Cardiopulmonary nematodes, Dirofilaria immitis and Angiostrongylus vasorum, are severe and life-threatening parasites increasingly reported in dogs throughout Europe. However, the lack of country-wide epidemiological data regarding these two vector-borne helminths in Italy could make more difficult the awareness by practitioners and thus the implementation of effective prevention and control strategies. According to this, the present thesis presented new epidemiological and diagnostic data on dirofilariosis and angiostrongylosis underlying their clinical impact in dogs in Italy as in other European countries. In order to reach our purpose, four objectives were fulfilled: (i) the prevalence of D. immitis and A. vasorum in kennel dogs from southern Italy by serological analysis; (ii) the extent and clinical relevance of A. vasorum infection in owned and stray dogs from southern Italy by using the FLOTAC technique; (iii) the prevalence of D. immitis through ELISA antigen testing before and after heat treatment, in dogs from eastern Romania; (iv) the evaluation of single and multiple vector-borne infections (Dirofilaria, Leishmania and Ehrichia) in dogs from Spain and Italy by ematological and serological diagnosis by also testing the possibility of a D. immitis quantitative ELISA test to reverse false negatives due to antigen-antibody complexes.
The PhD thesis entitled “Dirofilaria immitis and Angiostrongylus vasorum in dogs: epidemiological and diagnostic challenges” consists of two parts, according to the European standard requirements. The first part - entitled “Literature Review” - is divided into two chapters and summarizes information from literature about aetiology, epidemiology, clinical implications, diagnostic concerns and treatment approaches of heartworm diseases and angiostrongylosis in dogs. The second part entitled - “Own Research” - presents the general and specific aims of the thesis followed by four original studies conducted in Italy, Romania and Spain, focused on epidemiology, diagnosis and clinical relevance of Dirofilaria and Angiostrongylus infection in dogs, with conclusions and recommendations.
The literature review in Chapter 1 provides an overview of the main cardiopulmonary nematode (Dirofilaria immitis) infection in dogs in Europe. Data regarding aetiology, life cycle and other biological aspects, pathogenesis, clinical diagnosis, treatment and prevention are discussed in detail with emphasis on the geographical distribution and diagnostic concerns of the infection. Chapter 2 provides literature data on aetiology, life cycle and other aspects of the clinical, diagnostic and control challenges for canine angiostrongylosis. Subsequently, we also analyse the current distribution of canine angiostrongylosis in Europe and Italy. This review provided herein, indicates a lack of detailed studies on the prevalence of both D. immitis and A. vasorum pathogens in southern Italy, as well as data regarding the clinical importance of these nematodes.
Chapter 3 assesses the seroprevalence of D. immitis and the faecal presence of first stage larvae (L1) of A. vasorum in dogs from 68 kennels of the Campania region (southern Italy). Antigens of D. immitis were detected in 24/537 (4.4%) dogs in 6 out of the 68 kennels (8.8%). A. vasorum L1 were detected in dogs from 9 out of the 68 kennels (13.2%). Pooled faecal samples from 25 boxes (epidemiological unit in the kennel) out of the 1360 analyzed (1.8%) resulted positive to A. vasorum L1. The results indicated that cardiopulmonary nematodes are present in southern Italy in symptomatic dogs as well as in asymptomatic ones. Therefore, regular parasitological surveillance, appropriate treatment strategies and high-quality standard of hygiene are required to guarantee the health and welfare of kennel dogs.
Chapter 4 reports the findings of a survey conducted in southern Italy in order to evaluate the extent and clinical relevance of A. vasorum infection in owned and stray dogs using the FLOTAC technique. A. vasorum was detected in 29 out of 1499 faecal samples examined (1.9%). Out of 656 owned dogs examined, seven (4 females, 3 males) were positive for A. vasorum (1.1%); age ranged between 18 and 48 months; the LPG values detected varied between 10-560 (mean value = 216 LPG). Out of 843 stray dogs investigated, 22 (14 females, 8 males) were positive for A. vasorum (2.6%). Age ranged between 12 and 84 months and the LPG count varied between 10-668 (mean value = 254.8 LPG). The prevalence in stray dogs (2.6%) was significantly higher than the prevalence detected in owned dogs (1.1%). Seven dogs resulted positive to A. vasorum by FLOTAC showed clinical evidences compatible with the lungworm infection. As expected, clinical signs of A. vasorum infected animals were: gagging, coughing, anorexia, weight loss, exercise intolerance. The most consistent findings at the radiological exam were: generalized interstitial and alveolar pattern and enlargement of tracheobronchial lymph nodes. The findings showed a spread of A. vasorum in southern Italy, demanding the necessity of stimulating concern on this infection among vet practitioners, which should always include angiostrongylosis on differential diagnosis when signs are consistent.
Chapter 5 provides results of a study aimed to determine infection prevalence for D. immitis, through ELISA antigen testing before and after heat treatment, in dogs from eastern Romania where both D. immitis and Dirofilaria repens are endemic. Of 194 dogs sampled from four cities in Romania, D. immitis circulating antigens were found in 16 (8.2%) non-heated samples and in 52 (26.8%) heated samples. Of the 108 dogs examined by Knott test, 24 dogs (22.2%) were positive for circulating microfilariae (mf). Fifty% of dogs with circulating D. immitis mf had positive antigen tests before and after heating, while the other 50% reverted to positive only after heat treatment. Sixty% of dogs with mixed D. immitis/D. repens infection were antigen positive before and after heating, while the other 40% converted to positive after heating. Antigen testing for D. immitis in the 12 dogs with only D. repens mf gave conflicting results. Only two dogs (16%) were antigen negative both before and after heat treatment. Six dogs (50%) became antigen positive after heating and four dogs (30%) were antigen positive both before and after heat treatment. Results would suggest that: false negative result for antigen testing can be reverted by heating of the serum sample; dogs infected with D. repens may have also an occult infection with D. immitis; heat treatment of serum from D. repens-infected dogs can reveal an occult infection with D. immitis.
Chapter 6 aimed to evaluate the prevalence of single and multiple vector-borne infections (Ehrlichia, Dirofilaria and Leishmania) in dogs from Spain and Italy by ematological and serological diagnosis. In particular, a quantitative ELISA test was evaluated before and after serum heat treatment in order to: detect D. immitis antigen; verify the cross-reactivity with other pathogens; verify the possibility to reverse false negatives due to antigen-antibody complexes. A total of 46 blood samples (23 from Madrid, Spain and 23 from Molise region, Italy) were randomly collected from stray, hunting and owned dogs. All the dogs were screened for D. immitis, D. repens, Leishmania infantum and Ehrlichia canis, using the following laboratory techniques: Knott test, quantitative ELISA test (Petcheck®, Idexx) before and after heat treatment, antibody test (SNAP 4Dx Plus Test). The results of this study showed: i) the presence of L. infantum, E. canis and D. immitis in central Spain as well as in central Italy; ii) the lack of occult infection with D. immitis probably due to the fact that vector-borne pathogens other than D. immitis (e.g. Leishmania and Ehrlichia) even if induce hyper-gammaglobulinemia, not always can affect antigen test results as expected; iii) the increase of OD in heartworm positive samples after heating, confirming the hypothesis that canine serum and plasma from some dogs may contain inhibitors of D. immitis antigen detection, so the heat treatment of these samples prior to testing could improve the sensitivity of these assay in some dogs.
In chapter 7, the present assessments and future perspectives on dirofilariosis and angiostrongylosis in dogs are discussed with particular focus on the epidemiological and diagnostic challenges. Promoting awareness among practitioners and dog owners is one of the priority areas for an integrated parasite control in pets as recommended by the European Scientific Counsel Companion Animal Parasites (ESCCAP) and the European Society of Dirofilariosis and Angiostrongylosis (ESDA)
