First report of Spirocerca vulpis in red foxes (Vulpes vulpes) in Portugal.

Recent studies have described Spirocerca lupi-like nematodes in the stomach of red foxes (Vulpes vulpes) in Europe. A phylogenetic analysis of those specimens using mitochondrial DNA and their morphological reexamination allowed their characterization as a different species, Spirocerca vulpis. Between the years of 2010 and 2017, roundworms were collected from seven red foxes of northeastern Portugal found at necropsy with nodular lesions on their stomach wall. Histopathological analysis of four foxes revealed granulomatous lesions of the gastric nodules. On morphological assessment, by light microscopy, nematodes revealed the presence of six triangular teeth-like buccal capsule structures, which are absent in S. lupi. Polymerase chain reaction was run to amplify a 551 bp partial fragment of the cytochrome c oxidase subunit 1 gene. Sequences were 99% similar to S. vulpis (85% coverage) of red foxes from Spain and Bosnia and Herzegovina, 99% similar (99% coverage) to sequences of Spirocerca sp. of red foxes from Denmark and 93% similar (99% coverage) to S. lupi from South Africa. This is the first report of S. vulpis in foxes or any other host from Portugal.

Unravelling Spirocerca vulpis from red foxes from Switzerland: a 20-year-old record.

Spirocerca vulpis is a parasitic nematode of red foxes associated with gastric nodule formation. Since its description in 2018, this species has been reported in red foxes from Spain, Bosnia and Herzegovina, Italy, and Portugal. We present here the analysis of uncharacterized nematodes obtained from gastric nodules of a red fox from Switzerland in 1999. The specimens were identified as S. vulpis based on the observation of teeth-like structures in the buccal capsule and a 99.4% sequence identity to S. vulpis DNA from Spain. Phylogenetic analysis demonstrated the clustering of the Swiss sequences in a different group from specimens of other geographical locations. Altogether, this study constitutes the first report of S. vulpis in Switzerland and a report of the oldest specimen of this species in the world. Our findings highlight the widespread distribution of S. vulpis in Europe which may be facilitated by the free-roaming nature of red foxes.

Occurrence, prevalence, and explanatory environmental variables of Spirocerca vulpis infestation in the foxes of western Spain.

The main aim of this study was to not only establish the prevalence of the recently described Spirocerca vulpis parasite in the wild-life cycle of carnivores in western Spain but to also elaborate a model to explain the risk of infestation based on 16 topo-climatic and habitat variables. During the period from June 2016 to November 2017, 1644 carcasses of red foxes (Vulpes vulpes) and another 105 wild mammals, legally hunted or killed in car accidents, were analyzed. Parasitic nodules of Spirocerca were found in 6% of the foxes, and the molecular analyses established a homology of our samples with the species S. vulpis. There were no differences in the occurrence of the infestation between sexes, but there were differences in terms of age, such that infestation was proportionally more frequent among young individuals. In terms of temporality, a higher percentage of positive cases was observed during the late-autumn and winter months, especially between December and February. This study provides new data on the factors that predispose S. vulpis infection in the red fox. Model results indicate that a spatial pattern exists in the occurrence and prevalence of this species in the studied area (higher probabilities to the west), and that this pattern seems to mainly be associated with topo-climatic variables.

Spirocerca vulpis sp. nov. (Spiruridae: Spirocercidae): description of a new nematode species of the red fox, Vulpes vulpes (Carnivora: Canidae).

Previous studies have reported nematodes of the Spirocercidae family in the stomach nodules of red foxes (Vulpes vulpes) described as Spirocerca sp. or Spirocerca lupi (Rudolphi, 1819). We characterized spirurid worms collected from red foxes and compared them to S. lupi from domestic dogs by morphometric and phylogenetic analyses. Nematodes from red foxes differed from S. lupi by the presence of six triangular teeth-like buccal capsule structures, which are absent in the latter. Additionally, in female worms from red foxes, the distance of the vulva opening to the anterior end and the ratio of the glandular-to-muscular oesophagus lengths were larger than those of S. lupi (P < 0.006). In males, the lengths of the whole oesophagus and glandular part, the ratio of the glandular-to-muscular oesophagus and the comparison of the oesophagus to the total body length were smaller in S. lupi (all P < 0.044). Phylogenetic analyses revealed that S. lupi and the red foxes spirurid represent monophyletic sister groups with pairwise nucleotide distances of 9.2 and 0.2% in the cytochrome oxidase 1 and 18S genes, respectively. Based on these comparisons, the nematodes from red foxes were considered to belong to a separate species, for which the name Spirocerca vulpis sp. nov. is proposed.

CHARACTERIZATION OF LESIONS INDUCED BY SPIROCERCA VULPIS (SPIRURIDAE: SPIROCERCIDAE) IN RED FOXES (VULPES VULPES).

Spirocerca lupi infection in dogs (Canis domesticus) is associated with esophageal lesions that may evolve to a neoplastic stage in the form of esophageal sarcoma. In the red fox (Vulpes vulpes) infected with the closely related Spirocerca vulpis, similar lesions may occur in the stomach, but neoplastic forms have not been reported. We characterize Spirocerca vulpis-induced lesions in the fox, using pathology and immunohistochemical (IHC) techniques. Seventy-one out of 163 Spirocerca vulpis-positive red foxes were selected and subjected to histopathological study. Lesions were classified as patchy or diffuse. Ten patchy and 10 diffuse lesion samples were studied using three IHC markers (CD68, CD3, and CD79α for macrophages, T lymphocytes, and B lymphocytes, respectively) and H&E stain for neutrophils and eosinophils. Intensity of necrosis, hemorrhages, and the presence of collagen was also analyzed. Of the S. vulpis-positive red foxes, 96.9% had S. vulpis nodules localized in the gastric area (wall and/or omentum), and 3.1% had nodules in the small intestine. All the samples had a moderate to severe lymphoplasmacytic infiltrate. Mild eosinophil infiltration was observed in both types of lesions, while neutrophil infiltration was significatively higher in the patchy than in the diffuse lesions. Fibrosis with mature collagen fibers was also predominant in the patchy lesions along with the presence of T lymphocytes and macrophages. Both the patchy and diffuse patterns had very few B lymphocytes. These findings suggest that the diffuse form is an earlier stage of the lesion, which eventually evolves into patchy forms. Neoplastic forms were not seen. Although more studies are necessary, this study describes the lesions, characterizes the inflammatory infiltrates, and establishes a possible evolution of the different pathological forms of S. vulpis infection in the red fox.

Morphological and molecular characterization of Spirocerca lupi (Nematoda: Spirocercidae) from domestic dogs in Vietnam with reference to Spirocerca vulpis.

Spirocerca lupi is a common parasitic nematode associated with esophageal cancer of canids. Recent surveys have revealed an increasing number of canids infected with Spirocerca spp. in Africa, the Americas, Europe and Western Asia, and described a new species, Spirocerca vulpis, from red foxes (Vulpes vulpes). However, in Southeast Asia, research on Spirocerca spp. is scarce. Therefore, the aim of this study is to explore Spirocerca infection in domestic dogs in Vietnam and to identify the Spirocerca species by analyzing their morphometric and molecular data. We found Spirocerca spp. specimens in 51 (17.7%) out of 287 dogs examined with the intensity of infection ranging from one to 29 nematodes per dog. These nematodes were morphologically and molecularly identified as S. lupi. For morphology, the presence/absence of teeth, the ratio of glandular to muscular esophagus lengths, and the position of the vulva opening of S. lupi and S. vulpis varied between reports, suggesting caution in identification of Spirocerca species based exclusively on morphological characteristics. The molecular analysis based on a partial cox1 sequence revealed that S. lupi from Vietnam is genetically close to those from India and China, but far different from those of Israel, South Africa, Peru and Hungary. Given high genetic and morphological variations, more extensive surveys on Spirocerca spp. from various mammalian hosts at a greater scale are necessary to elucidate the divergence of this nematode.

MOLECULAR INVESTIGATION OF VECTOR-BORNE PATHOGENS IN RED FOXES (VULPES VULPES) FROM SOUTHERN FRANCE.

Because of their free-ranging nature, the probability of wild animals being exposed to vector-borne pathogens is likely higher than that of humans and pets. Recently, the red fox (Vulpes vulpes) has been suspected as being a reservoir or host of several pathogens of veterinary and public health importance. We conducted a molecular survey on 93 red foxes hunted in 2008-18, in the departments of Bouches-du-Rhône and Var, in southeastern France, for pathogens including Leishmania infantum, Piroplasmida, Hepatozoon spp., nematodes, Coxiella burnetii, Borrelia spp., Rickettsia spp., and Anaplasmataceae. Spleen samples were screened for the presence of vector-borne pathogens by PCR followed by sequencing. Pathogens were detected in 94% (87/93) of red foxes, and coinfections were identified in 24% (22/93) of foxes. We identified DNA from Hepatozoon canis, L. infantum, and Babesia vogeli in 92% (86/93), 15% (14/93), and 3% (3/93) of red foxes, respectively. We also found DNA of nematodes in 3% (3/93) of foxes; Spirocerca vulpis was identified in one fox and Dirofilaria immitis in the two others. Interestingly, C. burnetii genotype 3, previously described in humans from the same region, was identified in 3% (3/93) of foxes and Anaplasma platys in 2% (2/93) of foxes. We did not detect DNA of Borrelia spp., Bartonella spp., or Rickettsia spp. In our study, the prevalence of pathogens did not vary by fox origin, sex, or tick carriage. Molecular evidence of B. vogeli, H. canis, S. vulpis, D. immitis, C. burnetii, and A. platys in red foxes has not previously, to our knowledge, been reported from southern France. We propose that red foxes are potential reservoirs for several pathogens, including major zoonotic agents such as L. infantum. They could be incidental hosts for pathogens, such C. burnetii. The high prevalence for H. canis suggests an important role of foxes in domestic dog (Canis lupus familiaris) infection. These animals may pose a threat to human and animal health.

First epidemiological data on Spirocerca vulpis in the red fox: A parasite of clustered geographical distribution.

This is the first study describing the epidemiology of Spirocerca vulpis after its description as a new species in 2018. During the period 2006-2013, a total of 286 red foxes (Vulpes vulpes) from the Valencian Community (southeast Spain) were necropsied. Based on data collected, S. vulpis prevalence and intensity were calculated, as well as the spatial distribution of this nematode. Influence of host (sex and age) and environmental factors on S. vulpis occurrence was evaluated. MAXENT software was used to model and predict the parasite distribution. Continuous and discrete prediction maps were built using ArcMap 10.6. The prevalence of S. vulpis was 22% (63/286; 95% CI: 17.4-27.3), and the median intensity was 5 (IQR 11) nematode specimens. No significant difference in term of intensity was found between males and females; regarding the host age, S. vulpis was found only in adult foxes, with the exception of one juvenile individual. The distribution of S. vulpis in foxes was skewed to the left, highlighting that parasite infection affects few individuals within a population, with parasitized animals being responsible to maintain the infection at the population level. The majority of parasitized foxes had a parasite burden lower than eight parasites/individual. S. vulpis distribution in Valencian Community presents sharply defined areas in which there are optimal environmental conditions for maintaining the life cycle of this parasite. Climatic variables and altitude are the main factors influencing the parasite presence. Our results indicate that S. vulpis has epidemiological characteristics similar to those of S. lupi and, therefore, based on the phylogenetic proximity of both nematode species, it is likely that coprophagous beetle species might play a key epidemiological role in the maintenance of this newly described Spirocerca species. Moreover, it is currently unknown if S. vulpis can infect the dog and other wild canid species apart from the red fox and, if so, what are the pathogenic effects on these host species. Therefore, it is necessary to continue investigating the epidemiology of this parasite in order to know the range of appropriate host species. This information will enable to know if S. vulpis endemic areas should be considered as health risk points for dogs, especially for the most exposed, such as those living in rural areas, and hunting dogs.

Phylogenetic analysis of Spirocerca lupi and Spirocerca vulpis reveal high genetic diversity and intra-individual variation.

BACKGROUND: Spirocerca lupi is a parasitic nematode of canids that can lead to a severe and potentially fatal disease. Recently, a new species, Spirocerca vulpis, was described from red foxes in Europe, suggesting a high genetic diversity of the Spirocerca spp. infecting canids. The genetic variation and phylogenetic relationships of S. lupi collected from naturally-infected domestic dogs from Australia, Hungary, Israel, Italy, India and South Africa, and S. vulpis from red foxes from Bosnia and Herzegovina, Italy and Spain, was studied using mitochondrial and rDNA markers. RESULTS: A high intra-individual variation was found in the first internal transcribed spacer (ITS1) locus in all Spirocerca spp., ranging between 0.37-2.84%, with up to six haplotypes per specimen. In addition, a combination of phylogenetic and haplotype analyses revealed a large variability between S. lupi specimens collected from different geographical locations using the ITS1 (0.37-9.33%) and the cytochrome c oxidase subunit 1 (cox1) gene (1.42-6.74%). This genetic diversity led to the identification of two S. lupi genotypes circulating among dogs (PTP support > 0.829), including genotype 1 found in S. lupi from Australia, India, Israel and South Africa, and genotype 2 represented by specimens from Hungary and Italy. These genotypes presented pairwise nucleotide distances of 0.14%, 8.06% and 6.48 ± 0.28% in the small rDNA subunit (18S), ITS1 and cox1 loci, respectively. Additionally, Nei's genetic distance in the ITS1 showed a further subdivision of genotype 1 worms into 1A (Israel and South Africa) and 1B (Australia and India). A morphological analysis of the anterior and posterior extremities of genotype 1 and genotype 2 worms using scanning electron microscopy did not show any differences between the specimens, contrary to the morphological differences between S. lupi and S. vulpis. CONCLUSIONS: These findings demonstrate the high genetic variability among Spirocerca spp. from different geographical locations, thereby expanding our understanding of the epidemiology, evolution and phylogenetic variability within the genus.