CIUCA, LAVINIA (2022) New insights into diagnosis and control of Dirofilaria infection in dogs. [Tesi di dottorato]

Anteprima

Testo Ciuca_Lavinia_35.pdf Download (2MB) | Anteprima

Abstract

Dirofilariosis are an important vector-borne diseases affecting primarily domestic dogs and wild canids, as well as cats, other species of wild mammals and humans. The most important species responsible of dirofilariosis are Dirofilaria immitis and Dirofilaria repens (Spirurida, Onchocercidae) which cause canine heartworm disease and subcutaneous dirofilariosis, respectively. The majority of dogs diagnosed with heartworm infection are asymptomatic (or have only mild symptoms such as intermittent cough) and go through adulticide therapy without complication. Complications that occur with heartworm infection and during its treatment are usually directly directly attributable to damage to the pulmonary vascular and parenchymal injury inflicted by D. immitis. Early diagnosis and treatment are essential for dogs with heartworm disease, as the risk of these complications increase with disease progression. Although D. repens is the main zoonotic species and its presence is increasingly reported in Europe, it has received less attention by researchers than D. immitis, therefore it is under-reported and still neglected. Diagnosis of D. repens in dogs is a challenge: many infected dogs are asymptomatic and there is a limited number of reliable diagnostic tools available. Indeed, diagnosis is mostly based on the detection of circulating microfilariae during patent infection by Knott’s test, followed by morphometric or molecular species identification. Heartworm disease is a parasitosis that is increasing its geographical range over time, for reasons related to the parasites, the hosts and the environment. Southern Italy, once considered low-risk, is increasingly becoming the site of autochthonous outbreaks. Currently, D. repens occurs in southern Italy (Campania and Molise regions), especially in the coastal areas of the two regions. However, the lack of country-wide epidemiological data on these two vector-borne filariosis in Italy could hamper the awareness of practitioners and thus the implementation of effective prevention and control strategies. In order to deepen the knowledge on D. immitis and D.repens infections in dogs, the present thesis entitled “New Insights into the Diagnosis and Control of Dirofilaria Infection in Dogs” had four specific objectives: i) to improve of the Knott's test method for identifying the microfilariae of Dirofilaria immitis and D. repens; ii) to advance the knowledge in the immune response of D. repens in experimentally infected dogs; iii) to evaluate the slow kill therapy with doxycycline and various commercially available formulations of moxidectin in dogs naturally infected with D. immitis; iv) to update the distribution of D. immitis and D. repens in dogs from an endemic area of southern Italy. The PhD thesis consists of two parts, according to the European standard requirements. The first part - entitled “Literature Review” - summarizes information from the literature about aetiology, epidemiology, clinical signs, diagnostic concerns and control approaches on dirofilaria infections in dogs. The second part entitled - “Own Research” - presents the general and specific objectives of the thesis followed by four original studies addressing the epidemiology, diagnosis, clinical relevance and treatment of Dirofilaria spp. infections in dogs. The literature review in Chapter 1 provides an overview of both pathogens Dirofilaria immitis and D. repens 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 diagnostical concerns of the infection. This review provided herein, indicates a lack of detailed studies on the prevalence of both D. immitis and D. repens pathogens in southern Italy, as well as data regarding the clinical importance of these diseases. Moreover, the need to expand our current knowledge on D. repens biology and the immune response of the infected host is mandatory for a better prevention and an accurate diagnose of this canine infection. Chapter 2 reports the suitability of different reagents as safe alternatives to 2% formalin in the modified Knott’s test for the diagnosis of subcutaneous (D. 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). 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. 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. Chapter 3 provides new insights from experimental infections of dogs with D. repens, focusing on the evaluation of: 1) the pre-patent period and 2) the antibody response against D. repens somatic antigens and against the Wolbachia endosymbiont. Briefly, on Day 0, twenty purpose-bred Beagle dogs were experimentally infected with 50 infective larvae (L3) of D. repens. Starting from Day 58 until the last day of the study (Day 281), blood samples were collected on a monthly basis for detection of antibodies against D. repens (Dr) and recombinant Wolbachia surface protein (rWSP) by non-commercial IgG- ELISAs. Additional samples were collected on Days 220, 245 and 281 for the detection of microfilariae (mff) using the modified Knott’s test and biomolecular analysis, following two PCR protocols: Gioia et al. (2010; protocol A) and Rishniw et al. (2006- protocol B). Overall, the outcome of the study revealed that out of the 20 dogs experimentally infected with D. repens, 16 (80 %) were microfilaraemic, 17 (85 %) were positive at DNA detection in the blood, 18 (90 %) had D. repens antibodies and 16 (80 %) had Wolbachia antibodies on the last day of the study. The overall k agreement between Knott’s and PCR protocol B was 0.442 (P = 0.0001) and increased throughout the study, reaching 0.828 (P = 0.0001) on Day 281. Results would suggest that the development of an immunological response to infection could lead to application in epidemiological studies, risk assessment and as an aid in the diagnostic approach in dogs, in particular for early infections without mff. Chapter 4 aimed to evaluate the adulticide effect 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. A total of 30 dogs with naturally acquired D. immitis infection were divided in three groups (G) and treated either with oral moxidectin (G1) once a month for 9 consecutive months, topical moxidectin (G2) once a month for 9 consecutive months or with an 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 concentration in 1 ml (mff/ml) of 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) exams performed at Day -15 (before treatment) and Day 180. In general, mff loads decreased markedly in all dogs from all groups at Day 30, and all but one dog were negative at Day 60. Results from the present study suggest that efficacy (evaluated at Day 270) is related to the moxidectin formulation used and that injectable moxidectin showed superior efficacy compared to topical and oral injectable formulations. Overall, the treatment with moxidectin and doxycycline combination was effective and almost all the dogs from the treatment groups were cleared of pulmonary abnormalities by six months from the beginning of treatment (p-value=0.000). Although the therapy proved to be an effective adulticide, the echocardiographic parameters studied were not able to show a marked improvement of cardiac function after the treatment. Chapter 5 aimed to investigate the occurrence of Dirofilaria spp. in southern Italy. For this purpose, a local dog shelter in Castel Volturno (Caserta province, southern Italy) was selected and dogs were screened for the presence of Dirofilaria spp. A total of 260 blood samples were examined for identification of microfilariae (mff) and for detection of Dirofilaria immitis antigens. Moreover, all the dogs that showed co-infections with both D. immitis and D. repens mff were confirmed with molecular analyses. In addition, data regarding the length of stay of the dogs in the shelter at the time of sampling were also recorded. The dogs were divided into four age classes (class 1: dogs ≤ 2 years; class 2: dogs > 2 ≤ 6 years; class 3: dogs > 6 ≤ 10 years; class 4: dogs > 10 years old) and three groups of dogs based on the length of stay in the shelter at the time of sampling (group 1-new arrivals: dogs that have been received in the shelter in the last four months; group 2- dogs that were housed in the shelter for more than four months up to 2 years; group 3-dogs that were housed for more than 2 years). The modified Knott’s test revealed that 188 dogs (72.3%; 95%CI=66.4-77.6) were positive for circulating mff of Dirofilaria spp. Specifically, 113 (60.1%; 95%CI=52.7-67.1) dogs were positive for D. immitis mff and 75 (39.9%; 95%CI=32.9-47.3) were positive to D. repens mff. In addition, 58 (30.8%; 95% CI=24.4-38.1) dogs presented both D. immitis and D. repens mff. Antigen testing showed 98/260 (37.7%; 95%CI=31.8-43.9) dogs positive to D. immitis. However, 13% (95%CI=6.5-19.2) of the dogs with D. immitis mff were antigen-negative. PCR testing confirmed the co-infections with both pathogens in all 58 dogs. The prevalence was almost twice as high in males (65.4%; 95%CI=58.1-72.1) as in females (34.6%; 95%CI=27.9-41.8). As expected, prevalence was lowest in age class 1 (16.5%; 95%CI=11.6-22.7) and higher in age classes 2, 3 and 4 (25.5%; 95%CI=19.5-32.5; 27.6%; 95%CI=21.5-34.7 and 30.3%; 95%CI=23.9-37.5 respectively) but these differences were not significant. There was a significant difference in relation to the length of stay of the dogs in the shelter, reflecting mainly an increase in prevalence in the group 1 (45.2%; 95%CI=39.0-53.7; P=0.012) in which all dogs were new arrivals in the shelter since four months and their origin was from different localities of the Campania region, including the city center of Naples. We conclude that dog shelters from southern Italy constitute hot spots for Dirofilaria spp. transmission and we strongly recommend education and veterinary guidance regarding regular testing and systematic treatments. The thesis ends with an overall discussion based on the four studies presented herein with particular emphasis on epidemiological status of Dirofilaria spp. infection in dogs in southern Italy, diagnostic challenges and slow-kill treatment of Dirofilaria spp. infection in dogs.

Downloads

Actions (login required)

Modifica documento