identified at higher altitudes. The ENM correctly classified most of the areas where D. immitis seropositive cats were found, with 80.4% occurring in high and very high-risk areas. Conclusions: Results of the present study suggest that cats in Italy are exposed to D. immitis infection, and that routine prevention should be part of the general health care protocols in cats. Moreover, the resulting risk maps indicate that areas with a suitable habitat for Ae. albopictus may put cats at risk of exposure to D. immitis.

Efficacy of topical administration of prallethrin-permethrin-piperonyl butoxide (Bronco® Equine Fly Spray) for the treatment and control of flies and other nuisance insects of horses.

Numerous biting and nuisance insects are a noted cause of discomfort and stress to horses. Pyrethrins and pyrethroids have been used for many years in numerous formulations for the control of insect pests in animals, humans and environment. There are, however, few studies reporting their field efficacy in horses. The aim of the present study was to evaluate the repellent activity of a spray formulation based on prallethrin and permethrin synergized with piperonyl butoxide (BRONCO® Equine Fly Spray, Farnam Companies, Inc., USA) against annoying and harmful insects for horses in field conditions. Nine horses of mixed breed were divided into 2 groups (treatment and control). Pre-treatment insect counts were compared to daily counts for 4 days post-treatment (pt). One minute after the administration of the product (day 0), all the horses were negative for the presence of insects. All counts up to the 6-h pt check remained negative for Hippobosca equina, tabanid flies and Simulium spp., showing 100% efficacy. This remained above 90% throughout the study. For the H. equina, the repellent efficacy remained > 99.7% for all 4 days pt, for tabanid flies > 93.3% and for Simulium spp. > 97.4%. The efficacy against Musca spp. decreased from 82.2% at day 0 to 62.2% at day 3. Treatment was well-tolerated. In conclusion, despite the low number of tested horses, Bronco® has demonstrated high insecticide and repellent efficacy and a good persistence, maintained for up to 4 days post-treatment, against the most common species of insects harmful for horses.

How Italian practitioners manage dirofilariosis in dogs and cats? Data of a second national survey.

The zoonotic nematodes Dirofilaria immitis and Dirofilaria repens are endemic in many European countries and their distribution is changing. A correct diagnosis is the starting point to reduce their spread and veterinary practitioners represent the first line of defense against these two vector-borne diseases in pets and humans. With the aim to better understand current knowledge by veterinarians and to compare them with the previous survey, an online questionnaire was sent to Italian companion animal veterinary facilities. The overall response rate was 22.3%. Approximately 50% and 5% of facilities have diagnosed at least one case of Dirofilaria spp. in the last year in dogs and cats, respectively. In agreement with international guidelines, of the 665 facilities that responded to the questionnaire, 16.8% used Knott's test in combination with serology (Antigenic test-Ag-) to diagnose heartworm infection in dogs. For the diagnosis of D. repens infection, 13.8% used Knott's test in dogs and cats, and 10.5% used Knott's test only in dogs. Most facilities recommended beginning prevention of canine heartworm disease in April and ending in November. Injectable moxidectin and oral ivermectin were the two most commonly used preventives for D. immitis in dogs, while selamectin was most frequently used in cats. The drug most commonly used for heartworm adulticide treatment in dogs was melarsomine followed by the combined ivermectin-doxycycline treatment. The results of this study suggest that, on the one hand, veterinarians are aware of the recent developments in adulticide therapy, while on the other hand, diagnosis is not always made according to what is recommended by international guidelines and that the start and end times of prevention may not be optimal. Furthermore, the data show an increase in cases of coinfection compared to the previous survey.

Diversity and geographic distribution of haplotypes of Dirofilaria immitis across European endemic countries.

BACKGROUND: Dirofilaria immitis, also known as heartworm, is one of the most important parasitic nematodes of domestic dogs, causing a potentially serious disease, cardiopulmonary dirofilariosis, which can be lethal. This species seems to be less 'expansive' than its sister species Dirofilaria repens, and it is believed that climate change facilitates the spread of this parasite to new non-endemic regions. METHODS: In total, 122 heartworm isolates were analysed from nine endemic countries in Europe (Portugal, Spain, Italy, Greece, Hungary, Romania, Slovakia, and Ukraine) and a single isolate from Bangladesh by amplification and sequencing of two mitochondrial (mt) DNA markers: cytochrome c oxidase subunit 1 (COI) and dehydrogenase subunit 1 (NADH). The main aim of the current study was to determine the genetic diversity of D. immitis and compare it with D. repens haplotype diversity and distribution. DNA was extracted from adult heartworms or microfilariae in blood. Most isolates originated from dogs (Canis lupus familiaris) while 10 isolates originated from wildlife species from Romania, including eight isolates from golden jackals (Canis aureus), one isolate from a Eurasian otter (Lutra lutra) and one isolate from a red fox (Vulpes vulpes). RESULTS: Median spanning network analysis was based on the combined sequence (1721 bp) obtained from two mt markers and successfully delineated nine haplotypes (Di1-Di9). Haplotype Di1 was the dominant haplotype encompassing 91 out of the 122 sequences (75%) from all nine countries and four host species. Haplotype Di2 was the second most common haplotype, formed solely by 13 isolates from Italy. The remaining sequences were assigned to Di3-Di9 haplotypes, differing by 1-4 SNPs from the dominant Di1 haplotype. There was evidence for geographical segregation of haplotypes, with three unique haplotypes associated with Italy and four others associated with certain countries (Di4 and Di7 with Slovakia; Di8 with Greece; Di6 with Hungary). CONCLUSION: Diversity in D. immitis mt haplotypes was lower by half than in D. repens (9 vs. 18 haplotypes in D. immitis and D. repens, respectively), which may be associated with the slower expansion of heartworm in Central and NE Europe. NADH gene appears to be conserved in Dirofilaria sp. by showing lower genetic diversity than the analysed COI gene.

Diagnostic challenges for Aelurostrongylus abstrusus infection in cats from endemic areas in Italy.

BACKGROUND: The lungworm Aelurostrongylus abstrusus infects wild and domestic feline species worldwide and is considered a primary respiratory parasite of cats. Definitive diagnosis is based on the identification of first-stage larvae (L1s) released in faeces approximately 5 to 6 weeks after infection. More recently, serology has been shown to be a diagnostic alternative for A. abstrusus infection in cats. The present study aimed at evaluating the diagnostic performance of serological antibody detection compared to faecal examination for A. abstrusus infection in a population of cats with known infection status from endemic areas in Italy and to identify factors (larval scores, age, co-infections with other helminths) that may influence test sensitivity and specificity of serology. METHODS: All cats resulting positive using the Baermann technique (n = 78) were tested with the A. abstrusus ELISA. An additional 90 serum samples from cats living in three geographical areas with infection prevalence > 10%, but that resulted negative on Baermann, were also tested. RESULTS: Among 78 cats copromicroscopically positive for L1s of A. abstrusus (Group 1), 29 (37.2%) were seropositive in ELISA. Of the 90 cats from Group 2 (cats living in three geographical areas in Italy with A. abstrusus prevalence > than 10%, but negative on Baermann examination), 11 (12.2%) were positive on ELISA. The overall seroprevalence was 23.8%. There was no statistical difference either between average optical density (OD) values of cats excreting > 100 L1s vs. cats excreting < 100 L1s (0.84 vs. 0.66; P value = 0.3247) or comparing the OD values with age of infected cats. Few Baermann-negative cats positive for Toxocara cati or hookworms were seropositive, supporting lack of cross-reactivity to these nematodes. CONCLUSIONS: Results from the present study suggest that relying solely on faecal examination may underestimate prevalence of A. abstrusus infection in cats and that field surveys based on antibody detection are useful for establishing true prevalence of infected and/or exposed animals.

A New Method for Ivermectin Detection and Quantification through HPLC in Organic Matter (Feed, Soil, and Water).

Ivermectin is a macrocyclic lactone widely used in veterinary medicine for its broad-spectrum antiparasitic properties. It has been proven to be effective and safe. The purpose of this study was to develop a high-performance liquid chromatography method with a diode array detector for ivermectin screening in feed and water for animal consumption. Furthermore, the objective was to quantify ivermectin levels that were higher than 0.5 mg/kg in solid matrixes and 0.1 mg/kg in water. Doramectin was used as process standard. Samples were extracted using solid phase extraction with silica and C-18 columns. The method involved the use of high-performance liquid chromatography (HPLC) with a diode array detector (DAD). The results were interpreted using a calibration curve built with ivermectin standards at multiple concentrations (0.5, 1, 2, 5, and 12.5 mg/kg). Statistical evaluation of data was done using ANOVA. The data analysis showed that the linear regression was highly significant (P < 0.001), the intercept values were not significantly different from zero, and the correlation coefficient values (>0.999) indicated excellent linearity. Further tests demonstrated that this method is also useful when studying soil matrixes. The soil was dried and analyzed in the same way as feed; the same recoveries were realized on the spiked samples. The method is easy, inexpensive, precise, and repeatable; it requires very small amounts of sample.

Efficacy of oral, topical and extended-release injectable formulations of moxidectin combined with doxycycline in Dirofilaria immitis naturally infected dogs.

BACKGROUND: Several studies in both experimentally and naturally infected dogs have reported the adulticide effect of a combination of macrocyclic lactones and doxycycline against Dirofilaria immitis, showing that these protocols can be used as an alternative to melarsomine. The present study evaluated the efficacy of oral, topical and extended-release injectable formulations of moxidectin when combined with doxycycline in dogs naturally infected with D. immitis from a shelter located in southern Italy. METHODS: Thirty dogs with naturally acquired D. immitis infection were divided in three groups (G) and treated with oral moxidectin (G1) once a month for 9 consecutive months, topical moxidectin (G2) once a month for 9 consecutive months or extended release moxidectin injectable (G3) at enrolment and again at 6 months (Day 180). All treatment groups received doxycycline for the first 30 days. Microfilarial concentrations in 1 ml (mff/ml) blood were determined monthly for 9 months with the modified Knott's test. A clinical scoring system was employed for each dog enrolled in the study based on thoracic radiography and cardiac ultrasound (CU) examinations performed at Day - 15 (before treatment) and at Day 180. RESULTS: Results from the present study suggest that the majority of dogs from all treatment groups became antigen negative, as evaluated at Day 270: 9/10 dogs (90.0%) from G1, 6/10 dogs (60.0%) from G2 and 8/10 dogs (80.0%) from G3. Improvement of radiographic alterations was observed in all treatment groups, and almost all dogs were cleared of pulmonary abnormalities by 6 months from the beginning of treatment (P = 0.000). Cardiac ultrasound examination showed a progressive improvement of cardiac function in a limited number of animals (4/30). CONCLUSIONS: The combination of doxycycline and three different formulations of moxidectin leads to antigen-negative status in naturally infected dogs.

Seroprevalence of Ascaris suum compared to milk spot prevalence at slaughter in Italian fattening pigs.

Ascaris suum is one of the most important parasites of pigs. Apart from liver condemnation due to lesions caused by migrating larvae ("milk spots"), A. suum infections can compromise weight gain, feed conversion efficacy, as well as meat quality. The true prevalence of infection depends on the diagnostic test used and is often underestimated. We compared liver inspection at slaughter with serology, based on the recognition of a purified A. suum haemoglobin or complete homogenate of the 3rd stage larvae isolated from lungs, in nine pig farms in northern Italy. Liver lesions were found on all farms with prevalence ranging from 3.8% to 98.3%. All farms were also positive for circulating antibodies against As-Hb and As-Lung-L3, with prevalence among pigs on each farm ranging from 36.4-100% and 54.5-100%, respectively. Seroprevalence was consistently higher when compared to the prevalence of milk spots at slaughter. The higher sensitivity of the ELISA tests combined with their ease of use makes them an interesting tool to evaluate A. suum infection levels.

Transporter gene expression and Wolbachia quantification in adults of Dirofilaria immitis treated in vitro with ivermectin or moxidectin alone or in combination with doxycycline for 12 h.

Due to their marked larvicidal activity, macrocyclic lactones (MLs) are used for the prevention of heartworm disease ( Dirofilaria immitis) in dogs. They have also been shown to eliminate adult parasites after long-term administration, with a so-called "slow-kill" effect. In addition, recent studies have established that a combination of doxycycline, which eliminates the endosymbiont Wolbachia, and MLs has superior adulticide effects when compared to MLs alone. It has been hypothesized that the apparent synergism between doxycycline/MLs may be due to interaction with drug efflux transport proteins. The aim of the present study was to evaluate gene expression of several transport proteins in D. immitis adults treated in vitro either with doxycycline alone, ivermectin alone, moxidectin alone, or a combination of ivermectin or moxidectin with doxycycline for 12 h. Quantitative PCR analysis showed a sex-dependent response to treatments. In female worms, Dim-pgp-10, Dim-haf-1 and Dim-haf-5 were upregulated compared to controls with doxycycline alone and when combined with ivermectin. Moxidectin did not induce any changes in gene expression. In males, moxidectin administered alone induced a slight increase in Dim-pgp-10, Dim-pgp-11and Di-avr-14, while ivermectin in combination with doxycycline produced significant upregulation of the ML receptor Di-avr-14. These results suggest possible synergism between the two drug classes and different susceptibility of males vs. females to adulticide effects.

Successful treatment of pleural and peritoneal larval mesocestoidosis with fenbendazole in two dogs.

Mesocestoides spp. are common intestinal tapeworms of dogs and cats. The presence of numerous larval stages in the pleural or peritoneal cavities causes larval mesocestoidosis, that is a potentially life-threatening disease in massive infection. In vivo diagnosis of larval mesocestoidosis is challenging and confirmed diagnosis is often made post-mortem. For this reason, therapeutic recommendations are empirical and there is currently no data on the best treatment options in dogs and cats. Both fenbendazole and praziquantel have been used to treat canine larval mesocestoidosis but failure of complete clearance of larvae has been described for both. Treatment with fenbendazole at 100 mg/kg orally twice a day, for 1-3 months has been reported as being effective. However, it is known that administration of high, off-label doses of fenbendazole can lead to adverse reactions. Here, we report the efficacy of fenbendazole at a lower dosage (50 mg/kg twice a day) in two dogs with pleural and peritoneal mesocestoidosis.

Prevalence and risk factors associated with cat parasites in Italy: a multicenter study.

BACKGROUND: Parasites that infect cats include protozoa, helminths and arthropods, many of which are transmissible to humans. Effective control relies on a good knowledge of parasite distribution and the risk factors for infection. The present study was aimed at evaluating the prevalence of major feline parasites in Italy and the risk factors associated with their occurrence. METHODS: Over a 12-month study period, feces, hair and ectoparasites from naturally infected cats from feral colonies, shelters and private households were analyzed at 13 study centers across Italy. Samples from these cats (n = 987) were analyzed at all centers using the same diagnostic methods. Prevalence values and risk factors were evaluated statistically for the identification of predictors of risk. RESULTS: The overall prevalence of gastro-intestinal and broncho-pulmonary (BP) nematodes was 35.9% (354/987). Toxocara cati was the most prevalent species (253/987; 25.6%), followed by Ancylostomatidae (98/987; 9.9%). Among BP nematodes, Aelurostrongylus abstrusus was the most common (76/987; 7.7%). Approximately 35.7% (352/987) of the study population was infested by ectoparasites, of which the most common were fleas (29.4%, 290/987), followed by ear mites Otodectes cynotis (9.8%, 97/987). Predictors of risk for parasite infection included age, a predominantly or exclusively outdoor lifestyle, geographic area and lack of antiparasitic treatment. CONCLUSIONS: Both ecto- and endoparasites are still common in cats throughout Italy, many of them being of zoonotic concern and vectors of pathogens to humans. Given the presence of parasites throughout the entire study period, year-round treatment should be considered. Furthermore, data confirm the need to protect the human-animal bond using proper endo- and ectoparasiticides to reduce the risk of human infection, in application of the One-Health concept.

Evaluation of alternative reagents on the performance of the modified Knott's test.

The aim of the present study was to evaluate the suitability of different reagents as safe alternatives to 2% formalin in the modified Knott's test for the diagnosis of subcutaneous (Dirofilaria repens) and cardiopulmonary (D. immitis) dirofilariosis. A total of 61 blood samples from dogs naturally infected with D. immitis and D. repens were collected and analysed in two different laboratories (Lab 1, University of Parma and Lab 2, University of Napoli). For each blood sample the modified Knott's method was performed to identify and measure the mean length and width of the microfilariae (mfs) using 2% formalin (A), 2% acetic acid (B), 2% glacial acetic acid (C), 10% saponin (D) and distilled water (E). When compared to 2% formalin, there was no significant difference (P > 0.05) among the mean length and width of either D. immitis or D. repens mfs with distilled water (E). The lengths and widths of mfs, however, were significantly reduced (P < 0.05) when using B, C, D likely due to more pronounced parasite dehydration. Despite differences in measurements, the morphological features of the head and tail of the two species were maintained, suggesting that all the solutions tested could be a suitable alternative to formalin. All alternative reagents caused more marked haemolysis compared to formalin, improving readability of slides. The values of the mean length and the mean width of D. immitis and the mean width of D. repens mfs obtained with formalin and distilled water were statistically different (P < 0.005) between the two laboratories. The difference in mf measurements between the two labs could be due to the use of reagents purchased from different manufacturing companies. Results suggest that distilled water could replace formalin in the modified Knott's test, as a safer reagent that allows morphology-based species differentiation of Dirofilaria spp.

A survey on canine leishmaniosis: Best practice and guideline awareness among Italian veterinary practitioners.

Canine leishmaniosis (CanL) is a severe, potentially life-threatening disease of dogs and is considered one of the most important vector-borne zoonoses in Europe, where there is evidence that it is currently spreading into previously unaffected areas. Veterinary practitioners represent the first-line defence against CanL and in recent years, several guidelines have been developed to assist veterinarians in the correct diagnosis, management and prevention of the disease. The present study was aimed at surveying Italian practitioners on how they manage CanL and compare results to recommendations from guidelines developed by the European Scientific Council for Companion Animal Parasites (ESCCAP®), by the Leishvet® organization and by the Canine Leishmaniasis Working Group (C.L.W.G.). Approximately 14.0 % of veterinarians contacted replied to the survey. Results suggest that Italian practitioners are aware of currently available guidelines for the management of CanL and adherence to recommendations is good. Interestingly, 16.0 % carry out year-long prevention, even though recent data suggest that transmission in Italy continues to be seasonal. In conclusion, the high awareness among practitioners reported here should be considered an excellent opportunity for guideline developers to continue updating their recommendations to include any new information that can support the correct management of CL.

Effectiveness of Fenbendazole and Metronidazole Against Giardia Infection in Dogs Monitored for 50-Days in Home-Conditions.

A field trial performed in-home conditions was conducted on 24 dogs naturally infected with Giardia, in order to compare the efficacy of fenbendazole and metronidazole. Animals were allocated in groups randomly in order to obtain two groups of 12 dogs each with similar parasitic loads of Giardia cysts: dogs in Group A were treated with fenbendazole (Panacur®, Intervet Italia Srl) administered at the dose of 50 mg/kg orally once a day for 5 consecutive days, dogs in Group B were treated with metronidazole (Flagyl®, Zambon Italia Srl) administered orally at the dose of 50 mg/kg, once a day for 5 consecutive days. All the dogs that were shedding Giardia cysts after the first treatment (Day 0) were retreated (either at Day 7 or at Day 14 or at Day 21) until a negative result was obtained with the same treatment. Additionally, all the dogs were re-examined at Day 50. All the dogs were tested for the presence of Giardia cysts using a fecal flotation method (FLOTAC). The percent efficacy of the treatments (A and B) was calculated at each sampling point (Days 7, 14, 21, and 50) as reduction in mean Giardia cysts. After the first therapy, on day 7, 4/12 (33.3%) dogs tested positive for Giardia cysts in the Group A and 5/12 (41.7%) in the Group B. Efficacies at (Days 7, 14, 21, and 50) of the treatments against Giardia infection were 80.9, 94, 100, and 97% in the Group A and 70.8, 99, 100, and 97.1% in the Group B. Statistically significant differences were not observed between the efficacy of Fenbendazole and Metronidazole against infection by G. duodenalis (P = 0.686). Molecular analysis revealed full homology (i.e., 100% with JN416550) with the canine specific assemblage D in six positive dogs. Different hypotheses might explain the re-appearance of the Giardia cysts in some dogs after treatment, e.g., re-infection from the home environment, the correct medication given by the owners, the diet, as well as treatment failure, but also biological issues related to the intermittent excretion of Giardia cysts.

First report of Echinococcus granulosus sensu lato genotype G6/G7 in domestic pigs from mainland Italy.

Cystic echinococcosis (CE; Echinococcus spp.) is widespread in many domestic animal species in Italy, with the G1-G3 genotype predominating. The G7 genotype ("pig strain"), which is much less common, has only been reported in pigs and wild boar from the island of Sardinia and in wild boars from southern mainland Italy. Ten pig livers with hydatid cysts were identified in a slaughterhouse in northwestern Italy. Multiplex PCR for Echinococcus granulosus gave positive results for two of these and subsequent sequencing confirmed the species as Echinococcus granulosusu s.l. G6/G7. Affected pigs came from an intensive farm in northeastern Italy. This is, to the authors' knowledge, the first report of CE by Echinococcus granulosusu s.l. G6/G7 in the domestic pig in mainland Italy. E. granulosus s.l. G6/G7 is zoonotic and its circulation in Italy should be of concern for public health.

Toxoplasma gondii in the Eurasian kestrel (Falco tinnunculus) in northern Italy.

BACKGROUND: Identifying factors that sustain parasite transmission is important for understanding their spread and emergence, including how changes in biodiversity may affect parasite prevalence and spread. Toxoplasma gondii is a protozoan parasite infecting humans and animals. Birds can acquire T. gondii infection through ingestion either of oocysts from the ground or of tissue cysts present in infected prey and are therefore suitable indicators of the presence of T. gondii in the natural environment. METHODS: The aim of the study included the evaluation of T. gondii seroprevalence in clinically healthy Eurasian kestrels (Falco tinnunculus) using a modified agglutination test. Birds were captured in a small area of Parma (northern Italy) for two consecutive years (2016-2017), sex and age determined and serological study carried out. Food sources for the birds were also evaluated, in particular rodent and grasshopper population estimates in the study area. The biomass of rodents and grasshoppers per hectare was estimated in order to directly compare food availability. Statistical analyses were performed in order to evaluate factors influencing the probability of kestrels being T. gondii-seropositive using R 3.4.4 fitting linear mixed-effect models with the 'glmer' function of the package lme4, 'lsmean' in package lsmean for pair-wise post-hoc comparisons using differences of least square means (DLSM) and the 'betareg' function of the package betareg for beta regression. RESULTS: Seroprevalence for T. gondii was 33.3% (49/147) in 2016, while in 2017 seroprevalence decreased to 14.3% (13/91). An increase in the probability of kestrels being T. gondii-seropositive was associated with a higher rodent biomass in the environment, suggesting a positive feedback of the biotic factors driving infection risk. CONCLUSIONS: These results underline the need for multidisciplinary studies aimed at better understanding pathogen-host relationships and for predictions in disease ecology.

Chimeric symbionts expressing a Wolbachia protein stimulate mosquito immunity and inhibit filarial parasite development.

Wolbachia can reduce the capability of mosquitoes to transmit infectious diseases to humans and is currently exploited in campaigns for the control of arboviruses, like dengue and Zika. Under the assumption that Wolbachia-mediated activation of insect immunity plays a role in the reduction of mosquito vectorial capacity, we focused our attention on the Wolbachia surface protein (WSP), a potential inductor of innate immunity. We hypothesized that the heterologous expression of this protein in gut- and tissue-associated symbionts may reduce parasite transmission. We thus engineered the mosquito bacterial symbiont Asaia to express WSP (AsaiaWSP). AsaiaWSP induced activation of the host immune response in Aedes aegypti and Anopheles stephensi mosquitoes, and inhibited the development of the heartworm parasite Dirofilaria immitis in Ae. aegypti. These results consolidate previous evidence on the immune-stimulating property of WSP and make AsaiaWSP worth of further investigations as a potential tool for the control of mosquito-borne diseases.

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.

Efficacy of imidacloprid 10%/moxidectin 2.5% spot on (Advocate®, Advantage Multi®) and doxycycline for the treatment of natural Dirofilaria immitis infections in dogs.

Heartworm infection (also known as dirofilariosis due to Dirofilaria immitis) in dogs causes chronic pulmonary disease that, if left untreated, can lead to right-side congestive heart failure. Currently, the only registered drug for adulticide therapy in dogs with heartworm disease (HWD) is melarsomine dihydrochloride. The recent targeting of the bacterial endosymbiont Wolbachia, through antibiotic therapy of the infected host, has offered an interesting alternative for the treatment of HWD. Recent reports of the adulticide activity of an ivermectin/doxycycline combination protocol has lead the American Heartworm Society (AHS) to include in its guidelines that, in cases where arsenical therapy is not possible or is contraindicated, a monthly heartworm preventive along with doxycycline for a 4-week period might be considered. In the present study, 20 dogs with confirmed natural D. immitis infection were included following owner consent. Fourteen dogs were treated with a topical formulation containing 10% w/v imidacloprid and 2.5% w/v moxidectin (Advocate®, Advantage Multi®, Bayer), monthly for nine months, associated to doxycycline (10 mg/kg/BID) for the first 30 days. Six dogs were treated with melarsomine (Immiticide®, Merial) (2.5 mg/kg) at enrollment, followed one month later by two injections 24 h apart. The presence of circulating antigens and the number of microfilariae (mf) were evaluated at the moment of enrollment and then at 1, 2, 3, 4, 5, 6, 7, 8, 12, 18, 24 months post enrollment. Echocardiogram and radiographs were performed at month 0, 6, 12, 18, 24. Monthly moxidectin combined with 30 days of doxycycline eliminated circulating microfilariae within one month, thus breaking the transmission cycle very quickly. Furthermore, dogs treated with the combination protocol started to become negative for circulating antigens at 4 months from the beginning of treatment and all except one were antigen negative at 9 months. All dogs treated with melarsomine were antigen negative by 5 months from the beginning of the treatment. No dogs showed worsening of pulmonary patterns or criteria indicative of pulmonary hypertension 12 to 24 months after. For the criteria mf concentration, antigen concentration, radiography and echocardiography at 12, 18 and 24 months the non-inferiority for the moxidectin group could be proven for a non-inferiority margin of 15% for the rate difference. Dogs treated with moxidectin and doxycycline became negative for microfilariae and antigens sooner when compared to melarsomine in the present study and to dogs treated with doxycycline combined with ivermectin in studies previously published.

Differential ABC transporter gene expression in adult Dirofilaria immitis males and females following in vitro treatment with ivermectin, doxycycline or a combination of both.

BACKGROUND: Combination doxycycline/macrocyclic lactone (ML) protocols have been shown to provide a more rapid adulticidal and microfilaricidal effect than either MLs or doxycycline alone, although female worms were reported to have a higher tolerance to treatments compared to male worms. The present study aimed to evaluate how ABC transporters may be involved in the synergic effect of the combination treatment. Adult worms of D. immitis were treated in vitro for 24 hours with doxycycline (DOXY), ivermectin (IVM) and a combination of both, and changes in the modulation of ABC transporter genes were measured. Levels of doxycycline inside different treatment media, post-treatment, were determined through HPLC analysis. RESULTS: Quantitative RT-PCR analysis showed the presence of changes in the modulation of ABC transporter genes evaluated in this study. In particular, in female worms, the combination treatment induced a substantial increase in gene expressions, especially of Dim-pgp-10 and Dim-haf-4; whereas in male worms, the greatest increase in gene expression was observed for Dim-pgp-10 and Dim-pgp-11 when treated with DMSO + IVM and DMSO + DOXY/IVM. HPLC analysis of the DOXY concentrations in the media after in vitro treatments of male worms showed a slight difference between the DMSO + DOXY samples and the combination (DMSO + DOXY + IVM), while no difference was observed among females. CONCLUSIONS: Further studies are required to explain whether the modulation of cellular efflux plays a role, even partially, in the adulticide effect of doxycycline/macrocyclic lactone combinations in heartworm-infected dogs. To the authors' knowledge, this is the first study to evaluate P-gp expression in adult D. immitis.