Microfilaremia by Cercopithifilaria bainae in a dog from the central western region of Brazil: case report

[Microfilaremia por Cercopithifilaria bainae em um cão da região Centro-Oeste do Brasil: relato de caso]

ABSTRACT

Cercopithifilaria bainae is a nematode belonging to the family Onchocercidae that parasitizes the subcutaneous tissue of dogs. Its transmission occurs through the tick Rhipicephalus sanguineus and its geographical distribution overlaps that of this vector. The present study reports the detection of microfilaremia by C. bainae in an eight-year-old male dog that presented anorexia, hyperthermia, motor incoordination, mydriasis, a nodule in the left testicle and concomitant infection by Ehrlichia sp. Blood samples were analyzed using microscopy, PCR and DNA sequencing. Microfilariae measuring 150±5.5μm in length and 7±1.8μm in width were retrieved. The DNA sequence exhibited 98% identity with C. bainae sequences available in Genbank. This is the first report of microfilaremia by C. bainae in a dog in the central western region of Brazil.

Keywords:
dog; microfilariae; Brazil; DNA

RESUMO

Cercopithifilaria bainae é um nematoide pertencente à família Onchocercidae, que parasita o tecido subcutâneo de cães. Sua transmissão ocorre pelo carrapato Rhipicephalus sanguineus, e sua distribuição geográfica se sobrepõe ao espalhamento desse vetor. O presente estudo relata a detecção de microfilaremia por C. bainae em um cão macho de oito anos que apresentava anorexia, hipertermia, incoordenação motora, midríase e nódulo no testículo esquerdo e infecção concomitante por Ehrlichia sp. A coleta de sangue foi realizada, e o material analisado por meio dos exames de microscopia, PCR e sequenciamento de DNA. Microfilárias medindo 150±5,5μm de comprimento e 7±1,8μm de largura foram recuperadas. A sequência de DNA obtida mostrou 98% de identidade com sequências de C. bainae disponíveis no Genbank. Este é o primeiro relato de microfilaremia de C. bainae em um cão na região Centro-Oeste do Brasil.

Palavras-chave:
cão; microfilárias; Brasil; DNA

INTRODUCTION

Filarioids of the family Onchocercidae include species with microfilariae that circulate in the bloodstream (Dirofilaria immitis and Acantocheilonema reconditum) or parasitize the subcutaneous tissue of vertebrate hosts (Cercopithifilaria sp. and Onchocerca lupi) (McCall et al., 2008). Among these nematodes, those of the genus Dirofilaria and Acantocheilonema have been extensively studied throughout the world and their pathogenic role is well defined (McCall et al., 2008). Conversely, species that inhabit subcutaneous tissue (Cercopithifilaria and Onchocerca) have been under-investigated.

The genus Cercopithifilaria comprises 28 different species of filarioids, three of which (Cercopithifilaria grassi, C. bainae and Cercopithifilaria sp. II sensu) parasitize dogs. Among these species, C. bainae is considered the most common in dogs and its biology has been studied in recent years (Otranto et al., 2012). The distribution of this species is believed to be strictly associated with that of its main arthropod vector - the tick Rhipicephalus sanguineus sensu lato (Brianti et al., 2012). The first report of C. bainae in dogs was published more than thirty years ago (Almeida and Vicente, 1984), but this nematode has since been largely ignored. In recent years, C. bainae has been described in dogs and/or ticks in several countries (Latrofa et al., 2014; Solinas et al., 2014; Santos et al., 2017).

C. bainae has been considered a filarioid of minor pathogenic relevance. However, dermatological conditions, such as perivascular interstitial dermatitis, have been associated with infection by this parasite (Otranto et al., 2012). Moreover, a case of chronic polyarthritis has been described in a European dog (Gabrielli et al., 2014).

In Brazil, information on the occurrence of C. bainae is available in dogs and Rhipicephalus sanguineus only for the southeastern (Almeida and Vicente, 1984) and northeastern regions (Latrofa et al., 2014; Ramos et al., 2016), however there is no information of this filarioids in other regions of the country, despite the wide distribution of the tick vector. This paper reports a case of C. bainae in a dog in the central western region of Brazil (state of Mato Grosso do Sul) showing atypical microfilaremia.

CASUISTRY

In September 2016, an eight-year-old male poodle in the municipality of Bandeirantes (19º55'04" S and 54º21'50" W) in the state of Mato Grosso do Sul (central western region of Brazil) was admitted to a private veterinary practice with the complaint of vomiting and diarrhea. The clinical examination revealed hyperthermia, motor incoordination, mydriasis, anorexia and an increase in left testicular volume. Blood sampling was performed for the blood cell count. The hematological analysis revealed hypochromic normocytic anemia, neutrophilia, eosinophilia, lymphopenia and thrombocytopenia. Other findings included morulae of Ehrlichia sp. and microfilaria in the blood with a mean size of 150.42±5.5μm in length and 7.09±1.8μm in width (Figure 1).

Figure 1
Microfilaria in blood sample from dog.

The animal was treated with febantel 15mg/kg, pyrantel 14.4mg/kg, praziquantel 5mg/kg, ivermectin 0.006mg/kg (Endogard® - Virbac) and doxycycline 10mg/kg and was kept under observation, but died after five days due to clinical complications.

At necropsy, a marked increase in the volume of both testicles was observed. The right testicle exhibited diffuse suppurative necrotizing orchitis of an undetermined origin and a diagnosis of seminoma was made in the left testicle. In the liver, plasma extravasation was found, with inflammatory infiltrate of neutrophils and plasma cells, fibrinoid necrosis in the paracentral region and fibrillar eosinophilic material partially occluding the blood vessel lumen. In the kidneys, we found increased cellularity in the glomerular base, predominantly inflammatory infiltrate of lymphocytes and peri-glomerular plasma cells, and interstitial foci adjacent to the renal pelvis. In the spleen, we found multiple foci of fibrinoid necrosis in the white pulp, abundant fibrillar eosinophilic material interspersed with lymphocytes, plasma cells and macrophage populations, which were attributed to infection by Ehrlichia sp.

For the identification of the microfilaria, genomic DNA was extracted from the blood sample following protocol described by Araújo et al. (2009). The integrity and quantity of the DNA was evaluated by 1% agarose gel electrophoresis and spectrophotometry (A260/A280), respectively.

A PCR was carried out using the primer set Fila12SF (5’-CGGGAGTAAAGTTTTGTTTAAACCG-3’) and Fila12SR (5’ -CATTGACGGATGGTTTGTACCAC-3’) (Otranto et al., 2012) developed to amplify a 330-bp fragment of 12S rRNA gene common to filarioids of the genera Acanthocheilonema, Cercopithifilaria and Dirofilaria. The PCR assay was performed in a final volume of 25µL containing 10mM tris-HCL (pH 8.3), 50mM of KCl, 1.5mM of MgCl₂, 0.2mM of each deoxynucleotide (dNTP), 1.25U of Taq DNA polymerase (Invitrogen), 10pmol of each primer, approximately 100ng of genomic DNA and H₂O to a final volume of 25uL. The thermocycling conditions were 80°C for one minute, 95°C for five minutes, followed by 40 cycles at 94^oC for one minute, annealing at 58^oC for one minute and extension at 72^oC for one minute. A final extension step was performed at 72ºC for one minute.

The amplification products were viewed under an ultraviolet light after electrophoresis on 2% agarose gel stained with GelRed® Biotium) following the manufacturer's instructions. DNA from C. bainae (characterized by Ramos et al., 2016) and nuclease-free water were used at the positive and negative control reactions, respectively.

The amplicon (approximately 330pb) was purified with CleanSweep PCR Purification Reagent (Thermo Fisher Scientific) following the manufacturer's instructions and submitted to DNA sequencing in both directions using the Sanger method in an ABI-3130 automatic sequencer (Applied Biosystems). The chromatograms were evaluated and edited using the Contig Editor program (Gene Studio) v. 2.2.0 and the consensus sequence (272bp) was submitted to BLASTn analysis. Sequences of C. bainae showed 98% homology to those available in GenBank database (accession numbers KX156956, MG793438 and MG793436). The consensus DNA sequence obtained in the present study was deposited in Genbank under accession number MH972532.

DISCUSSION

Among the filarioids that infect dogs, those transmitted by culicid vectors (Dirofilaria immitis and Dirofilaria repens) have been extensively studied (McCall et al., 2008). Attention has recently been given to species of the genus Cercopithifilaria, the microfilariae of which can be detected under the skin of dogs, but little is known regarding its pathogenic potential (Santos et al., 2017).

Due to the wide distribution of its vector R. sanguineus (Brianti et al., 2012), the distribution of filarioids is also extensive (Latrofa et al., 2014; Solinas et al., 2014; Ramos et al., 2016), but the prevalence in dogs varies. Ramos et al. (2016) found a rate of only 0.96% (1/104) among animals in northeastern Brazil. In contrast, Solinas et al. (2014) found a rate of 9.4% (17/180) in dogs in Italy. The low prevalence of the filarioids in some regions may account for the lack of veterinarian knowledge and little clinical importance attributed to the nematode, making it difficult to obtain information on its actual pathogenic potential.

In Brazil, R. sanguineus sensu lato is the most prevalent tick species in dogs and is responsible for the transmission of several pathogenic microorganisms, such as Ehrlichia canis, Babesia canis vogeli, Hepatozoon canis and even C. bainae (Santos et al., 2017). In the present report, although no ticks were found during the clinical examination, the transmission of the filarioid to the dog likely occurred through this route, since morulae from Ehrlichia sp. were found during the hematological analysis. This pathogen is common among dogs in the region and is known to be transmitted by R. sanguineus (Soares et al., 2017).

Cercopithifilaria sp., (Ie. C. bainae and C. grassii) was reported in R. sanguineus sensu lato by Latrofa et al. (2014) in 9,6% (17/177) from several country’s samples, including three positive in Pernambuco- Brazil, Santos et al. (2017) founded C. bainae in 2.68% (51/1906) of dissected ticks at the same region. However, there are no studies of this filarioids in R. sanguineus in other regions of Brazil, despite the wide distribution of this vector.

Adult nematodes of C. bainae as well as their larvae (microfilariae) are found in the subcutaneous tissue and are difficult to detect, unlike D. immitis and A. reconditum, which are found in the blood (Ramos et al., 2016). The dog in the present report had microfilariae in the blood, which may be considered an ectopic location, based on the information currently available on the lifecycle of the pathogen described by Brianti et al. (2012). Other aberrant locations of C. bainae microfilariae have been reported in dogs, such as the synovial fluid (Gabrielli et al., 2014). The ectopic location of other members of the family Onchocercidae has also been reported, such as Onchocerca lupi found in the larynx of a dog (Alho et al., 2016).

The differentiation between types of filariasis is important for the prescription of the treatment and the establishment of a prognosis. D. immitis leads to severe heart disease and requires expensive, potentially aggressive therapy (McCall et al., 2008). In contrast, infection by C. bainae is usually an accidental finding, with no clinical signs (Otranto et al., 2012). The animal in the present report presented microfilaremia, which occurs with A. reconditum and D. immitis and could therefore have led to improper treatment.

The microfilariae species can be differentiated by morphological observation. Studies by Ramos et al. (2016) show thatD. immitishas 301.2±7.6μm long and 5.9±0.8μm wide on average and features a conical front end and a straight posterior end. Microfilariae ofA. reconditumare 272.3±4.3μm in length and 4.1±0.3μm in width, and are characterized by a blunt front end. Conversely, microfilariae ofC. bainaehas 180.2±2.3μm in length and 4.6±0.2μm in width rounded head, short dorsal-ventrally flattened body and thick cuticle presenting transverse striations. The morphometric analysis of the microfilariae found in this study revealed measures somewhat lower than those reported by Ramoset al. (2016). These variations may be attributed to differences in the stage of larval development. Another possible explanation would be genetic variation among nematode strains.

Due to the complexity of the clinical condition found in the dog of the present report, it is difficult to establish the actual pathological contribution of C. bainae. However, as observed in other studies, the ectopic location of the microfilaria likely triggered a local immune response capable of causing damage to adjacent tissues. Thus, it is essential to conduct studies involving the experimental infection of animal models to clarify the pathological potential of this filarioid.

This report is the first description of C. bainae in a dog from central western Brazil. The atypical case of microfilaremia underscores the importance of considering this aspect in the differential diagnosis in order to avoid misdiagnosis and inadequate treatment.

ACKNOWLEDGMENT

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001

REFERENCES

• ALHO, A.M.; CRUZ, L.; COELHO, A. et al. Aberrant laryngeal location of Onchocerca lupi in a dog. Parasitol. Int., v.65, p.218-220, 2016.
• ALMEIDA, G.L.G.; VICENTE, J.J. Cercopithifilaria bainae sp. n. parasita de Canis familiaris (L.) (Nematoda, Filarioidea). Atas Soc Biol., v.24, p.18, 1984.
• ARAUJO, F.R.; RAMOS, C.A.N.; LUIZ, H.L. et al. Avaliação de um protocolo de extração de DNA genômico a partir de sangue total. Campo Grande: Embrapa Gado de Corte, 2009. 120p. (Comunicado Técnico, n.5).
• BRIANTI, E.; OTRANTO, D.; DANTAS-TORRES, F. et al. Rhipicephalus sanguineus (Ixodida, Ixodidae) as intermediate host of a canine neglected filarial species with dermal microfilariae. Vet. Parasitol., v.183, p.330-337, 2012.
• GABRIELLI, S.; GIANNELLI, A.; BRIANTI, E. et al. Chronic polyarthritis associated to Cercopithifilaria bainae infection in a dog. Vet. Parasitol. v.205, p.401-404, 2014.
• LATROFA, M.S.; DANTAS-TORRES, F.; GIANNELLI, A.; OTRANTO, D. Molecular detection of tick-borne pathogens in Rhipicephalus sanguineus group ticks. Ticks Tick Borne Dis., v.5, p.943-946, 2014.
• MCCALL, J.W.; GENCHI, C.; KRAMER, L.H. et al. Heartworm disease in animals and humans. Adv. Parasitol., v.66, p.193-285, 2008.
• OTRANTO, D.; BRIANTI, E.; LATROFA, M.S. et al. On a Cercopithifilaria sp. transmitted by Rhipicephalus sanguineus: a neglected, but widespread filarioid of dogs. Parasit. Vectors, v.5, p.1, 2012.
• RAMOS, R.A.; OLIVEIRA, RÊGO, A.G.; FARIAS FIRMINO, E.D. et al. Filarioids infecting dogs in northeastern Brazil. Vet. Parasitol., v.226, p.26-29, 2016.
• SANTOS, M.A.B.; SOUZA, I.B.; MACEDO, L.O. Cercopithifilaria bainae in Rhipicephalus sanguineus sensu lato ticks from dogs in Brazil. Ticks Tick Borne Dis., v.8, p.623-625, 2017.
• SOARES, R.; PEDROSO, T.; BABO-TERRA, V. et al. Molecular survey of Anaplasma platys and Ehrlichia canis in dogs from Campo Grande, Mato Grosso do Sul, Brazil An. Acad. Bras. Ciênc., v.89, p.301-306, 2017.
• SOLINAS, C.; VARCASIA, A.; BRIANTI, E. et al. Cercopithifilaria spp. in dogs in Sardinia Island (Italy). Parasitol. Res., v.113, p.675-679, 2014.

Publication Dates

• Publication in this collection
  08 May 2020
• Date of issue
  Mar-Apr 2020

History

• Received
  30 Nov 2018
• Accepted
  27 June 2019