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.

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.

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.

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.

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.

The prevalence of Dirofilaria immitis and D. repens in the Old World.

Dirofilaria immitis and D. repens are endemic throughout Europe and southern eastern regions of Asia and reported with increasing frequency in Africa. Nevertheless, the increased awareness of veterinary practitioners, even in countries where the prevalence is low, has led to a decrease D. immitis prevalence in dogs, especially in previously endemic/hyper-endemic areas. Prevalence has significantly increased, however, in areas where heartworm has apparently spread more recently, such as Central and North Eastern Europe. Furthermore, autochthonous cases have been observed in Siberia. Low seroprevalence has been reported in Croatia, while in Romania it has reached 14%. In Greece, the prevalence ranges between 0.7% and 25% whilst in Turkey is 0-18%. Data for canine dirofilariosis in Africa is scarce, and most are case reports. Overall, the dominant species is Achanthocheilonema dracunculoides, although both D. immitis and D. repens have been reported from some countries. In the Far East, the prevalence ranges from 2% to 15% in northeastern of China. In Hong Kong a novel species has been found in dogs and humans (Candidatus Dirofilaria hongkongensis). In India, the prevalence ranges from 4.7%-29.5% in Northeastern states. The main factors that have influenced the spreading of Dirofilaria infections are the climate changes and the introduction of new, invasive, competent mosquito species such as Aedes albopictus and Ae. koreicus. Other factors include relocation and insufficient prevention in dogs, manly in the new areas of colonization. Feline heartworm infection has been diagnosed in every European country when diagnosed either by the Knott test or by serology for circulating antibodies and antigens of the parasite. However, prevalence is much lower than in dogs. In spite of the continuing spreading of heartworm infection, D. repens is the main concern in Europe, mostly for physicians, while the infection is nearly always asymptomatic in dogs. The infection is spreading from Portugal to the Southeastern regions of Finland and Siberia, and in some areas its prevalence overlaps that of D. immitis. Many reasons make more difficult the control of D. repens than D. immitis: the frequent lack of clinical symptoms of suspicion, the specific diagnosis being possible only by blood examination and the inefficacy of some macrocyclic lactones.

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.

Immunoproteomic approach for identification of Ascaris suum proteins recognized by pigs with porcine ascariasis.

Ascaris suum, the causative agent of porcine ascariasis, is responsible for marked economic losses in pig farms worldwide. Despite recent advances in research, including the characterization of the genome of A. suum, knowledge about the parasite/host relationship in porcine ascariasis at the molecular level is scarce and chemotherapy is the only effective option for parasite control. The aim of this study was to identify immunogenic proteins of A. suum somatic antigens associated with the pathogenicity/survival mechanisms of the parasite, by using two-dimensional (2-D) electrophoresis, 2-D Western blot and mass spectrometry (MS). A total of 24 parasite proteins recognized by serum samples from pigs naturally infected with A. suum were identified. Most of them (23/24) were identified as being involved in parasite survival mechanisms, including functions related to energy generation (12 proteins) and redox processes (5 proteins). These results may aid the search for effective chemo-therapeutic targets in porcine ascariasis. Further studies are needed, however, to illustrate the effect of the host immune response on the survival mechanisms of A. suum.