Canine Filariasis Outbreak in Southwestern Colombia: A Molecular and Epidemiological Study

Different nematodes affect canines, however, Dirofilaria immitis is the most prevalent filariae. D. immitis causes heartworm disease that can be fatal for dogs and potentially transmitted to humans. Climate change, animal migration, among other factors have changed the dynamics of vector-borne diseases, including filariasis. In the last four years, a sudden increase of dogs with microfilaremia was reported by different veterinary centers in Cali, the main city of Southwest Colombia. The objective of this study was to molecularly identify the etiologic agent of this filariasis outbreak reported in this area from 2018-2019, using a novel PCR–RFLP method. Those filariasis cases were initially detected by microscopic and serological tests. A total of 82 canine filariasis cases were molecularly analyzed, identifying 55 (67%) of them as Acantacheilonema reconditum. PCR-sequencing was performed in eight cases confirming this finding. The filariasis cases were statistically associated with male dogs who had clinical signs of anemia, with low levels of hemoglobin and hematocrit (p &lt;0.0001), and high levels of plasma proteins (p &lt;0.001). This emerging canine disease constitutes an important public health concern among clinicians and advises active surveillance to explore its zoonotic potential.


Introduction
Filariasis is a parasitic disease caused by nematodes of the Onchocercidae family that affect a wide variety of mammals. Microfilariae is the first larval stage of the nematode that can be observed circulating in peripheral blood, representing the parasite diagnostic stage [1,2]. Among these parasites, Dirofilaria immitis is the most widely known since it causes heartworm disease in canines. geographic regions of this country, varying from less than 0.5% in the Andean region to about 20% in the Caribbean region during the last 18 years [8][9][10][11][12]. In the Southwest of Colombia, there are not recent studies about the distribution of filarial nematodes among dogs. The last study reported in 1967 showed a prevalence of 5% for D. immitis in canines from the city with the largest metropolitan area of Southwest Colombia: Cali [2,13].
The transmission of filaria infection in dogs is influenced by the causing parasitic species as well as external environmental factors [14]. Depending on the nematode´s species, different arthropods have been identified as biological vectors, generating different transmission dynamics.
For instance, D. immitis is transmitted by mosquitoes such as Aedes spp, Culex spp, and Anopheles spp [1]; while A. reconditum is transmitted by fleas such as Ctenocephalides felis, C. canis, and lice such as Heterodoxus spiniger [3]. Besides, climate change, constant human and animal migration and commercial trades have a direct effect on the incidence, distribution, and control of endemic and emerging vector-borne diseases [8,[15][16][17][18]. These events impose the need for constant surveillance of canine filariasis, as a vector-borne disease with zoonotic potential [1,19]. Veterinary diagnostic centers of Cali, Colombia, recently reported an unusual increase in the number of microfilariasis in dogs since 2016 compared with previous years (unpublished data), suggesting a canine filariasis outbreak in the Southwestern region of this country. Interestingly, most cases (detected by microscopy) were negative for commercial serologic tests for D. immitis, generating some concerns about the etiology of the increasing filaria cases observed in Cali. The latter emphasizes the need for novel diagnostic approaches to specifically identify the filarial species involved in this outbreak to establish directed and more efficient therapeutic options and control measures. For this reason, here we conducted a prospective study that aims to propose a novel molecular method to identify the circulating filariae in canines from Southwest Colombia and epidemiologically characterize the cases registered for one year.

Microscopic and Serologic test
From August 2018 to August 2019, 2,971 blood samples from different canines were analyzed for the presence of hemoparasites. A total of 102 (3.4%) patients were identified with microfilariae by microscopic examination (Figure 1), but only 82 met the inclusion criteria. Out of the 82 filaria cases, only three were positive for the D. immitis antigen. Moreover, 43 healthy individuals were included as controls with negative results for both microscopic and serologic tests.

Demographic Data Analysis
The median age for both cases and controls was 5 years, with an interquartile range (IQR) of 2-9 years for filaria cases and 3-8 years for controls. Most of the cases were male (64/82), having filariasis significantly associated with male sex (p<0.00001, Table 1). Regarding the breed, the mestizo/mixed predominated among cases and controls, followed by the Poodle and Schnauzer (Table 1).  Interestingly, about 39% (32/82) of owners reported their dogs having infestation with one or more types of ectoparasites (fleas, ticks, or lice).

PCR-Sequencing and serological testing
Only three cases (cases No. 30, 46, 67) had positive results for the D. immitis antigen test. However, all these cases had been identified as A. reconditum by PCR-RFLP. PCR-sequencing of these three cases confirmed our PCR-RFLP results (Table 4). Additionally, five additional cases (from No. 01 to 05) were PCR-sequenced, confirming the presence of A. reconditum in our sample set and the PCR-RFLP results. With the PCR-sequenced cases (n=8), an NJ phylogenetic analysis was performed, identifying four unique species and two clusters for the A. reconditum species (Table 4).

Discussion
The presence of microfilariae in dogs from Southwestern Colombia was investigated for one American countries, that could represent a public health concern in the context of a zoonotic disease in a globalized world. Additionally, this finding rises concerns regarding the distribution of nematodes species and potentially other vector-borne pathogens that can reach human beings and other animal species.
Interestingly, the sequencing results showed a high identity and coverage of the eight analyzed strains with the nematode strains reported from the East of Brazil (  (Table 4). Although the antigen test applied in the study has a sensitivity of 94% and specificity of 100% (according to the manufacturer), these discrepant results may possibly be due to cross-reactivity with A. reconditum antigens. Cross-reactivity between D.
immitis and Acantacheilonema species such as A. reconditum and A. odendhali have been previously described [36,37]. Shared epitopes have been reported even between D. immitis and other nematodes species such as Toxocara canis and Angiostrongylus vasorum [36,[38][39][40] A. reconditum is known for its low pathogenicity in canines and association with mild clinical manifestations mainly at the subcutaneous tissue and skin [6,41]. Although, most of the analyzed cases reported here did not have clinical signs of disease, they exhibited a significantly reduced level of hemoglobin and hematocrit compared to controls ( Table 2). This finding highlights anemia as an important clinical manifestation of filariasis, most likely caused by A. reconditum (confirmed for 67% of cases). Additionally, the high levels of plasmatic proteins in canine filariasis suggested an inflammatory response in these dogs. A reduction in hemoglobin concentration and an increase in total protein levels have been previously reported among A. reconditum cases, attributing those findings to the destruction of erythrocytes and the increase of antibody response to parasitic antigens [42]. This high protein levels could be further evaluated in order to find a potential biological marker specific for A. reconditum, looking at either host or parasite´s proteins. Interestingly, 84.2% of the males that were evaluated in this study were positive cases for canine filariasis (64/76). In fact, our study found that the risk of acquiring microfilaria infection is nine times higher in males than females (p <0.00001, Table 1). Two previous studies carried out in Colombia did not find a significant relationship between male dogs and microfilariae infection [9]. In the same sense, a study done in Brazil between 2015 and 2017 reported a higher prevalence of A.
reconditum in males, albeit without significant differences in relation to the sex of the canine [43].
However, a study carried out in Vesuvius, Italy, in 2000 was in line with our findings, showing that male dogs have a significantly higher risk for A. reconditum infection [44]. In the same sense, previous a study attributed a higher prevalence of A. reconditum in male canines (85.7%) possible to hormonal effects [42].
We did not observe an association of the filaria cases with the age or breed of the dogs.
However, a higher proportion of young adult mestizo canines was observed among filariasis cases (Table 1). Few articles referred to the relationship between age and microfilariae infection, for instance, there was a report from Barcelona that showed a significant association of D. immitis with dogs older than 5 years [45]. Similarly, a significant relationship between A. reconditum with adult canines was also reported in dogs infected with A. reconditum [44]. Therefore, age should be considered when suspecting filariasis, as it could be possible that older dogs have an increased likelihood of exposure to different filarial vectors. Moreover, despite the high percentage of mestizo dogs positive for microfilaria infection (59%, 48/82), we did not find breed as a factor associated with filaria cases ( Table 1). Some of these mestizo dogs had been rescued directly from the street or living in shelters. This is important considering that A. reconditum is transmitted by vectors such as fleas (Ctenocephalides felis) and lice (Heterodoxus spiniger) [3] that commonly infest stray or refuged dogs rather than "pure" breed dogs [41,46]. In fact, a study in Zambia, reported a prevalence of 7.6% microfilariasis (different from D. immitis) in mestizo dogs and not any dog of "pure" breed with these nematodes among 272 dogs studied [47].
We observed a higher concentration of cases in the Southwest of Cali, identified as a red area in the map (Figure 4). This cluster of canine filariasis coincides with the location of the biggest dog refugee centers as well as recent informal and formal urbanization projects. This area of current development has more distribution of vegetation (compared with the other parts of the city), with small of water bodies (such as Melendez, Lili, and Pance rivers and small pounds, Figure 4 and S1).
Additionally, according to the local environmental administration reports, the Southwest of Cali has a higher distribution of urban wetlands and has the lowest surface temperatures in the urban area of the city (ranging from 17.4°C-23.8°C, Figure S1) [48,49]. These environmental conditions may favor the growth and dissemination of different vectors for the filariae. It is necessary to explore the presence of previously known (fleas and lice) and potentially new vectors for A. reconditum in the city that could affect the geographic distribution of this filariae.
We acknowledge some limitations of our study, such as the need to expand the PCR-sequencing analysis for more samples, however, the number of sequenced strains was given by the budget restrictions. Additionally, we did not quantify the parasitemia, which could have helped to explain the reason for low sensitivity for some cases and the detection limit of the PCR test after the standardization in our lab. We propose to expand the number of subjects to include dogs from other areas of Colombia and potentially identify other filariae in order to build a diagnostic algorithm, based on the species and clinical findings identified.

Sample collection and study setting
Positive canine filariasis cases detected by microscopy from August 2018 to August 2019 were selected for this prospective study. The canines were either initially diagnosed at a local veterinary center or sent from another veterinary laboratory/clinic from Cali, Colombia, or the neighboring cities. Animals owners were asked to bring the animal to enroll in the study, provide a new blood sample and, fill out an informed consent before the canines were included in the study. In parallel, samples from healthy dogs of different sex and breeds were collected as a control group.

Variables
A case report format (CRF) was filled out including the clinical and sociodemographic characteristics of the canines such as the canine age, breed, address during the last year, presence of ectoparasites (lice, fleas, and/or ticks) and, any noticeable clinical sign such as weakness, malnutrition or cachexia, skin lesions, bleeding, ulcers, respiratory distress, seizures, among others.
Participants were identified with a code to ensure data confidentiality.

Clinical laboratory testing
In the initial diagnosis stage, blood smears were stained with Wright and a thick drop stained with Giemsa or Knott´s test for a microscopic examination that allowed us to recruit positive cases. All cases detected by microscopy were also analyzed with a serological test that specifically detects the secreted 14KDa antigen of D. immitis [50] (Canine Heartworm Ag 2.0 Bionote, Inc, Korea).
These microscopic and serological tests were also performed for the control group. Additionally, a comprehensive hemogram examination was performed for cases and controls, that included the determination of hemoglobin, hematocrit, white and red blood cell counts, using the KT3800 Vet Auto Hematology analyzer (Kindle, China). Blood cell population and morphological features were confirmed by blood smear microscopic analysis by trained personnel. Total plasmatic proteins were analyzed using a refractometer.

Molecular tests 4.4.1 DNA extraction
After the positive cases and controls were enrolled in the study, a sample of venous blood was collected with ethylenediamine tetraacetic acid (EDTA, as an anticoagulant) from both groups.
The time between the initial diagnosis and the second sampling for DNA isolation was from hours up to seven days. Genomic DNA (gDNA) was extracted using 200 µl of EDTA anticoagulated blood from positive cases (previously detected by microscopy) and controls, using the commercial kit (Thermo Scientific™ GeneJET ™ DNA Purification Kit), following the manufacturer's instructions.
DNA concentration and quality were determined using a nanodrop (Thermo Scientific NanoDrop One). DNA of three filariae species (D. immitis, A. reconditum and D. repens) donated by academic institutions from Italy and Argentina, were used as positive controls to validate our molecular tests.

Polymerase Chain Reaction (PCR)
We conducted a PCR amplification of fragments of the 5.8S-ITS2-28S ribosomal subunits of different filariae, using the pan-filarial primer pair as well as the cycling conditions reported by of Taq buffer (Promega), completing a final volume of 20uL with ultrapure water. About 60 ng/ul of DNA was added to the mixture. After the cycling conditions, PCR products were analyzed in 1.5% agarose gels and stained with SYBR® safe.

Restriction Fragment Length Polymorphisms (RFLP)
We used the in silico predicted sequence of the PCR products for the three most reported filaria species among dogs (D. immitis, D. repens and A. reconditum), using the parasite sequences, the pan-filarial primer pair defined previously by Rishniw et al [51] and the software SnapGene®

PCR-Sequencing
Sanger sequencing by capillary electrophoresis was performed using the 3500 Genetic Analyzer and the BigDye™ Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific), in order to confirm the RFLP findings. Data analysis was performed using the software BioEdit version 7.2.5, MEGA X version 10.1.7 and, the Basic Local Alignment Search Tool (BLAST) [53]. MEGA X was also used to build a neighbor-joining (NJ) phylogenetic tree.

Conclusions
The microscopic test combined with the "pan-filarial" PCR and the RFLP test using MwoI, allowed the detection of microfilariae species circulating in the South-western cities of Colombia.
This study led us to identify A. reconditum as the most frequent causing species of the canine filariasis outbreak in Cali during 2018-2019. It is possible that climate change together with animal migration and commercial trades (including animal trade: pets and exotic animals), provide the conditions that allow the proliferation of vectors that transmit this nematode and other emerging infectious agents.
Infection with A. reconditum has consequences in the canine's health with alterations in hemoglobin, hematocrit, and total plasmatic protein levels. Our study did not identify the presence of other species, however, the possibility of D. immitis cases in the region cannot be ruled out. Although A.
reconditum is not a highly pathogenic species, this study highlights the importance of including it in the differential diagnosis of anemias and skin problems in canines from Colombia. At the public health level, it is necessary to alert the medical community about this canine pathogen since it has the potential to infect humans, as demonstrated in a human case of A. reconditum already reported in Australia, causing eye discomfort and irritation [28].

Supplementary Materials:
The following are available online at www.mdpi.com/xxx/s1, Figure S1: