Emerging risk of Dirofilaria spp. infection in shelter dogs in southern Italy

In southern Italy, the number of autochthonous cases of Dirofilaria immitis in dogs has increased considerably. This also occurs in the Campania region, particularly in coastal areas, where infections with D. immitis and Dirofilaria repens have been reported more frequently. Therefore the aim of the present study was to better investigate the occurrence of Dirofilaria spp. in a local dog shelter in Castel Volturno (Campania region, southern Italy). Briefly, a total of 260 blood samples were analysed for identification of microfilariae (mff) and detection of Dirofilaria immitis antigen. Dogs were classified according to their age (1–3  years; 4–6  years; 7–11  years; > 11  years) and length of stay in the shelter at the time of sampling (dogs that entered in the shelter in the last 4  months; dogs housed in the shelter for more than 4 months up to 2  years; dogs housed for more than 2  years). The modified Knott’s test revealed that 195 dogs (75.0%) were positive for circulating mff of Dirofilaria spp. Specifically, 104/260 (40.0%) dogs were positive for D. immitis and 91/260 (35.0%) were positive for D. repens. In addition, 72/260 (27.7%) dogs had both D. immitis and D. repens mff. Antigen testing revealed that 78/260 (30.0%) dogs were positive for D. immitis. However, 26/104 (25.0%) of the dogs with D. immitis mff were antigen-negative. The overall k concordance between the modified Knott’s test and the antigenic test was ≤0.2 (poor) (p = 0.000). The results of the logistic regression model showed a significant association between Dirofilaria exposure and the period of time the dogs had spent in the shelter at the time of sampling. Dogs housed in the shelter for 4 months (group 1) and between 4 months and 2 years (group 2) had higher Dirofilaria positivity than dogs in group 3 (housed for more than 2 years) (80.4% vs. 79.6% vs. 62.4%, respectively). Moreover, male dogs and older dogs (between 7 and 11 years of age) were more likely to be infected with Dirofilaria spp.


Introduction
Heartworm disease and subcutaneous dirofilariosis caused by Dirofilaria immitis and D. repens (Spirurida, Onchocercidae), respectively, are important vector-borne diseases, especially for dogs and cats (1)(2)(3)(4).In addition, zoonotic infections, mostly due to D. repens, are a major public health problem (4).The epidemiological situation of canine dirofilariosis based on data reported in recent studies has shown that the prevalence of both D. immitis and D. repens is increasing in Europe and the southerneastern regions of Asia and Africa (1,4,5).However, there are studies that continuously report changes in the prevalence of both D. immitis and D. repens pathogens, identifying non-endemic areas with increased prevalence and previously endemic/hyper-endemic areas with decreasing prevalence (6)(7)(8).For example, the prevalence 10.3389/fvets.2023.1112036Frontiers in Veterinary Science 02 frontiersin.org of D. immitis in northern Italy has decreased from >40% (9) to 8% in owned dogs over the last three decades (6,7), due to the increased awareness of veterinary practitioners and better prevention strategies.
In southern Italy, the number of autochthonous cases and foci of D. immitis in dogs has increased considerably (10,11).This also occurs in the Campania region, particularly in coastal areas, where infections with D. immitis and D. repens have been reported more frequently and the prevalence of D. repens has increased from 2% (12) to 10% (13).As with D. immitis, hunting dogs, stray dogs, and dogs housed in kennels are more exposed, with seroprevalence ranging from 0.2 to 4.4% (14)(15)(16).The results of the questionnaire survey on heartworm and subcutaneous dirofilariosis addressed to veterinary practitioners throughout Italy (7) showed that at least one clinical case per year of cardiopulmonary dirofilariosis was diagnosed in dogs with frequent co-infestation by D. immitis and D. repens in Campania.
Recent studies confirm the presence of autochthonous cases of D. immitis with a prevalence of approximately 0.1% (13,17).Based on the last two reports of two cases of heartworm disease (HW) detected during post-mortem examination of two roaming dogs ( 17) and a screening study in which 10% of dogs tested positive for D. repens (13), both studies from the urban area of Castel Volturno in the Campania region of southern Italy, the aim of the present study was to describe an outbreak of infection in a dog shelter from the same area.

Materials and methods
The study was conducted in a dog shelter located in Castel Volturno (Figure 1), a deltaic coastal plain of the Volturno River, an area where veterinary practitioners have reported a high incidence of D. repens infection (13).

Ethics aspects
The study started in July 2020 and ended July 2021.The protocol of this study was approved by the Ethical Committee for Animal Experiments of the Department of Veterinary Medicine and Animal Production, University of Federico II Naples, Italy (Approval Number 0093204/2022).Informed consent was obtained from the shelter owner before starting the study.

Animals and collection of blood samples
Screening for Dirofilaria spp. was performed on all shelter dogs, with a few exceptions, as follows.Dogs younger than 9 months of age and dogs that have received doxycycline in the last month at the time of sampling, were excluded from the study.The shelter housed a total of 285 dogs, and based on the above exclusion criteria, a total of 260 dogs (96 females, 164 males, age range: 2-14 years) were selected and sampled.The dogs were housed in outdoor boxes, each box hosting 3-4 dogs.The management of the shelter in terms of parasitic disease diagnosis and treatment strategy was as follows: i) all dogs (new arrivals and others with symptoms) were tested for Ehrlichia/Anaplasma and Leishmania infantum; ii) all dogs were treated with a topical formulation (based on fipronil) against ectoparasites.Moreover, no screening for Dirofilaria spp., was performed until June 2020 (the start of the study) and no prophylactic treatment against Dirofilaria spp. was perfomed in the shelter.Anamnestic data were collected for the dogs studied, including age, sex, health status (i.e., activity level, appetite, health problems, skin abnormalities).Data were also collected on the length of stay of the dogs in the shelter at the time of sampling.

Laboratory analyses
All samples (N = 260 EDTA blood and serum samples) were examined for identification of circulating microfilariae (mff) using the modified Knott's test (18) and for detection of D. immitis antigen using the Petcheck Canine Heartworm test (IDEXX).Moreover, molecular analyses [PCR protocol as described by Rishniw et al. (19)] were used to confirm the diagnosis of dirofilariosis in case of co-infections with both D. immitis and D. repens.

Modified Knott test and molecular analysis
A modified Knott's test was used for the detection of circulating mff of Dirofilaria spp. as follows.One mL of EDTA blood was mixed with 9 mL of distilled water and centrifuged at approximately 500 g for 3-5 min.The supernatant was removed from the tube and the content was stained with 1-2 drops of 1% methylene blue.One drop was placed on a microscope slide covered with a coverslip and observed under an optical microscope at 100X (18) to assess the level of microfilariaemia (mff/mL).The Dirofilaria negative/positive status of each dog included in the study was based on the examination of the entire contents of the sediment resulted from the modified Knott's test.For molecular identification of microfilaria species, genomic DNA was extracted from 200 microliters of each blood sample using the DNeasy ® Blood and Tissue kit (Qiagen, Germany), according to the manufacturer's instructions.Molecular analyses were performed according to the multiplex PCR protocol for simultaneous detection of the different Dirofilaria species, described by Rishniw et al. (19).Amplification of the internal transcribed spacer 2 (ITS2) region was carried out using the primers DIDR-F1 and DIDR-R1 with expected amplification product sizes of 542, 484, 578 and 584 bp for D. immitis, D. repens, A. reconditum and A. dracunculoides, respectively.The PCR reactions were increased to 25 μL of total volume, and included 5 μL of genomic DNA for each sample amplification, respectively (3).

Statistical analysis
The data of the dogs included in the study -i.e.gender, age, length of stay in the shelter at the time of sampling -were analysed by univariate statistical analysis using the Pearson's Chi-square test for independence, to test the association with positivity for D. immitis and D. repens.For this purpose, dogs were divided into the following age classes: class 1) 1-3 years (n = 76); class 2) 4-6 years (n = 75); class 3) 7-11 years (n = 81); class 4) > 11 (n = 28) years.In addition, the dogs were divided into three groups based on the length of stay in the shelter at the time of sampling as follows: group 1) dogs that entered in the shelter in the last 4 months (n = 51); group 2) dogs that were housed in the shelter for more than 4 months up to 2 years (n = 108); group 3) dogs that were housed for more than 2 years (n = 109).The Kappa (k) statistic was used to measure the concordance between the antigenic test and the modified Knott's test using the following criteria (20): ≤0.2 = poor; 0.21-0.40= fair; 0.41-0.60= moderate, 0.61-0.80= good and ≥ 0.80 = very good.Therefore, all the dogs were divided into four groups based on the number of microfilariae/ml (0 mff = negative (group 1); 1-100 mff (group 2); 101-500 mff (group 3); 501 > mff (group 4).Moreover, multivariable (logistic regression) statistical analyses were performed using the Dirofilaria spp.exposure (positive/ negative) as the dependent variable.Only the independent variables that showed significance (p < 0.01) in the univariate test were used for the logistic regression model.When an interaction between variables was suspected, the logistic regression model was run with and without these variables to assess possible effect modification on their behalf (21).The significance level was set at a p value <0.05.Data analysis was performed using SPSS version 17 software, Chicago, IL, United States.

Discussion and conclusions
The data presented in this manuscript on the occurrence of Dirofilaria spp. in a shelter dog from southern Italy (Campania region) are consistent with those endemic/hyperendemic areas in the Mediterranean region (4,8,10,11,(23)(24)(25).The present study revealed a high prevalence of both D. immitis (40%) and D. repens (35%) in a dog shelter located in a semi-urban area of Castel Volturno, representing a high risk of infection not only for companion animals, but also for humans.Although the screening included only one dog shelter in the Campania region, the survey of dirofilariosis status was conducted for the entire dog population housed in the shelter.In addition, two previous studies conducted in the same area -one reporting two cases of HW disease at post-mortem examination of two roaming dogs, and the second showing the presence of both D. immitis and D. repens in owned dogs (13,17) -had underscored the need for further evaluation of dirofilariosis risk in dogs in the coastal area of Castel Volturno, which provides suitable climatic and environmental conditions to sustain year-round populations of mosquito vectors and Dirofilaria pathogens (13,26).
The overall prevalence of D. immitis and D. repens (microfilariae) in the studied dog population in the Campania region was 75%.This result may be indicative of an increased risk of dirofilariosis in the studied area, although further studies with larger dog populations (both stray and owned dogs), should be investigated for Dirofilaria spp.However, this study is consistent with other studies recently conducted in other regions of southern Italy, where a high prevalence of D. immitis has been reported [e.g., (10,11,(27)(28)(29)].
The apparent emerging risk of D. immitis infection in the Campania region raises important questions.There are many factors  related to the vectors (presence of competent mosquito species) and the hosts (owner compliance with chemoprophylaxis, presence of wild canid reservoirs) that may influence the prevalence of HW disease in different areas of southern Italy.There are few studies in the literature on the prevalence of D. immitis in the Campania region that showed an increase in seroprevalence in dogs over time (11,12,14,30).
Dirofilaria repens is considered one of the most widespread zoonotic pathogens in Europe, and the occurrence of zoonotic cases goes hand in hand with infection in dogs.Currently, D. repens infection occurs in southern Italy, especially in coastal areas.The results of the questionnaire survey on dirofilariosis addressed to freelance veterinarians throughout Italy (7) showed clinical cases of D. repens and/or D. immitis in dogs in the Campania region.The prevalence of D. repens ranges from 1.5 to 10% in Campania (13,17) and 0.8% in the neighboring Molise region (31).Our study showed prevalence values of D. repens (35%) higher than those reported in the above studies.However, sheltered dogs are generally more exposed to canine vector-borne diseases (32,33), and therefore future investigations will need to be conducted on owned dogs in the area to better understand the epidemiology of Dirofilaria spp.
Given the zoonotic potential of both D. repens and D. immitis, vector control and systematic treatment of domestic and stray dogs should be considered to reduce the risk of human infection.Humans are usually dead-end hosts, although there have been cases with fertile adults and/or microfilariaemia due to D. repens (34-37).In most patients, D. immitis locates in the lungs and causes pulmonary dirofilariosis, whereas D. repens appears as nodules in subcutaneous tissues or as free worms under the conjunctiva (ocular dirofilariosis).Pulmonary dirofilariosis may be confused with pulmonary neoplasms (benign, carcinoma, metastasis), tuberculosis, or mycotic infections (37) and presents a diagnostic challenge.
The lack of entomological surveys in the Campania region is an important gap in the knowledge of Dirofilaria transmission of in this coastal area of southern Italy, which has several favorable environmental characteristics for the survival and development of the vector and, consequently, for the spread of both D. immitis and D. repens (26).
In the present study, almost half of the positive dogs had been infected before their arrival at the shelter.In addition, statistical analysis showed that male dogs were more exposed to D. immitis than females.To date, there is no evidence of an actual predisposition of sex to these pathogens, although many studies have reported similar results (38)(39)(40).On the other hand, age is considered an important risk factor for D. immitis infection.In fact, the prevalence in older dogs is generally higher than in younger dogs, which is related to the increasing duration of exposure to mosquitoes (41,42).Accordingly, our study showed the highest prevalence in dogs in the age classes >11 years (89.3%) and 7-11 years (86.4%) and the lowest in dogs in the age classes 1-3 years (64.5%) and 4-6 years (68%).
From a diagnostic point of view, our results showed that the antigen test alone (which is usually the most commonly used for the diagnosis of D. immitis) was not sufficient, as the modified Knott test detected a higher number of positive samples.This finding is not consistent with other studies (43, 44), in which the proportion of antigen-positive dogs was higher than that of Knott-positive dogs.However, one study showed the same pattern with dogs resulted positive with the modified Knott test but negative with the antigen test (45).The disagreement between the modified Knott test and the antigen test in the present study could be due to the low concentration of free antigen available for detection by serological tests.Also, it has been reported that the immune-complex formation can lead to falsenegative antigen test results and that pre-heating serum samples can disrupt immune-complexes, leading to a positive test result (28,46,47).However, the authors chose not to pre-heat the serum samples because there were also dogs co-infected with D. repens.Moreover, it has been previously reported that mono-infection with D. repens can lead to false-positive antigen tests for D. immitis (48,49).In addition, blocked antigens are more likely to occur in microfilaraemic dogs, in which a higher antibody response occurs compared to amicrofilaremic dogs (43, 50); this may also justify our results from the present study.
In this study, the antigen of D. immitis was detected using only one test (Petchek HW antigen test).However, as reported in a previous study by Constantinoiu et al. (43), double checking of antigen-negative samples from dogs with mff of D. immitis, using a different antigen test might be beneficial for accurate diagnosis of HW disease.Several studies have demonstrated that molecular methods can be a highly sensitive and specific analytical tool for simultaneous diagnosis and characterization of infections, and provide more reliable data compared to serological and parasitological methods (3,51).In the present study, there were no differences between the modified Knott test and molecular tests.Similar results were obtained in studies in which the modified Knott test was found to be effective, sensitive, and compatible with PCR (3,52).
In conclusion, HW disease is spreading over time for the reasons mentioned above, and southern Italy, once considered a low-risk area, is increasingly becoming the site of autochthonous outbreaks.We conclude that dog shelters in southern Italy are hotspots for Dirofilaria spp.transmission and strongly recommend education and veterinary advice for systematic treatment and the use of a diagnostic strategy with multiple tests to detect positive dogs in a given population (10,11,33).Proper management of these infections should be based on an effective and correct approach to diagnosis and up-to-date therapy, as well as practical prophylactic measures to protect animals and humans.Di mono-inf.= D. immitis mono-infection; Dr moni-inf.= D. repens mono-infection; "−"not performed; "+" performed.

FIGURE 1
FIGURE 1Shelter located in Castel Volturno, southern Italy.