We analyzed the internal transcribed spacer 1 (ITS1) and 12S loci of circulating microfilariae collected from blood samples of raccoons in Manuel Antonio National Park (Quepos, province of Puntarenas) and surrounding areas in Costa Rica. Raccoons were captured for routine health evaluation during 2019–2022. Wildlife specialists captured raccoons by using Tomahawk traps in areas where local persons and park rangers reported a high frequency of raccoons. Researchers then sedated the raccoons and collected blood samples. Sample collection, procedures, and analyses were approved under permits R-CM-UNA-005–2021-OT-CONAGEBIO and SINAC-PNI-ACOPAC-021–2019. Laboratory technicians stored the blood at 4°C prior to subsequent analysis. Raccoons were released once they fully recovered from the anesthesia.

We performed microscopic assessment of all blood samples by using the Knott modified technique (Appendix). We then extracted DNA from blood samples by using the DNeasy Blood and Tissue Kit (QIAGEN, https://www.qiagen.com), according to the manufacturer’s instructions. We then amplified mitochondrial 12S rRNA by using 12S.C345.F and 12S.C345R primers (6) and amplified the nuclear ITS1 loci with rDNA2 and rDNA158S primers (7). All reactions included Toxocara canis DNA as a positive control and a nontemplate control with PCR-grade water. We examined obtained amplicons on 1.5% agarose gels, purified positive reactions with Exo-SAP, and sequenced in both directions according to the Sanger method using the ABI 3730xl System (Macrogen Inc., https://dna.macrogen.com).

We used MEGA 7.0 software (https://www.megasoftware.net) to inspect and trim ITS1 sequences and aligned our sequences with other Mansonella spp. sequences available in GenBank by using the MUSCLE algorithm. We constructed a Bayesian inference tree by using the BEAST package (https://beast.community) and drew a Templeton Crandall-Sing haplotype network. Finally, we calculated Nei’s genetic distance for the ITS1 sequences (Appendix).

We observed microfilariae in 23.5% (4/17) of the raccoon blood samples in concentrations ranging 200–800 microfilariae/mL (Table). We noted the morphology of the observed microfilariae to be compatible with M. ozzardi original descriptions: 180–190 µm long and 3–4 µm wide; no sheath; tail elongated and slender without nuclei (Appendix) (8).

DNA could be isolated from 17 blood samples. In molecular analyses, 3 of 4 raccoons scoring positive at Knott’s test also demonstrated positive PCR tests, and 4 of 7 raccoons that tested negative at Knott’s test drew positive results by ITS-1 and 12S PCRs. Five of the 17 blood samples tested positive in the ITS1 PCR, and 4 of the 17 samples tested positive in the 12S PCR (Table). ITS1 sequences obtained from our analyses were 397–441-bp long and 97.0%–99.1% similar, with 100% of coverage with M. ozzardi isolated from humans in Brazil (GenBank accession no. MN432519). The 12S sequences were 107–151-bp long and 97.1%–99.1% similar to M. ozzardi from Brazil (GenBank accession no. LT623914.1). We deposited our new sequences into GenBank (accession nos. OR636492–6 for ITS1 and OR700019–22 for 12S).

Figure

Figure. Phylogenetic and haplotypic analysis of Mansonella ozzardi ITS1 sequences obtained from racoons (Procyon lotor) in Costa Rica, 2019–2022 (black diamonds). A) Bayesian inference phylogenetic tree of ...

Mansonella spp. clustered according to the nominal species in the ITS1 phylogenetic tree. Moreover, the M. ozzardi sequences we generated clustered in a separate clade in the BI tree, haplotype network and Nei’s genetic distance principal component analysis. The haplotype network revealed 6 M. ozzardi haplotypes: 1 from Costa Rica, 2 from Japan, 2 from Brazil, and 1 from Argentina (Figure).

M. ozzardi is a filarioid nematode affecting humans in the Americas. Human mansonellosis is not completely harmless, and ocular impairment has been reported in humans from Panama (9). The lack of specific symptoms associated with mansonellosis usually leads to underdiagnosed infections and inadequate treatment (10). Accurate and prompt detection of the disease is therefore critical to avoid complications.

In our study, as in prior reports, PCR provided higher sensitivity compared with microscopic methods because molecular tools target DNA sequences rather than whole parasitic structures (11,12). Our results were as expected except for 1 sample determined to be positive by Knott’s test and negative in PCRs, possibly due to a prolonged period at room temperature that might have affected DNA quality. We attempted amplification of larger fragments of other genes but failed due to the presence of fragmented DNA.

Our findings suggest that raccoons are potential definitive hosts of M. ozzardi. Confirming the role of these procyonids in the life cycle of this worm, however, requires investigation into the anatomic location of adult worms inside the animals, the reservoir capacity of raccoons, and the feeding preference of intermediate hosts to raccoons. The Simulium sanguineum black fly, found in Panama, is the only simulid species reported thus far in Central America to act as intermediate host of M. ozzardi, but this simulid has not been reported in Costa Rica (13). Nevertheless, other Simulium spp. flies have been detected in this country, suggesting a need for more research to track infection status of such species and to determine the potential intermediate hosts used by M. ozzardi in Costa Rica.

Sympatry of raccoons and humans in densely populated regions has become a public health problem that can increase pathogen spillover to humans (14). Costa Rica is not the only country facing the expansion of raccoon populations into urban areas, largely due to human invasion of wildlife habitats resulting in ecosystem fragmentation. Several countries in Europe have reported a rise in the number and geographic range of racoons into human-inhabited environments. This expansion may increase pathogen spillover and the transmission of perilous diseases or parasites, such as M. ozzardi, to both domestic animals and humans (15). In Costa Rica, raccoons are reservoirs and carriers of such pathogens as Salmonella (14) and B. procyonis, one of the causing agents of visceral larva migrans (4). The encroachment of raccoons into urban areas thus represents a patent zoonotic risk (4,14).

Because our findings implicate raccoons as potential reservoirs of the M. ozzardi parasite, and given the scarcity of information on intermediate hosts, we recommend active surveillance of M. ozzardi infections in humans. Our findings also support the need for histopathological studies to determine the location of adult M. ozzardi parasites in infected procyonids and further investigation into possible intermediate hosts and their feeding preferences to P. lotor. Such investigations will be crucial to providing a full understanding of the lifecycle of M. ozzardi in Costa Rica.