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Cited by 44Parasitic Diseases Research and TreatmentHelminth infection and controlResearch on Leishmaniasis Studies

Dirofilaria immitis and D. repens show circadian co-periodicity in naturally co-infected dogs

Open access
Ionică, Angela Monica, Matei, Ioana Adriana, D’Amico, Gianluca, Bel, Lucia Victoria, Dumitrache, Mirabela Oana, Modrý, David, Mihalca, Andrei Daniel·2017·Parasites & Vectors
DOI 10.1186/s13071-017-2055-2PMC PMC5331712
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Abstract

BACKGROUND: Dirofilaria immitis and Dirofilaria repens are mosquito-borne zoonotic filarioids typically infecting dogs, causing a potentially fatal cardiopulmonary disease and dermatological conditions, respectively. The females are larviparous, releasing the larvae (microfilariae) into the bloodstream, which further develop in mosquito vectors. However, microfilaremia greatly fluctuates during a 24-h period. As the sampling time can greatly influence the accuracy of diagnosis, the aim of the present study was to assess the circadian periodicity of D. immitis and D. repens in naturally co-infected dogs in an endemic area of Romania and to investigate possible differences of periodicity between these two species. METHODS: Overall, four dogs harbouring natural co-infection with D. immitis and D. repens were selected and sampled every two hours for two consecutive days: two dogs in July 2014 and two in July 2015. At each sampling time, a 0.7 ml blood sample was taken. Modified Knott's test was performed on 0.5 ml, and the remaining 0.2 ml were used for DNA extraction and molecular amplification, both in single and duplex PCR reactions. Microfilariae of both species were morphologically identified and counted in each collected sample, microfilaremia was calculated, and fluctuation was charted. RESULTS: The dynamics of microfilaremia showed similar patterns for both Dirofilaria species. In all four dogs, D. immitis was present at all sampling times, with several peak values of microfilaremia, of which one was common for all dogs (1 am), while minimum counts occurred between 5 and 9 am. Similarly, for D. repens, one of the peak values was recorded in all dogs at 1 am, while minimum counts (including zero) occurred at 9 and 11 am. Single species-specific PCR reactions were positive for both D. immitis and D. repens in all collected samples, while duplex PCR failed to amplify D. repens DNA in many cases. CONCLUSIONS: Both Dirofilaria immitis and D. repens microfilariae are subperiodic, following a similar variation pattern, with peak values of microfilaremia registered during the night in Romania. Duplex PCR fails to identify the infection with D. repens in co-infected dogs when the ratio of microfilaremia is in favour of D. immitis.

AI snapshot

Written from the paper’s full text.

TL;DR

In four naturally co-infected dogs in Romania, *Dirofilaria immitis* and *D. repens* microfilariae showed similar subperiodic circadian patterns, with a shared peak at 1 am and morning minima. Duplex PCR missed *D. repens* when *D. immitis* microfilariae predominated, whereas species-specific PCR was positive for both species in all samples.

Key findings

  • In all four dogs, *D. immitis* was present at every sampling time, with one common peak at 1 am and minimum counts between 5 and 9 am; *D. repens* also had a common 1 am peak, with minimum counts including zero in dogs 1 and 2 at 9 and 11 am.
  • Microfilaremia dynamics correlated significantly in dog 1 (R = 0.853, P = 0.0004) and dog 4 (R = 0.732, P = 0.0067), but not in dog 2 (R = 0.204, P = 0.52) or dog 3 (R = 0.195, P = 0.54).
  • Single species-specific PCR reactions were positive for both *D. immitis* and *D. repens* DNA in all collected samples, while duplex PCR failed to amplify *D. repens* DNA in many cases.
  • Duplex PCR false negativity for *D. repens* correlated with the *D. immitis*:*D. repens* microfilaria ratio (R = 0.592, P = 0 for 12S rDNA; R = 0.242, P = 0.017 for cox1).

Methods

  • The study was conducted in Chilia Veche, Danube Delta, Romania, in July 2014 and July 2015; from 70 dogs tested by modified Knott’s test, four naturally co-infected dogs were enrolled and sampled every 2 h for 48 h (24 sampling events per dog).
  • At each sampling, 0.7 ml blood was taken: 0.5 ml for modified Knott’s test with morphological identification and counting of microfilariae, and 0.2 ml for DNA extraction and single/duplex PCR targeting 12S rDNA and cox1.
  • Microfilaremia was calculated and charted; Spearman’s rank correlation was used to evaluate relationships between microfilaremia values and between Knott test and duplex PCR results.

Limitations

  • The paper does not include a formal limitations section.
  • Evident from the text: the sample was very small (four dogs), all male, medium-sized mixed breed, from one rural locality, and sampled only in July 2014 and July 2015, limiting generalizability.
  • Evident from the text: each dog was sampled for only two consecutive days, and the exact mechanism of periodicity was not established.

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

Integrity signals

Registry

Crossref · 10.1186/s13071-017-2055-2

Cites

31 of 31 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 3 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Dirofilaria immitis microfilariae were present in all four dogs at all sampling times.

    “In all four dogs, D. immitis was present at all sampling times”
    • Also stated in: § Periodicity of microfilaremia · plain
      “Dirofilaria immitis microfilariae were present in all dogs at all sampling times”
    Quote in the paperNot a substantial claim
  • Findingown data

    The dynamics of microfilaremia showed similar patterns for both Dirofilaria species in all four naturally co-infected dogs.

    “The dynamics of microfilaremia showed similar patterns for both Dirofilaria species.”
    • Also stated in: § Periodicity of microfilaremia · plain
      “Overall, the dynamics of microfilaremia showed similar patterns for both Dirofilaria species, in all four dogs (Fig. 1).”
    Quote in the paperSubstantial claim
  • Findingown data

    In all four dogs, one peak of D. repens microfilaremia occurred at 1 am.

    “for D. repens, one of the peak values was recorded in all dogs at 1 am”
    • Also stated in: § Periodicity of microfilaremia · plain
      “one peak value of D. repens microfilaremia (1 am) was common for all animals.”
    Quote in the paperSubstantial claim
  • Findingown data

    Single species-specific PCR reactions were positive for both D. immitis and D. repens DNA in all collected samples, regardless of the target gene.

    “Single species-specific PCR reactions were positive for both D. immitis and D. repens in all collected samples”
    • Also stated in: § Molecular assays · plain
      “Single species-specific PCR reactions were positive both for D. immitis and D. repens DNA in all collected samples, regardless the target gene.”
    Quote in the paperNot a substantial claim
  • Findingown data

    In duplex PCR reactions, there was a statistically significant correlation between the ratio of microfilariae of the two species (D. immitis: D. repens) and false negativity for D. repens (R = 0.592, P = 0 for 12S rDNA; R = 0.242, P = 0.017 for cox1).

    “In the case of duplex PCR reactions, a statistically significant correlation between the ratio of microfilariae of the two species (D. immitis: D. repens) and the false negativity for D. repens was noted (R = 0.592, P = 0 for 12S rDNA; R = 0.242, P = 0.017 for cox1).”
    Quote in the paperNot a substantial claim
  • May combine two claims. 3
  • Classified as probably not a substantial claim. 1

  • May combine two claims.

    Findingown data

    In the other two dogs (dogs 2 and 3), the correlation between the dynamics of D. immitis and D. repens microfilariae was weak and not statistically significant (dog 2: R = 0.204, P = 0.52; dog 3: R = 0.195, P = 0.54).

    “In the other two dogs, a weak correlation with no statistical significance was noted (dog 2: R = 0.204, P = 0.52; dog 3: R = 0.195, P = 0.54).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Once a patent co-infection is established, D. immitis and D. repens microfilariae display similar circadian periodicity with no apparent influence on each other.

    “However, our results indicate that once the animal develops a patent co-infection, the microfilariae of the two species display a similar circadian periodicity, probably as a reaction to the same stimuli, with no apparent influences between each other.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    The night peaks of microfilaremia correspond to the peak biting activity of local mosquito species, such as Culex pipiens, which is an efficient vector for both Dirofilaria spp. and highly attracted to dogs, and to the sleeping behaviour of the sampled dogs.

    “This corresponds to the peak biting activity of local mosquito species, such as Culex pipiens, which is an efficient vector for both Dirofilaria spp. and highly attracted to dogs [25, 26], but also to the sleeping behaviour of the sampled dogs.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [25] Nicolescu G, Vladimirescu A, Ciolpan O. The distribution of mosquitoes in Romania (Diptera: Culicidae). Part I: Anopheles, Aedes and Culex. Eu Mosquito Bull. 2002;13:17–26.

      Not checked in the free run.

    • cites [26] Capelli G, Frangipane di Regalbono A, Simonato G, Cassini R, Cazzin S, Cancrini G, et al. Risk of canine and human exposure to Dirofilaria immitis infected mosquitoes in endemic areas of Italy. Parasit Vectors. 2013;6:60. source

      Not checked in the free run.

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

    Study design and conductnone given

    In each PCR reaction set, a positive control (DNA extracted from adult nematodes) and a sample with no DNA were included.

    “In each reaction set, a positive control (DNA extracted from adult nematodes) and a sample with no DNA were included.”
    Quote in the paperNot a substantial claim
4 of 4

  • The authors state this as an assumption.

    This is the first periodicity study performed on co-infected dogs.

    “To our knowledge, this is the first periodicity study performed on co-infected dogs.”

  • The paper relies on this without support.

    The four sampled dogs are representative of naturally co-infected dogs in Romania for circadian periodicity.

    “Four of the dogs which had D. immitis and D. repens co-infection were enrolled in the experiment”
    “with peak values of microfilaremia registered during the night in Romania.”
2 of 2

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • In Romania, prevalence between 7.58-9.26% was reported in golden jackals, 0.33% in red foxes, and 10% in feral cats.

    “In Romania, prevalence between 7.58–9.26% was reported in golden jackals, 0.33% in red foxes, and 10% in feral cats [140,141].”

    From What has happened to heartworm disease in Europe in the last 10 years? (2022) · cites this paper as [141]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • D. immitis does not exceed D. repens at every sampling time

    Minor

    The discussion says the microfilarial disproportion was always in favour of D. immitis. At 11 pm for dog 1, Table 1 instead reports 429 D. immitis and 479 D. repens microfilariae/ml.

1 of 1

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Authors
Ionică, A. M., Matei, I. A., D’Amico, G., Bel, L. V., Dumitrache, M. O., Modrý, D., & Mihalca, A. D.
Affiliations
  • University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca (RO)
  • University of Veterinary Sciences Brno (CZ)
Corresponding author
Dumitrache, Mirabela Oana
ORCID
Venue
Parasites & Vectors
Publisher
Springer Science and Business Media LLC
Published
2017-02-28
Volume
10
Issue
1
Pages
116-116
ISSN
1756-3305 (ISSN-L)
DOI
10.1186/s13071-017-2055-2
PMC
PMC5331712
OpenAlex
W2593711027
Cited by
43
References
30
Citations by year
2026: 2 · 2025: 1 · 2024: 4 · 2023: 8 · 2022: 7 · 2021: 5 · 2020: 11 · 2018: 4 · 2017: 1
FWCI
3.37
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Parasites & Vectors (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • ASEP (submittedVersion, public-domain)
Funders
UEFISCDI, Romania (TE299/2015)
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, circadian rhythm, microfilaremia, co-infection, dogs, duplex PCR, Romania, cardiopulmonary disease, skin diseases, sampling time
MeSH terms
Animals, Circadian Rhythm, Dirofilaria immitis, Dirofilariasis, Dog Diseases, Dogs, Romania, Species Specificity, Polymerase Chain Reaction, Parasitemia, Coinfection, Dirofilaria repens

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