First epidemiological survey of Angiostrongylus vasorum in domestic dogs from Spain

doi:10.21203/rs.2.19168/v2

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First epidemiological survey of Angiostrongylus vasorum in domestic dogs from Spain

https://doi.org/10.21203/rs.2.19168/v2

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Background: Angiostrongylus vasorum is the causative agent of canine angiostrongylosis, a disease that mainly affects domestic dogs and others wild carnivores. In Europe, the number of infected animals is increasing, being mainly located in central and southern countries. In Spain, several studies have reported high prevalences of A. vasorum in wild animals. However, there are no studies that address the current situation and distribution of the disease in domestic dogs in the country. Therefore, the objective of the present study was to evaluate the prevalence of A. vasorum in different areas of Spain.

Methods: Between November 2018 and October 2019, blood was sampled from a total of 2024 domestic dogs from 6 eco-epidemiological zones of Spain with climates that favour the presence of competent gastropod mollusks, and tested for the presence of A. vasorum circulating antigens using Angio Detect TM . All included dogs lived outdoors or had regular access to areas with vegetation.

Results: The prevalence of canine angiostrongylosis in the studied areas of Spain was 1.73%. No differences were found between males and females. By age, significant higher prevalances were found in dogs younger than 4 years. By eco-epidemiological areas, the highest prevalences were obtained in the north (2.74%) and northwest (1.86%) of Spain, and the lowest was described in the western area (0.93%).

Conclusions: The prevalence obtained can be considered high and demonstrates the presence of canine angiostrongylosis in Spain, in those areas where infected wild animals have been described and where climatic conditions are favourable for the establishment of the disease. Prophylactic measures should be prescribed for dogs at risk of infection and veterinarians should be aware of the importance of this disease.

Parasitology

Domestic dogs

Canine angiostrongylosis

Angiostrongylus vasorum

Prevalence

Epidemiology

Spain

Angiostrongylus vasorum is the causative agent of canine angiostrongylosis. It is a metastrongyloid nematode that mainly affects domestic dogs (Canis lupus) and wild carnivores such as foxes (Vulpes vulpes), wolves (Canis lupus), golden jackals (Canis aureus) and others in Europe [1]. Adult worms reside in the right side on the heart and the pulmonary arteries of the definitive host. Female worms shed first-stage larvae (L1) that are released in faeces. These are ingested by gastropod mollusks [2] and developed to L3; amphibians and birds can act as paratenic hosts as well. When the definitive hosts feed on intermediate or paratenic hosts, L3 penetrates through the intestinal wall, and migrate to the mesenteric lymph nodes where they mature to L5; once that stage is reached, they migrate to the right ventricle and pulmonary arteries through the mesenteric lymphatic vessels, portal vein, hepatic veins and caudal vena cava. The prepatent period ranges from 1 to 2 months [3]. Currently, this parasite is not considered zoonotic [4]. The parasite can cause a wide range of clinical signs in the dog, and three main clinical presentations have been described: cardiorespiratory, hemorrhagic diathesis and neurological disease, while some dogs may remain asymptomatic. The severity of some presentations can lead to the death of the host. The unspecific nature of the symptoms makes it difficult to diagnose this disease, being one of the reasons why canine angiostrongylosis is underdiagnosed [4,5].

In Europe it is considered an emerging disease. It has been described in southern France, Austria, Denmark, France, Greece, Germany, Hungary, Ireland, Italy, Portugal, Romania, Spain, Sweden, Switzerland, Turkey, and the United Kingdom, mainly in foxes and reaching prevalences of 80% in some regions [1,6,7,8,9,10]. In Spain, A. vasorum has been described in the Iberian wolf (2,1-21.6%) and red foxes (3-43.2%) in the northwestern [11,12,13]. It has also been described in red foxes in the north (33,3%) and in northeast (19,4-20.7%) [14,15,16]. In the east, A. vasorum has been described in red foxes (1.8%) and in badgers (6,4%) [17,18]. Isolated cases have also been reported in the west and in the center of the Iberian Peninsula in red foxes (personal communication). Furthermore, A. daskalovi has been described in badgers in the north (24%) [16]. However, in dogs only one case of canine angiostrongylosis has been published so far, in the north of Spain [19].

There are different techniques to carry out a specific diagnosis. Baermann's method of detecting L1 in stool samples is considered the gold standard; In addition, the FLOTAC technique has great sensitivity. However, these techniques have several drawbacks, for example, can be mistaken by other larvae of lung worms, Filaroides spp or larvae of free-living nematodes, unless morphological identification is performed by an experienced veterinarian. There are other specific methods, such as PCR detection and serological methods, which are used in epidemiological screenings. Currently, there is available a commercial blood test for the detection of A. vasorum antigens in domestic dogs with high sensitivity and specificity [1].

Given the presence and, in some areas, high prevalence of A. vasorum in wild carnivores, as well as the absence of studies in domestic dogs, the objective of this study was to determine the prevalence of canine angiostrongylosis in dogs in various areas of the Iberian Peninsula (Spain).

Climatic characteristics of the study area

The climate that predominates in Spain is the temperate Mediterranean. In the northern regions, rainfall and vegetation is abundant. In the eastern and southern regions, rainfall is scarce, so the vegetation is much smaller than in the north. In the west, only winters are rainy.

The survey was carried out in six different eco-epidemiological areas of Spain (Figure 1): a) Zone 1: Comprised by the autonomous region of Galicia, located in the northwestern area; has an oceanic climate, with a rainfall seasonality controlled by the subtropical climatic component; b) Zone 2: Include the autonomous regions of Asturias, Cantabria and Basque Country, all located in the northern area, which has an oceanic climate, with abundant rainfall spread throughout the year and mild temperatures in both winter and summer; c) Zone 3: Include the provinces of Salamanca and León; both located in the western area of the country, with a warm and temperate climate with significant rainfall in some regions; d) Zone 4: Comprised by the autonomous regions of Catalonia and Navarra, as well as the provinces of Zaragoza and Huesca. This zone is situated in the northeastern area of the country; has a Mediterranean climate and, in some areas, annual rainfall above 1,000 mm; e) Zone 5: Include the autonomous regions of Valencian Community and Murcia, located in the eastern; this region has a Mediterranean climate with very hot and humid summers, long and cold winters, and the climate is mainly dry throughout the year; f) Zone 6: Central area, comprised by the autonomous region of Madrid and the provinces of Toledo and Ciudad Real, which have a local steppe climate a dry climate without much rainfall.

Sample collection and analysis

Between November 2018 and October 2019, a total of 2024 domestic dogs from 6 zones of Spain were included in the study. Of them, 323 came from the zone 1, 438 from the zone 2, 216 from the zone 3, 539 from the zone 4, 407 from the zone 5, and 101 from the zone 6. Epidemiological data from each dog regarding age and sex were recorded.

The criteria for inclusion were: being over 3 months of age, never having received prophylactic treatment against A. vasorum, no previous history of angiostrongylosis, and owner consensus to participate in the survey. All the studied dogs lived outdoors or had regular access to areas with vegetation and none had traveled outside the study area of interest in the past year.

Blood samples collected from the cephalic or jugular vein, were placed in 3 ml serum tubes and centrifuged. Serum samples were kept at -20ºC until tests were performed. All samples were tested at the University of Salamanca for the presence of A. vasorum circulating antigens using Angio Detect™ (IDEXX Laboratories Inc., Westbrook, Maine, USA) following manufacturer's instructions.

Statistical analysis

Data were analyzed using SPSS Base 20.0 software for Windows (SPSS Inc./IBM, Chicago, Illinois, USA). Descriptive analysis of the considered variables was carried out considering the proportions of the qualitative variables. Chi-square and Fischer exact tests to compare proportions were performed. In all cases, the significance level was established at p<0.05.

The prevalences by groups of age in each eco-epidemiological area studied can be observed in Table 1. The prevalence of canine angiostrongylosis in the studied areas of Spain was 1.73%, with no statistically significant differences between females and males (1.70% and 1.66% respectively). By age, prevalence in dogs under 1 year was 3.10%, 2.6% for dogs aged 1–4 years, 1.45% for dogs aged 5–10 years, and 1.01% for those older than 10 years. There were statistically moderated differences between dogs <1 years and the rest of three groups of age. The mean age of the infected dogs was 5.66 years, being found infected dogs from 5 months to 10 years old.

By eco-epidemiological areas, the highest prevalences were obtained in the north and northwest of the country (2.74% and 1.86%, respectively), followed by the area of the east (1.72%), northeast (1.3%) and west (0.93%). The lowest prevalence was described in the central area of Spain (0.99 %) (Figure 1).

In the European continent, A. vasorum has been described both in wild animals (foxes, wolves, badgers) and in domestic dogs, being considered an emerging parasite. Until two decades ago, angiostrongylosis was a disease that was only described in wild animals in southern and central Europe, mainly in foxes. However, there are currently many studies that have described cases of infected domestic dogs in the same areas where previously infected wild animals had been reported [1,13]. Different diagnostic techniques have been used in these studies, based on the postmortem detection of adult parasites in the pulmonary artery or in the right ventricle [16], or based in serological techniques by the detection of circulating antigens and / or antibodies [1,20,21]. In both techniques, the results obtained low prevalences. When the Baermann funnel technique was used, reported prevalences were higher in endemic countries, such as Denmark and Germany [22].

The Angio Detect test has a reported sensitivity of 84.6-97.1% and a sensitivity of 98.9-100% [23,24]. However, in one canine study it was observed that the earliest positive results using this test were not observed until 9 weeks post infection, and that all studied sera were not positive until another 5 weeks later [23]. Therefore, the sensitivity to detect early infections seems to be diminished and it is possible that the actual prevalence was higher than that reported in this study.

Specific antibodies may be detected with an ELISA with a sensitivity and specificity of 85.7% and 98.8%, respectively [25]. They can be detected as early as at 3 weeks post infection; however, specific antibodies indicate the parasite exposure, and seropositive dogs may also be free of parasites (i.e. self-curing or administration of a broad-spectrum macrolactone effective against A. vasorum) since they persist up to 63 days after elimination of the parasite [25]. This, therefore, would explain that the seropositivity percentages in other studies where this technique has been used were higher than in those that have been used for the detection of circulating antigens [1,20,26]

In this study the current presence of A. vasorum in different regions of Spain has been evaluated, being, to date, the most complete work carried out. The average prevalence obtained was 1.73%, being the highest prevalences obtained in the northern, northwestern and eastern regions. It is precisely in these areas where the presence of A. vasorum in wolves, badgers and foxes had been previously described with high prevalences [11,12,13,14,15,16,17,18]. It should be noted that all sampled dogs lived outside the houses or had regular access to outdoor areas, and that the regions with the highest prevalence are areas where vegetation predominates and rainfall is abundant at different times of the year, promoting the presence of intermediate hosts.

In other countries, and using similar diagnostic techniques, the obtained prevalences were also low, being the highest published in Portugal (1.99%) [10] and in Hungary (1.76%) [26]. On the other hand, other countries such as Germany and Romania have reported low prevalences (0.15-0.19%) using the same techniques [1,20], so the prevalence obtained in this work can be considered high.

Age is considered a risk factor, with dogs under 18 months showing the highest infection risk [27]. In accordance with Morgan et al. [27], in this study the highest prevalences were found in dogs younger than 1 year, followed by those between 1 and 4 years. No statistically significant differences were observed by sex, so there seems to be no predisposition between males and females.

The prevalence obtained in this study is high and show that prophylactic measures should be prescribed for dogs that have regular access to the outside and / or contact with wildlife susceptible to the disease. This is especially true in young dogs and puppies, which seem to have the highest prevalences. Taking into account the results obtained, and that A. vasorum is considered an emerging parasite in domestic animals, more studies are needed to complete the epidemiological map of angiostrongylosis in Spain. In addition, veterinary clinicians should be aware of the importance of this disease, in order to carry out appropriate control campaigns and raise awareness among pet owners.

L1: Angiostrongylus vasorum first-stage larvae; L3: Angiostrongylus vasorum third-stage larvae; L5: Angiostrongylus vasorum fifth-stage larvae; PCR: polymerase chain reaction; ELISA: Enzyme-Linked ImmunoSorbent Assay.

Ethics approval and consent to participate

The present research was approved by the ethical committee of Veterinary Medicine Service of the University of Las Palmas de Gran Canaria (MV-2017/07) and was carried out in accordance with the current European legislation.

Consent for publication

Not applicable.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article.

Competing interests

The authors declare that they have no competing interests.

Funding

This study has been supported by Boehringer Ingelheim España, S.A.

Authors’ contributions

EC, RM and JAMA designed the study and wrote the manuscript. YF, JIM and NCR performed the fieldwork and collected the data. All authors participated in the discussion of the results, corrected, read and approved the final manuscript.

Acknowledgements

The authors would like to thank all veterinarians who kindly collaborated and provided samples for this research. Without their collaboration and dedication this study could not have been carried out. We would like to thank Ángela Lambea for her support in the laboratory.

Author details

¹Faculty of Veterinary Medicine, Research Institute of Biomedical and Health Sciences (IUIBS), University of Las Palmas de Gran Canaria, Trasmontaña s/n, Arucas, 35413, Las Palmas, Spain.²Group of Animal and Human Dirofilariosis, Parasitology Area, Faculty of Pharmacy, University of Salamanca, Campus Miguel Unamuno s/n, 37007, Salamanca, Spain.

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Table 1: Numbers of dogs sampled as well as prevalence of Angiostrongylus vasorum by age and gender from 6 zones of Spain.

Zone		1	2	3	4	5	6	TOTAL
Age	<1	1/21 (4.76%)	2/19 (10.53%)	0/9 (0%)	1/47 (2.13%)	0/32 (0%)	0/1 (0%)	4/129 (3.10%)
	1-4	3/71 (4.23%)	5/98 (5.10%)	1/44 (2.27%)	3/191 (1.57%)	2/102 (1.96%)	0/33 (0%)	14/539 (2.60%)
	5-10	1/123 (0.81%)	3/191 (1.57%)	1/68 (1.47%)	2/164 (1.22%)	3/172 (1.74%)	1/42 (2.38%)	11/760 (1.45%)
	>10	1/108 (0.93%)	2/130 (2.31%)	0/95 (0%)	1/137 (0.73%)	2/101 (1.98%)	0/25 (0%)	6/596 (1.01%)
Sex	Male	2/126 (1.58%)	6/220 (2.73%)	0/80 (0%)	3/227 (1.32%)	4/195 (2.05%)	1/65 (1.54%)	16/963 (1.66%)
	Female	4/197 (2.03%)	6/218 (2.75%)	2/136 (1.47%)	4/312 (1.28%)	3/212 (1.42%)	0/42 (0%)	19/1067 (1.70%)
TOTAL		6/323 (1.86%)	12/438 (2.74%)	2/216 (0.93%)	7/539 (1.30%)	7/407 (1.72%)	1/101 (0.99%)	35/2024 (1.73%)

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First epidemiological survey of Angiostrongylus vasorum in domestic dogs from Spain

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