Identification of in Domestic Dogs from Ecuador via Various Techniques

Introduction

Hookworms are among the most common soil-transmitted helminth infections, with 1.5 billion cases worldwide, and are responsible for 65 000 deaths per year. The disease prevails in places with adequate humidity and a lack of environmental sanitation. In humans, it can cause systemic diseases and cutaneous larva migrans, representing a serious public health problem [1].

Half of all Ancylostomas infections occur in Asia and the Pacific, in countries with tropical climates, where there are overcrowded populations and poor hygiene and sanitation. The infections cause malnutrition and anemia and are responsible for the loss of up to 4.1 million disability-adjusted life years annually. The population most at risk of contracting Ancylostomas infections are children (preschool and school children), people who walk barefoot in the countryside and on beaches, farmers, travelers returning from tropical countries, and people who have contact with infested dogs and cats [2].

The most common and pathogenic nematode in pets (dogs and cats) worldwide is Ancylostoma caninum, which can cause anorexia, anemia, malnutrition, hemorrhagic diarrhea, and other intestinal disorders, such as aphthous ileitis and eosinophilic enteritis. Therefore, A. caninum is a highly prevalent anthropozoonotic parasite worldwide and is potentially serious in domestic dogs and humans [3,4].

The zoonotic canine hookworms A. caninum, A. ceylanicum, A. brasiliense, and Uncinaria stenocephala are capable of causing cutaneous larva migrans in humans [4]. Necator americanus and A. duodenale are nematodes that cause hookworm infections in humans [5].

Adult male and female A. caninum parasites lodge in the mucosa of the small intestine of dogs and cats. They copulate and lay eggs, which then fall to the ground with the feces, later embryonating and hatching. Subsequently, rhabditiform larvae 1 are released, which after 2 to 3 days, molt into rhabditiform larvae 2. Then, on the eighth day, they molt into filariform larvae 3, which are the infective forms for animals, via the fecal-oral and percutaneous route, and humans, via the percutaneous route. In humans, when larvae 3 is acquired, it does not molt, but instead produces cutaneous larva migrans; however, in domestic dogs, larvae 3 inside the organism transforms in juvenile form to adult parasite [2].

With respect to diagnostic techniques, direct, flotation, and sedimentation with centrifugation using saline solution are used as screening methods for the identification of parasite eggs [6,7]. The modified Baermann and Harada-Mori techniques are used for the identification of hookworm larvae [8,9].

Morphometric studies are useful for confirming the presence of eggs and larvae of A. caninum [6,7,10,11]. Culturing [8,9] to test parasitic infection has demonstrated that eggs can be passed into larvae under controlled conditions, and molecular identification by polymerase chain reaction (PCR) and molecular sequencing allows accurate detection of species [12].

The molecular marker mitochondrial cytochrome c oxidase subunit 1 (cox1) is considered a segment of “barcode DNA” and is one of the most common molecular markers used in Ancylostomatidae (Ancylostoma, Necator, and Uncinaria) [13,14]. Likewise, Lima et al [15] reported that mitochondrial sequences can confirm genetic divergence between Ancylostoma specimens. Additionally, the cox1 sequence allows the identification of haplotypes in populations of A. caninum [14,16].

In Ecuador, there are no studies on the morphometry, culture, molecular identification, and phylogeny of A. caninum in domestic dogs, using the molecular marker cox1; therefore, this study is conducive to understanding the importance of this nematode in wildlife, animal, and public health. Likewise, it is important to highlight that the present study is a continuation of the work described by Coello et al [17], whose theme was “Ecoepidemiology of Ancylostoma spp. in Urban-Marginal and Rural Sectors of the Ecuadorian Coast and Prevalence of Cutaneous Larvae Migrans”.

The objective of this study was to confirm the presence of A. caninum in domestic dogs from the urban-marginal and rural sectors of the Ecuadorian coast through morphometry, culture, and molecular techniques.

Material and Methods

ETHICS STATEMENT:

This research was reviewed and approved by the Research Council of the Faculty of Veterinary Medicine and Zootechnics of the University of Guayaquil, Research Department of the University of Guayaquil, and Ethics Committee of the University of Guayaquil of Ecuador. This was an observational, cross-sectional study with a mixed approach.

SAMPLING AREAS:

Fecal samples of domestic dogs were collected through convenience sampling (not probabilistic) in 2 urban-marginal sectors of Guayaquil, Balerio Estacio and La Ladrillera, and 2 rural sectors, Loma Larga (city of Nobol) and Santa Rosa (city of Daule), all located in the province of Guayas on the Ecuadorian coast, with temperatures ranging between 20°C and 37°C and a tropical savanna climate.

POPULATION AND SAMPLE:

Owing to the lack of a canine census, the total human population of 38 057 inhabitants housed in 7212 households was used to determine the sample size of domestic dogs. To calculate the latter, the WinEpi program was used, with a confidence interval of 95%, sampling error of 5%, population size of 7212 households, and minimum expected prevalence of 5% [18]. According to the calculations, 59 households per area where 2 or more dogs per household were sampled were investigated.

RECOGNITION OF AREAS AND INFORMED CONSENT:

A survey of the areas was conducted, and a discussion was held with the inhabitants of the area, who were informed about the study and the risk of hookworms in animals and humans. Prior to sampling, informed consent was obtained from the owners of domestic dogs who agreed to participate in the research.

SAMPLE COLLECTION, TRANSPORT, AND ANALYSIS:

Between March and September 2023, we collected fecal samples from domestic dogs in sterile bottles delivered to the animals’ owners, whose address and contact information were noted. Additionally, a data form was completed. In cases in which the dog owners were unable to collect the sample, a technical team did so [19].

The stool samples were immediately transported at room temperature to the Laboratory of the Veterinary Clinic “Besito Vet Pet Lab” in the city of Guayaquil, where they were analyzed by coproparasitic screening methods: direct, modified flotation, sedimentation with centrifugation using saline solution for egg identification, and modified Baermann and Harada-Mori for the identification of rhabditoid L1 and L2 and filariform L3 larvae. The parasite samples were stained with Lugol’s iodine solution (5 g of iodine and 10 g of potassium iodide diluted in 85 mL of distilled water) and observed via optical microscopy at 10× and 40×.

Importantly, the coproparasitic methods mentioned above followed the procedures and criteria described by Botero and Restrepo [6], Romero Cabello [7], and Girard [8].

DIRECT METHOD: A drop of saline solution (physiological serum with a density of 2.16 g/cm3) was placed in the center of a slide, and 1 to 2 mg of fecal matter was added with a sterile wooden stick. Then, a drop of Lugol’s solution was dispensed with a plastic Pasteur pipette, it was mixed (with a wooden stick), and a cover slip was placed. Finally, the parasitic forms were visualized in a binocular microscope with a built-in LED screen (RAM 2G and ROM 16G) of the Better Scientific brand [9].

MODIFIED FLOTATION TECHNIQUE:

An amount of 1 to 3 g of feces was placed in a beaker (or sterile plastic cup), and 3 mL of saline solution was added and homogenized with an applicator. The material was filtered, and was left to rest for 10 to 20 min. The supernatant was taken with a plastic Pasteur pipette, and a drop was added to a slide. A drop of Lugol’s solution was dispensed, it was mixed, and the cover slide was placed. Finally, the observation was conducted under the microscope (technique provided by the first author).

CENTRIFUGATION SEDIMENTATION TECHNIQUE WITH USING SALINE SOLUTION: In a beaker, 1 to 3 g of feces was placed, and 3 mL of saline solution was added and homogenized with a stirrer. The material was sifted with a gauze contained in a small sterile plastic funnel, and the product was collected in a test tube. The product was centrifuged (at 2500 rpm for 5 min) for 3 to 5 times, with the supernatant always discarded. From the last centrifugation, the sediment was taken and a drop was deposited on a slide. A drop of Lugol’s solution was dispensed, it was mixed, and the coverslip was placed. Later, the observation was made under the microscope [6,9].

MODIFIED BAERMANN METHOD:

Distilled water (up to half full) was dispensed into a plastic champagne glass, and 5 to 10 g of feces was wrapped in sterile gauze (2 to 3 layers). The fecal matter was introduced into the glass and slightly submerged in distilled water. The gauze was adjusted to the edge of the glass using a sterile plastic rubber band. Meanwhile, distilled water was placed in the water bath (up to the measurement slightly above the level of the sample), the water was turned on, and the water bath was regulated between a temperature of 37°C and 41°C. Next, the champagne glass was introduced into the water bath, it was incubated for 12 to 24 h, and the sample was removed. The sediment was collected with a Pasteur pipette and placed in a test tube. The sediment obtained was centrifuged with a little saline solution at 2000 rpm for 2 to 3 min. The sediment was collected, and a drop was placed on a slide. A drop of Lugol’s solution was dispensed, it was mixed, and the slide was placed on the cover slip. Finally, the observation was conducted under the microscope (technique provided by the first author).

HARADA-MORI TECHNIQUE: The sample was broken up with saline solution using a mortar and pestle. Separately, 2 to 3 mL of distilled water or saline solution was added to a plastic Falcon tube. A strip of filter paper measuring 12-15×1.5-2 cm, depending on the diameter and size of the tube, was cut, and a notch was made at the bottom end. The sample was spread using a sterile aluminum applicator, adding 0.5 to 1 g of the stool sample over the middle third of the surface of the paper, leaving the ends free (it is preferable to make a fold at the top of the paper and secure it with the edge of the tube). The filter paper was carefully placed inside the tube, avoiding contact of the stool with the liquid. The tube was capped, placed in a rack, and incubated between 24°C and 37°C for 4 to 10 days. The water was checked daily and replaced in cases of evaporation, and the filter paper was removed. A small amount of the liquid was taken from the bottom of the tube. A drop of the liquid was placed on a microscope slide. Lugol’s solution was added, it was mixed, and the slide was covered with a cover slip. Observation was conducted under a microscope [8,9].

MORPHOMETRY OF THE PARASITIC FORMS:

All the samples of domestic dogs that were positive were confirmed by morphometry with respect to the eggs and L1 (rhabditoid) larvae, according to the criteria of Lucio et al [10], who reported measurements of 58 to 68 μm in length and 37.68 to 40.73 μm in width for A. caninum eggs and 152.69 to 171.29 μm in length and 1.63 to 10.13 μm in width for the L1 larvae. In addition, the eggs have rounded ends and are covered with a thin membrane, with an embryophore made up of more than 8 blastomeres. L1 has a blunt anterior end with a short oral cavity and a divided esophagus (body, isthmus, and bulb). No genital primordium is present in the middle or posterior third, and the posterior end is pointed [10].

L2 (rhabditoid) larvae are 250 to 300 μm in length and 17 to 24 μm in width. A long and narrow oral cavity is present at the anterior end, and the esophagus is particularly long and muscular, divided into the body, isthmus, and bulb. A complete intestine ending in the anus is present, a genital primordium is absent in the middle or posterior third, and the posterior end is pointed [6,7].

The L3 filariform larvae are 500 to 600 μm long and 25 to 30 μm wide. An inconspicuous oral cavity is present at the anterior end. The esophagus is straight without divisions, covers a third of the body and the tail, and joins the rudimentary intestine by a small dilation that ends in the anus. No genital primordium is observed in the middle or posterior third, and the posterior end is long and pointed [6,7,20].

CULTIVATION:

Positive samples were diagnosed with A. caninum eggs via coproparasitic techniques: direct, flotation, and sedimentation with centrifugation using saline solution (screening method). Samples were grown [8] on agar in plates, Müller-Hinton agar plates [21], and MacConkey agar plates [22]. In addition, the fecal samples were manually sown in artisanal media with sand and/or ravine soil, both sterile, and all were prepared in Petri dishes.

AGAR CULTURE IN PLATES: The medium was prepared (agar 15 g, peptone 10 g, meat extract 5 g, and sodium chloride 5 g in 1000 mL of distilled water). It was placed in a water bath until the substances were completely dissolved. The medium was sterilized in an autoclave at 121°C and 21 pounds of pressure, for 15 min. Next, 10 mL of medium was dispersed in a thin layer in the Petri dish, was allowed to harden overnight, and was stored in a sealed plastic bag at 4°C (before using each plate, it was left at room temperature for a while). An amount of 1 to 3 g of feces was placed in the center of the plate as widely as possible with a sterile popsicle stick (if the feces were hard, they were softened with a few drops of sterile distilled water). The plate was covered (tightened with tape) and was incubated at 37°C for 12 to 24 h. The plate was placed in the stereomicroscope and, without uncovering it, it was checked for larval paths and/or larvae on the surface of the medium. If no paths or larvae were observed, it was left to incubate for another 24 h. Next, 10% formalin or saline solution was added on the surface of the agar. The plate was tilted, and with a Pasteur pipette, the formalin was aspirated, placed in a previously identified test tube, and centrifuged at 1500 rpm for 2 min. The sediment was removed from the tube with a plastic pipette, and a drop was dispensed on an object holder plate. Lugol’s solution was added; it was mixed, covered with the cover slip, and observed under the microscope [8].

CULTURE ON MüLLER-HINTON AGAR PLATES: First, 38 g of the medium was prepared in 1 L of sterile distilled water, heated in a water bath until completely dissolved, and sterilized in an autoclave at 121°C and 21 pounds of pressure for 15 min. The medium was dispensed into the Petri dish and allowed to harden overnight. An amount of 1 to 3 g of feces was placed in the center of the plate as widely as possible with a sterile popsicle stick and incubated at 37°C for 12 to 24 h. Saline solution was added to the incubated medium. Then it was mixed, the plate was tilted, and the saline solution was aspirated with a Pasteur pipette and placed in a previously identified test tube. It was centrifuged at 1500 rpm for 2 min. The sediment was removed from the tube with a plastic pipette, and a drop was dispensed onto an object holder plate. Lugol’s solution was added, it was mixed, covered with a cover slip, and viewed under a microscope [21].

CULTIVATION ON MACCONKEY AGAR PLATES: An amount of 50 g of the medium was prepared in 1 L of sterile distilled water. the same procedures were conducted for the media mentioned above, concerning preparation, cultivation, and isolation of larvae [22].

CULTURE ON PLATES WITH STERILE SAND:

The sand was dug between 40 to 50 cm and was sterilized in an autoclave at 121°C and 21 pounds of pressure for 30 min. Distilled water or saline solution was added to moisten the sand; 5 g of feces was placed as widely as possible on the surface of the sand with a sterile popsicle stick and brought to room temperature for 24 h. An amount of 5 mL of saline solution was slowly added with a Pasteur pipette on the surface of the feces, which were mixed with the saline solution on one side of the Petri dish. The plate was tilted, the mixture was aspirated, and it was placed in a previously identified test tube and centrifuged at 1500 rpm for 2 min. The sediment was removed from the tube with a plastic pipette, and a drop was dispensed on an object-holder plate. Lugol’s solution was added; it was mixed and covered with a coverslip. Finally, it was viewed under a microscope (technique provided by the first author).

CULTIVATION IN PLATES WITH STERILE RAVINE SOIL:

The ravine soil was excavated between 40 and 50 cm and was sterilized in an autoclave at 121°C and 21 pounds of pressure for 30 min. Then, the same procedures as those mentioned above were conducted (technique provided by the first author).

DNA EXTRACTION, PCR, AND SEQUENCING:

The samples that were positive by the screening methods were processed with a DNA/RNA shield and 0.5 g of sample in 500 μL of solution [23] in the Besito Vet Pet Lab laboratory. Then, they were transported at −20°C to the Institute of Genetics and Microbiology of the Miguel Lillo Foundation (San Miguel de Tucumán-Argentina), where the eggs were lysed following the procedures of Mulinge et al [24]. DNA extraction was subsequently performed via the Qiagen DNeasy blood & tissue kit [25]. The sequences of the cox1 gene were amplified via the specific primers JB3 (5′-TTT TTT GGG CAT CCT GAG GTT TAT-3′) and JB4.5 (5′-TAA AGA AAG AAC ATA ATG AAA ATG-3′) [14].

PCR was conducted via the following reaction mixtures, with a final volume of 25 μL: 2 μL of genomic DNA template (100 ng/μL); 0.3 μL of 2 U TAQ T-plus polymerase; 2.5 μL of Taq 10× hot start buffer; 1.5 μL of 25 mM MgCl₂ (Thermo Scientific); 0.5 μL of the primers; 1 μL of 0.5 mM dNTPs; and 16.7 μL of ddH₂O. The DNA amplification was conducted in a NYX Technik ATC 201 thermocycler with the following PCR profile: 1 cycle at 94°C for 5 min; 36 cycles at 94°C for 40 s, 46°C for 40 s, and 72°C for 1.10 min; and finally, 1 cycle at 72°C for 5 min, followed by a hold at 4°C. The PCR products and the fragments obtained were analyzed via electrophoresis in 1.5% agarose gels with GelRed Nucleic Acid Gel Stain 10 000× and visualized via a UV transilluminator.

The PCR products were sequenced by Macrogen, Inc (Seoul, South Korea).

SEQUENCE IDENTITY AND ALIGNMENT:

The DNA of all fecal samples that were positive for A. caninum was compared with DNA obtained from adult parasites, as described by Avila et al [25]. Similarly, a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was performed, and the results were verified with those registered in GenBank, to confirm the identity of the species. DNA sequences were automatically aligned with SeaView 4 [26], and then manual alignment and comparison were conducted with MEGA 6 [27].

MOLECULAR DATA ANALYSIS:

The online FaBox toolbox [28] was used to determine the presence of haplotypes in the analyzed cox1 sequences. To perform the phylogenetic analyses, cox1 sequences from Ancylostoma, Necator, and Uncinaria obtained from the GenBank database and segments of A. caninum amplified in our laboratories were used (Table 1). Maximum parsimony (MP) and maximum likelihood (ML) analyses were conducted using MEGA 6 software [27]. To determine the most appropriate evolutionary model, the software JModelTest 2.1.10 [29] was used. In molecular phylogenies, 1 sequence per haplotype was used to avoid errors due to inconsistency in the number of sequences for each haplotype.

In addition, the following were chosen for an external group: Uncinaria sanguinis, accession NC_02526 [30]; U. sanguinis, KF924756 [30]; Necator americanus, LC036564 [31]; and N. americanus, AB793544 [32].

STATISTICAL ANALYSIS:

The data obtained were tabulated and are presented in tables generated using the Microsoft Excel 2010 program. In addition, the prevalence of canine ancylostomiasis was related to through inferential statistical analysis performed with RStudio statistical software [17].

Results

:

Of the 498 dogs analyzed in the 4 areas studied, 250 (50.20%) were positive for A. caninum; 80 (32%) were positive in Balerio Estacio, 53 (21.20%) were positive in La Ladrillera, 62 (24.80%) were positive in Santa Rosa, and 55 (22%) were positive in Loma Larga.

MORPHOMETRY:

The positive eggs presented rounded ends and a thin membrane with an embryophore made up of more than 8 blastomeres, with average measurements of between 63 and 64 μm in length and 39 and 40 μm in width.

The positive L1 (rhabditoid) larvae presented a blunt anterior end with a short oral cavity and a divided esophagus (body, isthmus, and bulb). In the middle or posterior third, no genital primordia were observed, and the posterior end was pointed and presented measurements of 161 to 165 μm in length and 8 to 10 μm in width.

The L2 (rhabditoid) larvae were elongated and had a long and narrow oral cavity at the anterior end, a particularly long and muscular esophagus divided into a body, isthmus, and bulb, and a complete intestine that ended in the anus. No genital primordium was observed in the middle or posterior third, and the posterior end was pointed. These larvae were between 250 and 270 μm long and 19 and 23 μm wide.

L3 (filariform) larvae were elongated and large, with a poorly visible oral cavity at the anterior end and a straight esophagus without divisions and covering a third of the body and the tail, joining the rudimentary intestine by a small dilation ending in the anus. No genital primordium was observed in the middle or posterior third, and the posterior end was long and pointed. These larvae were between 510 and 560 μm long and 25 and 28 μm wide.

CULTIVATION:

All egg samples positive in the parasite screening methods were grown into rhabditoid and filariform larvae on agar in plates, Müller-Hinton agar plates, and MacConkey agar plates, and on artisanal media with sand or ravine soil (both sterile) in plates. It is important to note that the paths left on the surface of the agar in plates, Müller-Hinton agar plates, and MacConkey agar plates by the larvae were observed as described by Girard [8] (Figure 1), allowing a very accurate diagnosis of hookworm infection corroborated by microscopy. Likewise, it was possible to corroborate the cultivation of A. caninum in the aforementioned artisanal media.

PCR AND DNA SEQUENCING:

PCR products were obtained from 250/250 samples via specific primers. All the DNA samples produced a PCR product (Figure 2).

SEQUENCE IDENTITY AND ALIGNMENT:

A region of approximately 400 bp was amplified from the cox1 gene from the fecal samples positive for A. caninum and from the control sample obtained from an adult worm, which was generously provided by Dr. Ávila (Figure 2).

Using the BLAST tool, the pairing of DNA segments of approximately 250 bases for the cox1 sequence of A. caninum was corroborated, confirming a similarity of 100% and 100% identity between Balerio Estacio and Ladrillera dogs and the sequences of the GenBank database (accession numbers ON980673.1, ON980666.1, and ON980665.1), whereas dogs from Loma Larga and Santa Rosa presented 99% similarity and 100% identity with the sequences in GenBank (accession numbers ON980651.1, ON980650.1, ON980668.1, and ON980669.1).

MOLECULAR DATA ANALYSIS:

The FaBox toolbox was used to establish the presence of 22 haplotypes among the 43 A. caninum sequences analyzed, 5 haplotypes among the 22 segments of A. ceylanicum, 9 haplotypes among the 11 sequences of A. duodenale, and 3 haplotypes among the 3 sequences of A. tubaeforme (Table 1).

The haplotypes were identified according to the nomenclature used by Quintana et al [14]. Among the 25 haplotypes recognized by this author in the United States, our analyses were able to identify only 11 (Table 2). For the samples from Ecuador, 2 haplotypes were identified: one derived from the populations of Balerio Estacio and Ladrillera (Ecu-Hap1), and the other corresponding to the populations of Loma Larga and Santa Rosa (Ecu-Hap2). The Ecu-Hap1 haplotype was indistinguishable from the US-Hap 8, 19, and 20 and Australian (Aus)-Hap1 haplotypes, according to Quintana et al [14], whereas the Ecu-Hap2 haplotype was in line with the US-Hap1, 5, 7, 17, 21, and 22 haplotypes (Table 2).

MAXIMUM PARSIMONY:

The MP analysis allowed us to distinguish 3 subclades, I, II, and III. Subclade I included a sample of A. caninum, indicating that the species was a natural group (bootstrap of 84); subclade II included samples of A. duodenale and A. tubaeforme (support of 100) and was a sister group of subclade I; and subclade III was composed of A. ceylanicum, which was polyphyletic at the base of the cladogram. Sequences of A. caninum from the United States, Australia, and Ecuador were randomly distributed throughout subclade I. This subclade consisted of 2 clusters, A and B. Cluster A, with bootstrap of 100, consisted of most of the A. caninum sequences, while cluster B showed Ecu-Hap1 related to the Australian haplotypes Aus-Hap1 and Aus-Hap4.

A. duodenale and A. tubaeforme were monophyletic in subclade II, which had 2 well-defined clusters. Subclade III included A. ceylanicum and consisted of 2 well-defined clusters.

MAXIMUM LIKELIHOOD: Subclades I, II, and III from the ML analysis were also observed; however, subclade I presented moderate support (bootstrap value of 84). In addition, this subclade presented a similar topology to that of the MP analysis, but it differed in that phylogenetic relationships between Aus-Hap 5, Jap-Hap 2, and US-Hap 10 in cluster A were not resolved, and the Aus-Hap18 haplotype was disassociated from Jap-Hap1 and Aus-Hap3, with a bootstrap value of 99. In subclade II, clusters C and D of the ML tree were recovered almost intact. On the other hand, subclade III conformed by A. ceylanicum presented the same topology as in the MP analysis (Figures 3, 4).

No significant difference was found in the presence of Ancylostomas in domestic dogs according to location. However, 3 cases of cutaneous larva migrans were determined in patients aged 13, 14, and 34 years [17].

Discussion

LIMITATIONS OF THE STUDY:

More domestic dogs from different climatic zones (coastal, Andean, Amazonian, and insular) from Ecuador should have been analyzed. For budget reasons, other diagnostic methods such as electron microscopy and flow cytometry could not be applied.

Conclusions

For the first time, the prevalence of A. caninum for Ecuador was determined using different methods that were easy, agile, and economical and, when used together, increased the sensitivity and specificity of the diagnosis. In turn, we recorded for this country, by PCR and DNA sequencing, 2 of the known haplotypes for the first time.