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Cited by 33Parasitic Diseases Research and TreatmentHelminth infection and controlResearch on Leishmaniasis Studies

Molecular characterization of Dirofilaria spp. circulating in Portugal

Open access
Ferreira, Cátia, Afonso, Ana, Calado, Manuela, Maurício, Isabel, Alho, Ana Margarida, Meireles, José, Madeira de Carvalho, Luís, Belo, Silvana·2017·Parasites & Vectors
DOI 10.1186/s13071-017-2180-yPMC PMC5438543
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Abstract

BACKGROUND: Dirofilariosis is a potentially zoonotic parasitic disease, mainly transmitted by mosquito vectors in many parts of the world. Data concerning the canine Dirofilaria species currently circulating in Portugal is scarce. Thereby, a large-scale study was conducted to determine the Dirofilaria spp. present in Portugal, based on a molecular approach, and also to optimize a reliable and highly sensitive species-specific polymerase chain reaction (PCR) assay that could be used for the simultaneous detection and differentiation of Dirofilaria immitis, Dirofilaria repens, and other concurrent filarial species in animal reservoirs. METHODS: Blood samples were collected from three districts of Portugal (Coimbra, Santarém and Setúbal) between 2011 and 2013. Samples were tested using rapid immunomigration tests (Witness® Dirofilaria), modified Knott's technique and acid phosphatase histochemical staining. In addition, molecular analysis was performed by amplification of the internal transcribed spacer (ITS) region using two different PCR protocols, specific for molecular screening of canine filarial species. RESULTS: Of the 878 dogs sampled, 8.8% (n = 77) were positive for D. immitis circulating antigen and 13.1% (n = 115) positive for microfilariae by the modified Knott's technique. Of the 134 samples tested by acid phosphatase histochemical staining, 100 (74.6%) were positive for D. immitis. Overall, 13.7% (n = 120) were positive by PCR for D. immitis by ITS2, of which 9.3% (67/720) were also positive by ITS1. ITS2 PCR was the most sensitive and specific method, capable of detecting mixed D. immitis and A. reconditum infections. Heterozygosity, in the form of double peaks, was detected by sequencing of both ITS regions. No D. repens was detected by any of the diagnostic methods. CONCLUSIONS: The present study confirmed D. immitis as the dominant species of the genus Dirofilaria infecting Portuguese dogs, based on sequencing of ITS1 and ITS2 PCR fragments. Additionally, ITS2 PCR was the most adequate method for diagnosis and prevalence estimation.

AI snapshot

Written from the paper’s full text.

TL;DR

This study molecularly screened 878 dogs from three Portuguese districts (2011–2013) and found *Dirofilaria immitis* to be the dominant filarial species, with no *D. repens* detected by any diagnostic method. ITS2 PCR was the most sensitive and specific assay and also detected *Acanthocheilonema reconditum* and mixed infections.

Key findings

  • Among 878 dogs, 8.8% (77) were positive for *D. immitis* circulating antigen by Witness, 13.1% (115) had microfilariae by modified Knott’s, and 13.7% (120) were positive by ITS2 PCR; of 134 tested by acid phosphatase, 100 (74.6%) were positive for *D. immitis*.
  • In 720 dogs tested by both PCR targets, ITS1 detected 67 (9.3%) *D. immitis*, 1 (0.1%) *A. reconditum*, and 0 mixed infections, whereas ITS2 detected 93 (12.9%) *D. immitis*, 5 (0.7%) *A. reconditum*, and 2 (0.3%) mixed infections.
  • ITS2 PCR had higher analytical sensitivity than ITS1 PCR: 0.09 vs 4.5 pg DNA from female adult *D. immitis*, 200 vs 118 pg DNA from *D. repens*-infected dog blood, and 2.5 vs 250 pg DNA from a *D. immitis*-infected dog; statistical sensitivity and specificity were significantly higher for ITS2 (McNemar test, P < 0.05).
  • No *D. repens* was detected by any diagnostic method; ITS2 PCR prevalence was higher in males (15.8% vs 11.9%, P = 0.032), dogs > 6 years (17.8% vs 12.1% in > 3–6 years and 6.4% in 0.5–3 years, P = 0.01), and Setúbal (18.7% vs 13.8% Santarém and 10.4% Coimbra, P = 0.016).

Methods

  • Blood samples were collected from 878 dogs in kennels in Coimbra, Santarém, and Setúbal, Portugal, during 2011–2013; only dogs older than 6 months and resident in kennels for at least 6 months were included.
  • Samples were tested by Witness® *Dirofilaria* antigen assay, modified Knott’s technique, and acid phosphatase histochemical staining; molecular analysis used ITS1 semi-nested PCR and ITS2 PCR, followed by sequencing, BLAST, haplotype inference, and phylogenetic analysis.
  • Statistical comparisons used Pearson’s Chi-square and Fisher’s exact tests, with Cohen’s kappa for agreement; P < 0.05 was considered significant.

Limitations

  • The authors state that it was not possible to determine whether double peaks/heterozygous ITS sequences represented parasite heterozygosity or mixed infections in the dog; PCR on individually isolated microfilariae was suggested to clarify this.
  • The authors note that some ITS1 sequences in databases had been erroneously labelled as *D. repens* when they actually correspond to *D. immitis*, raising questions about earlier *D. repens* occurrence or prevalence estimates based on that target.
  • Evident from the text: sampling was restricted to kennels in three Portuguese districts, so findings may not represent all dogs in Portugal; the paper does not include a formal limitations section.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1186/s13071-017-2180-y

Cites

42 of 42 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 34 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    DNA from D. immitis and Acanthocheilonema spp. was detected by ITS2-PCR in two samples.

    “DNA from species (D. immitis and Acanthocheilonema spp.) was detected by ITS2-PCR in two samples”
    Quote in the paperNot a substantial claim
  • Findingown data

    Of 878 dogs sampled in Portugal, 8.8% (77/878) were positive for D. immitis circulating antigen by Witness.

    “Of the 878 dogs sampled, 8.8% (n = 77) were positive for D. immitis circulating antigen”
    • Also stated in: § Results · plain
      “Out of the 878 samples tested, D. immitis circulating antigen was detected in 77 (8.8%) by WT”
    Quote in the paperNot a substantial claim
  • Findingown data

    No D. repens was detected by any diagnostic method in the study.

    “No D. repens was detected by any of the diagnostic methods”
    • Also stated in: § Results · plain
      “Dirofilaria repens was not identified in blood smears through any method”
    Quote in the paperSubstantial claim
  • Findingown data

    Agreement between ITS2-PCR and AP: K=0.513, P=0.088.

    “Acid phosphatase | 134 | 97 (97.0) | 14 (43.8) | 0.513 | 0.088”
    Quote in the paperNot a substantial claim
  • Findingown data

    Using ITS2-PCR, it was possible to amplify both Dirofilaria spp. and A. reconditum in canine blood.

    “Using ITS2-PCR it was possible to amplify, not only both species of Dirofilaria spp., but also A. reconditum in canine blood”
    Quote in the paperSubstantial claim
  • May combine two claims. 24
  • Classified as probably not a substantial claim. 7
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data

    Analytical sensitivity of ITS1-PCR was 4.5 pg DNA from female adult D. immitis, 118 pg DNA from D. repens-infected dog blood, and 250 pg DNA from D. immitis-infected dog.

    “The analytical sensitivity of ITS1-PCR and ITS2-PCR were, respectively, 4.5 and 0.09 pg DNA from female adult D. immitis, 118 and 200 pg DNA from D. repens-infected dog blood, and 250 and 2.5 pg DNA from a D. immitis-infected dog”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Agreement between ITS2-PCR and KN: K=0.930, P=0.018.

    “Knott | 878 | 107 (93.0) | 750 (98.3) | 0.930 | 0.018*”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Five samples had H2 as the other haplotype, which has no correspondence in the database, and two samples had haplotype H4, which was present in sequences from China (red panda) and Iran (dogs).

    “Five samples had H2 as the other haplotype, which has no correspondence in the database, and two samples had haplotype H4, which was present in sequences from China (red panda) and Iran (dogs)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    All heterozygous Portuguese samples included haplotype H18, which was present in sequences from India and Brazil (dog).

    “All heterozygous Portuguese samples included haplotype H18, which was present in sequences from India and Brazil (dog)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    ITS2-PCR and KN presented the highest level of agreement (Cohen's kappa coefficient), which was lower, but also statistically significant, between ITS2-PCR and WT.

    “ITS2-PCR and KN presented the highest level of agreement (Cohen’s kappa coefficient), which was lower, but also statistically significant, between ITS2-PCR and WT”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    ITS2 sequences obtained were most similar to D. immitis and quite distinct from D. repens sequences present in GenBank, as revealed by phylogenetic analysis.

    “the ITS2 sequences obtained here were most similar to D. immitis and quite distinct from D. repens sequences present in GenBank, as revealed by phylogenetic analysis”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Heterozygosity, in the form of double peaks, was detected by sequencing of both ITS regions.

    “Heterozygosity, in the form of double peaks, was detected by sequencing of both ITS regions”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Two samples that were ITS1-PCR-positive for D. immitis were characterized by ITS2-PCR-RFLP as A. reconditum and were not included in the calculations.

    “Two samples that were ITS1-PCR-positive for D. immitis, were characterized by ITS2-PCR-RFLP as A. reconditum (accession number ENA: HG964682–HG964684) and were not included in the calculations”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    ITS2 PCR was the most sensitive and specific method among those tested, capable of detecting mixed D. immitis and A. reconditum infections.

    “ITS2 PCR was the most sensitive and specific method, capable of detecting mixed D. immitis and A. reconditum infections”
    • Also stated in: § Discussion · plain
      “ITS2-PCR was the most sensitive method”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Three out of nine ITS1 sequences analysed from PCR products obtained from canine blood were found to have a string of double peaks, as did a female D. immitis worm.

    “Three out of nine ITS1 sequences analysed from PCR products obtained from canine blood were found to have a string of double peaks, as did a female D. immitis worm”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Analytical sensitivity of ITS2-PCR was 0.09 pg DNA from female adult D. immitis, 200 pg DNA from D. repens-infected dog blood, and 2.5 pg DNA from D. immitis-infected dog.

    “The analytical sensitivity of ITS1-PCR and ITS2-PCR were, respectively, 4.5 and 0.09 pg DNA from female adult D. immitis, 118 and 200 pg DNA from D. repens-infected dog blood, and 250 and 2.5 pg DNA from a D. immitis-infected dog”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Agreement was strongest and statistically significant between PCR-ITS2 and KN test, but the molecular assay has the advantage of detecting filarial DNA in co-infected animals.

    “Agreement was strongest and statistically significant between PCR-ITS2 and KN test, but the molecular assay has the advantage of detecting filarial DNA in co-infected animals”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ITS2-PCR had higher analytical sensitivity and specificity than ITS1-PCR, particularly in samples with low microfilaremia (<5 Mf per 20 μl), for which ITS1 amplification failed or gave non-specific results.

    “In the present study, the ITS2-PCR had higher analytical sensitivity and specificity than the ITS1-PCR, particularly in samples with low microfilaremia (< 5 Mf per 20 μl of blood), for which ITS1 amplification failed or gave non-specific results”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ITS2-PCR was the most sensitive method, but with very similar analytical sensitivity to KN, followed by WT.

    “ITS2-PCR was the most sensitive method, but with very similar analytical sensitivity to KN, followed by WT”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    One adult worm presented the same heterozygous profile for ITS2, and the same ITS heterozygous patterns had been observed in the PCR product from a mosquito in Portugal, Aedes detritus (s.l.).

    “However, one adult worm presented the same heterozygous profile for ITS2, and the same ITS heterozygous patterns had been observed in the PCR product from a mosquito in Portugal, Aedes detritus (s.l.)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The ITS2-PCR assay detected mostly D. immitis single infections, but also 5 (0.6%) cases of A. reconditum and 2 (0.2%) of mixed infections (D. immitis + A. reconditum) (Table 2).

    “The ITS2-PCR assay detected mostly D. immitis single infections, but also 5 (0.6%) cases of A. reconditum and 2 (0.2%) of mixed infections (D. immitis + A. reconditum) (Table 2)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There were significant differences (P=0.01) in D. immitis prevalence between age groups; highest in dogs >6 years (76/426; 17.8%), followed by >3-6 years (32/265; 12.1%) and lowest in 0.5-3 years (12/187; 6.4%).

    “There were also significant differences (P = 0.01) in prevalence between age groups; the highest was found in dogs > 6 years of age (76/426; 17.8%), followed by the group with > 3–6 years of age (32/265; 12.1%) and the lowest in the 0.5–3 years age group (12/187; 6.4%)”
    Quote in the paperNot a substantial claim

  • May combine two claims. The quote only partly states this claim.

    Interpretationown data

    The present results showed that ITS2-PCR performs better in sensitivity, specificity and species identification than parasitological and serological methods.

    “the present results showed that ITS2-PCR performs better in different aspects (sensitivity, specificity and species identification)”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Dirofilaria repens was recently detected for the first time, in a dog, presenting as mixed infection with D. immitis.

    “Dirofilaria repens was recently detected for the first time, in a dog, presenting as mixed infection with D. immitis [26]”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26] Maia C, Lorentz S, Cardoso L, Otranto D, Naucke TJ. Detection of Dirofilaria repens microfilariae in a dog from Portugal. Parasitol Res. 2016;115:441–3. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The occurrence of autochthonous infections in domestic animals and the numbers of notified human cases of dirofilariosis, mainly attributed to D. repens, have increased substantially in several European countries in recent years.

    “the occurrence of autochthonous infections in domestic animals and the numbers of notified human cases of dirofilariosis, mainly attributed to D. repens, have increased substantially in several European countries in recent years [5, 27, 28]”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [5] Simón F, López-Belmonte J, Marcos-Atxutegi C, Morchón R, Martín-Pacho JR. What is happening outside North America regarding human dirofilariasis? Vet Parasitol. 2005;133:181–9. source

      Not checked in the free run.

    • cites [27] Pampiglione S, Rivasi F, Gustinelli A. Dirofilarial human cases in the Old World, attributed to Dirofilaria immitis: a critical analysis. Histopathology. 2009;54:192–204. source

      Not checked in the free run.

    • cites [28] Genchi C, Kramer LH, Rivasi F. Dirofilarial infections in Europe. Vector Borne Zoonotic Dis. 2011;11:1307–17. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    Dirofilaria immitis and Dirofilaria repens are widely present in the Mediterranean basin and are the causative agents of cardiopulmonary and subcutaneous dirofilariosis, respectively.

    “The species Dirofilaria immitis and Dirofilaria repens (Filarioidea, Onchocercidae) are widely present in the Mediterranean basin and are the causative agents of cardiopulmonary and subcutaneous dirofilariosis, respectively”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The ongoing north- and eastward expansion of both Dirofilaria species has been mainly attributed to global warming, as well as environmental changes, which promote the expansion of mosquito vectors, along with the increased international mobility of infected vertebrates.

    “Such expansion has been mainly attributed to global warming, as well as environmental changes, which promote the expansion of mosquito vectors, along with the increased international mobility of infected vertebrates [27, 39–41]”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [27] Pampiglione S, Rivasi F, Gustinelli A. Dirofilarial human cases in the Old World, attributed to Dirofilaria immitis: a critical analysis. Histopathology. 2009;54:192–204. source

      Not checked in the free run.

    • cites [39] Genchi C, Rinaldi L, Mortarino M, Genchi M, Cringoli G. Climate and Dirofilaria infection in Europe. Vet Parasitol. 2009;163:286–92. source

      Not checked in the free run.

    • cites [40] Morchón R, Carretón E, Grandi G, González-Miguel J, Montoya-Alonso JA, Simón F, et al. Anti-Wolbachia surface protein antibodies are present in the urine of dogs naturally infected with Dirofilaria immitis with circulating microfilariae but not in dogs with occult infections. Vector Borne Zoonotic Dis. 2012;12:17–20. source

      Not checked in the free run.

    • cites [41] Fuehrer HP, Auer H, Leschnik M, Silbermayr K, Duscher G, Joachim A. Dirofilaria in humans, dogs, and vectors in Austria (1978–2014) - from imported pathogens to the endemicity of Dirofilaria repens. PLoS Negl Trop Dis. 2016;10:e0004547. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Canine dirofilariosis due to D. immitis is known to be endemic and widely distributed in Portugal, with prevalence ranging between 0.9 and 27.3% in mainland regions to over 30% in Madeira Island.

    “Canine dirofilariosis due to D. immitis is known to be endemic and widely distributed in Portugal, with prevalence ranging between 0.9 and 27.3% in mainland regions to over 30% in Madeira Island [22–25]”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [22] Araujo AM. Canine and human Dirofilaria immitis infections in Portugal. A review. Parassitologia. 1996;38:366.

      Not checked in the free run.

    • cites [23] Vieira AL, Vieira MJ, Oliveira JM, Simões AR, Diez-Baños P, Gestal J. Prevalence of canine heartworm (Dirofilaria immitis) disease in dogs of central Portugal. Parasite. 2014;21:5. source

      Not checked in the free run.

    • cites [24] Cardoso L, Mendão C, Madeira de Carvalho L. Prevalence of Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi sensu lato, Anaplasma spp. and Leishmania infantum in apparently healthy and CVBD-suspect dogs in Portugal - a national serological study. Parasit Vectors. 2012;5:62. source

      Not checked in the free run.

    • cites [25] Alho AM, Landum M, Ferreira C, Meireles J, Gonçalves L, Madeira de Carvalho L, et al. Prevalence and seasonal variations of canine dirofilariosis in Portugal. Vet Parasitol. 2014;206:99–105. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Dirofilaria repens has been implicated in the increasing number of reports of human dirofilariosis in Europe.

    “this species has been implicated in the increasing number of reports of human dirofilariosis in Europe [28]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] Genchi C, Kramer LH, Rivasi F. Dirofilarial infections in Europe. Vector Borne Zoonotic Dis. 2011;11:1307–17. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Molecular protocols have been developed for reliable detection and differentiation of filarial species, in particular, a species-specific PCR assay and multiplex PCR and restriction fragment length polymorphism (RFLP) assays for simultaneous detection of different Dirofilaria spp., either in the vector or in blood.

    “Molecular protocols have been developed for reliable detection and differentiation of filarial species, in particular, a species-specific PCR assay and multiplex PCR and restriction fragment length polymorphism (RFLP) assays for simultaneous detection of different Dirofilaria spp., either in the vector or in blood [12, 14–21]”
    Quote in the paperSubstantial claim0 of 9 sources checked
    • cites [12] Casiraghi M, Bazzocchi C, Mortarino M, Ottina E, Genchi C. A simple molecular method for discriminating common filarial nematodes of dogs (Canis familiaris). Vet Parasitol. 2006;141:368–72. source

      Not checked in the free run.

    • cites [14] Mar PH, Yang IC, Chang GN, Fei AC. Specific polymerase chain reaction for differential diagnosis of Dirofilaria immitis and Dipetalonema reconditum using primers derived from internal transcribed spacer region 2 (ITS2). Vet Parasitol. 2002;106:243–52. source

      Not checked in the free run.

    • cites [15] Nuchprayoon S, Junpee A, Poovorawan Y, Scott AL. Detection and differentiation of filarial parasites by universal primers and polymerase chain reaction-restriction fragment length polymorphism analysis. Am J Trop Med Hyg. 2005;73:895–900. source

      Not checked in the free run.

    • cites [16] Gioia G, Lecová L, Genchi M, Ferri E, Genchi C, Mortarino M. Highly sensitive multiplex PCR for simultaneous detection and discrimination of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood. Vet Parasitol. 2010;172:160–3. source

      Not checked in the free run.

    • cites [17] Gasser RB, LeGoff L, Petit G, Bain O. Rapid delineation of closely-related filarial parasites using genetic markers in spacer rDNA. Acta Trop. 1996;62:143–50. source

      Not checked in the free run.

    • cites [18] Watts KJ, Courteny CH, Reddy GR. Development of a PCR- and probe-based test for the sensitive and specific detection of the dog heartworm, Dirofilaria immitis, in its mosquito intermediate host. Mol Cell Probes. 1999;13:425–30. source

      Not checked in the free run.

    • cites [19] Latrofa MS, Weigl S, Dantas-Torres F, Annoscia G, Traversa D, Brianti E, et al. A multiplex PCR for the simultaneous detection of species of filarioids infesting dogs. Acta Trop. 2012;122:150–4. source

      Not checked in the free run.

    • cites [20] Nuchprayoon S, Junpee A, Nithiuthai S, Chungpivat S, Suvannadabba S, Poovorawan Y. Detection of filarial parasites in domestic cats by PCR-RFLP of ITS1. Vet Parasitol. 2006;140:366–72. source

      Not checked in the free run.

    • cites [21] Rishniw M, Barr SC, Simpson KW, Frongillo MF, Franz M, Dominguez Alpizar JL. Discrimination between six species of canine microfilariae by a single polymerase chain reaction. Vet Parasitol. 2006;135:303–14. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    D. immitis has also been reported in three Eurasian otters, Lutra lutra, in Portuguese natural freshwater habitats and, recently, in a collection of pinnipeds from Algarve.

    “Dirofilaria immitis has also been reported in three Eurasian otters, Lutra lutra, in Portuguese natural freshwater habitats [45, 46] and, recently, in a collection of pinnipeds from Algarve [47]”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [45] Torres J, Feliu C, Fernández-Morán J, Ruíz-Olmo J, Rosoux R, Santos-Reis M, et al. Helminth parasites of the Eurasian otter Lutra lutra in southwest Europe. J Helminthol. 2004;78:353–9. source

      Not checked in the free run.

    • cites [46] Saraiva AL, Sousa S, Silva J, Andrade S, Botelho N, Canavarro I, et al. Dirofilaria immitis in an Eurasian Otter (Lutra lutra). J Comp Pathol. 2013;148:88. source

      Not checked in the free run.

    • cites [47] Alho AM, Marcelino I, Colella V, Flanagan C, Silva N, Correia JJ, et al. Dirofilaria immitis in pinnipeds and new host record. Parasit Vectors. 2017;10:142. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In a national serological survey conducted in red foxes, 8.5% were positive for D. immitis circulating antigen, with positive animals found in northern and southern areas of Portugal.

    “in a national serological survey conducted in red foxes, 8.5% were positive for D. immitis circulating antigen, with positive animals found in northern and southern areas of Portugal [44]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44] Alho AM, Cortes H, Lopes AP, Vila-Viçosa MJ, Cardoso L, Belo S, et al. Detection of Dirofilaria immitis antigen in red foxes (Vulpes vulpes) from Portugal. Parasit Vectors. 2016;10 Suppl 1:A16.

      Not checked in the free run.

27 of 27

  • The paper relies on this without support.

    The dogs sampled from kennels in Coimbra, Santarém, and Setúbal are representative of the canine population in Portugal for Dirofilaria species.

    “canine surveys were conducted in kennels (run by local authorities or animal protection associations) in three districts of Portugal: Coimbra (northern-Centre region), Santarém (central-Centre region) and Setúbal (southern-Centre region)”
    “The present study confirmed D. immitis as the dominant species of the genus Dirofilaria infecting Portuguese dogs”

  • The paper relies on this without support.

    Dogs residing in kennels for at least 6 months were infected locally (autochthonous infection).

    “Only dogs older than 6 months of age and residing in the kennels for at least 6 months were included”
2 of 2

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Cites

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1 of the 43 references couldn’t be matched to a known paper.

Research works printed but not linked: 4

  1. Venco L, Genchi C, Simón F. La filariosis cardiopulmonar (Dirofilaria immitis) en el perro. In: Simón F, Genchi C, Venco L, Montoya MN, editors. La filariosis en las especies domésticas y en el hombre. Barcelona: Merial Laboratorios; 2011. p. 19–60.
  2. Araujo AM. Canine and human Dirofilaria immitis infections in Portugal. A review. Parassitologia. 1996;38:366.
  3. Carvalho-Varela M, Marcos MVM. A helmintofauna da raposa (Vulpes vulpes silacea) em Portugal. Acta Parasitol Port. 1993;1:73–9.
  4. Alho AM, Cortes H, Lopes AP, Vila-Viçosa MJ, Cardoso L, Belo S, et al. Detection of Dirofilaria immitis antigen in red foxes (Vulpes vulpes) from Portugal. Parasit Vectors. 2016;10 Suppl 1:A16.

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Authors
Ferreira, C., Afonso, A., Calado, M., Maurício, I., Alho, A. M., Meireles, J., Madeira de Carvalho, L., & Belo, S.
Affiliations
  • Universidade Nova de Lisboa (PT)
  • University of Lisbon (PT)
Corresponding author
Belo, Silvana
ORCID
Venue
Parasites & Vectors
Publisher
Springer Science and Business Media LLC
Published
2017-05-19
Volume
10
Issue
1
Pages
250-250
ISSN
1756-3305 (ISSN-L)
DOI
10.1186/s13071-017-2180-y
PMC
PMC5438543
OpenAlex
W2615597439
Semantic Scholar
5d005b743f702e9000321bad2b988eac5025492e
Cited by
33
References
43
Citations by year
2026: 1 · 2025: 1 · 2024: 3 · 2023: 10 · 2022: 7 · 2021: 3 · 2020: 5 · 2019: 2 · 2018: 1
FWCI
1.68
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Parasites & Vectors (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion, other-oa)
Funders
Fundação para a Ciência e Tecnologia, FCT, Portugal (PTDC/SAU-SAP/113523/2009)
Topics
  • Health Sciences › Medicine › Infectious Diseases › Parasitic Diseases Research and Treatment
  • Health Sciences › Veterinary › Small Animals › Helminth infection and control
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Research on Leishmaniasis Studies
Keywords
Dirofilaria immitis, Dirofilaria repens, canine dirofilariasis, ITS1-PCR, species-specific diagnosis, heterozygosity, prevalence, Portugal
MeSH terms
Animals, Dirofilaria immitis, Dirofilariasis, Dog Diseases, Dogs, Microfilariae, Portugal, Species Specificity, Zoonoses, Prevalence, Polymerase Chain Reaction, Sequence Analysis, DNA, DNA, Ribosomal Spacer, Dirofilaria repens

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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