First Molecular Identification of Dirofilaria repens in a Dog Blood Sample from Guanajuato,Mexico

Introduction

D irofilaria repens is the causative agent of subcutaneous dirofilariosis in some carnivores including dogs and cats, and humans. It is transmitted by several mosquito species belonging to a wide range of genera such as Culex, Aedes, and Anopheles (Simon et al. 2012). Adult D. repens worms are generally found in the subcutaneous tissues of definitive hosts, although in humans it is common to find them in subcutaneous nodules and even in the ocular region (De et al. 2012, Sævik et al. 2014). D. repens is recognized as the etiological agent of relevant metazoonotic diseases in many countries in Europe, Asia, and Africa (Simon et al. 2012). In the Americas, only a few sporadic cases of subcutaneous filariosis have been reported, raising questions concerning the etiology of canine dirofilariosis and suggesting that further studies are needed to obtain a more reliable data regarding the species and genetic variants of Dirofilaria circulating among humans and domestic animals (Dantas-Torres and Otranto 2013). During a study carried out at the University of Guanajuato, a positive result for D. repens was obtained through conventional PCR (Salas 2014). However, further studies were needed to confirm that finding, which is the aim of this study.

Materials and Methods

Blood samples were obtained from the Biological Material Bank of the Anatomopathology Veterinary Educational Program of the Division of Science of the University of Guanajuato, Mexico. Samples had been collected during 2013 and 2014 from dogs in the municipalities of Silao (20°47′N and 101°35′W) and Irapuato (20°38′N and 101°40′W), Guanajuato, Mexico. Both municipalities have semiwarm subhumid climates with a mean temperature of 20°C and an annual rainfall of 750 mm.

A total of 177 blood samples were tested using the modified Knott's technique (Ranjbar-Bahadori et al. 2007). Circulating microfilariae were not identified nor differentiated. This study was reviewed and approved by the Ethics Committee of our Institution, and procedures were performed pursuant to national laws.

DNA was extracted from positive samples using the commercial kit UltraClean^®, BloodSpin (Mo Bio Laboratories), following the manufacturer's instructions.

A conventional PCR for the detection of the ITS2 gene was performed using the panfilarial primer set DIDR-F1 (5′ ACT GCG AAT TGC AGA CGC ATT GAG 3′) and DIDR-R1 (5′ AGC GGG TAA TCA CGA CTG AGT TGA 3′). On samples that tested positive, a second conventional PCR protocol was applied for the detection of the 5SrRNA gene of D. repens using the species-specific set of primers D.rep-F1 (5′ TGT TTC GGC CTA GTG TTT CGA CCA 3′) and D.rep-R1 (5′ ACG AGA TGT CGT GCT TTC AAC GTG 3′) (Rishniw et al. 2006).

PCR amplifications were carried out in a total volume of 25 μL, including ±100 ng of genomic DNA for each sample amplification, 200 mM Tris HCI (pH 8.4), 500 mM KCl, 1.5 mM MgCl₂, 0.2 mM dNTPs, 5 U of Taq polymerase (Qiagen, Inc., Mexico, D.F., Mexico), and 200 pM of each of the four primers.

The PCR procedure for the panfilarial primers consisted of a denaturing step at 95°C for 3 min and 40 cycles of denaturing (30 s at 94°C), annealing (60 s at 65°C), and extension (60 s at 72°C), followed by a final extension (10 min at 72°C). The procedure for the specific primers consisted of a denaturing step at 94°C for 3 min and 34 cycles of denaturing (30 s at 94°C), annealing (70 s at 56.5°C), and extension (90 s at 72°C), followed by a final extension (5 min at 72°C). Amplified products were separated, placed on 1.5% agarose gel electrophoresis, and observed under UV light. Fragments of the predicted size were extracted and purified using the Gene Clean Kit (Qiagen, Inc.) and sequenced by LAMBAMA-IPICYT. The sequence was compared with other similar sequences available in the GenBank, as identified through BLAST.

Results

Fifteen out of the 177 (8.4%) blood samples analyzed using the modified Knott's technique tested positive for microfilariae. The conventional PCR assay carried out using the panfilarial primer set showed that only 2/15 samples produced the expected bands. Specific primers for D. repens then applied confirmed only one of them (from Silao) as positive, obtaining a sample yielded a fragment of 247bp. The sequence derived from this 247-bp amplicon had 100% identity to the reference sequences (accession number AJ242966.1) of D. repens.

Discussion

After the first report of the presence of D. repens in Guanajuato (Salas 2014), in which the origin and number of microfilaraemic samples were unknown, the present result confirms the presence of D. repens in a dog blood sample from the municipality of Silao, Guanajuato, Mexico.

Notwithstanding based on only a positive dog and, that is more, lacking beneficial and necessary morphological data on the microfilariae that were found, this finding is significant. Indeed, in the Americas the most important Dirofilaria species reported to date are D. immitis, D. tenuis, and D. ursi, all etiological agents of zoonotic diseases (Simon et al. 2012). Although the American continent is currently recognized as a D. repens-free zone (Simon et al. 2012), and to our knowledge, no clinical cases attributed to D. repens have been reported to date in Mexico, there have been some reports that suggest its presence in other American countries, such as the report of D. repens-like microfilariae, confirmed by PCR but with a sequence match of only 95% in a community in Chile (Lopez et al. 2012). In addition, there is a report of a human clinical case near that area, where an unidentified filarial worm was extracted from a nodule in the arm of a girl (Perez and Arce 2007).

A possible explanation of the presence of exotic filaria (in Chile as in Mexico) is that traveling with pets is increasingly common, not only in local areas but also across countries, thus increasing the risk of healthy dogs becoming infected and the spread of pet-borne diseases such as dirofilariosis in countries where vector mosquitoes are present.

To raise the epidemiological significance of D. repens in Mexico, we suggest considering D. repens when making microscopic blood analysis, to identify morphological features of the microfilariae at a low cost. Moreover, veterinary practitioners should also consider this species in the differential diagnosis of canine and feline cardiopulmonary and skin diseases, to avoid its spread throughout the Americas, not only in animals but also in the human population.

Conclusions

This study confirms, by means of molecular methods, the presence of D. repens, never reported in Mexico thus far, and draws general attention to public health risks.