A Simple Fecal Flotation Method for Diagnosing Zoonotic Nematodes Under Field and Laboratory Conditions

Summary

This work describes the use of a flotation method to identify Toxocara canis and Ancylostoma spp. detected in fecal samples collected from dogs in Mexico from 2017 to 2021 under field conditions.

Abstract

Diagnosis of canine parasites with zoonotic potential such as Toxocara canis and Ancylostoma caninum under field conditions is usually difficult due to limited access to a laboratory in rural and suburban areas in Mexico. This study aimed to detect T. canis and Ancylostoma spp. in fecal samples collected from dogs in Mexico from 2017 to 2021 under field conditions. Sample size calculation resulted in a target enrollment of 534 dogs across the country.

Samples were collected directly from the rectum or the ground after defecation. Samples were stored in individual, tightly sealed, plastic bags at 4 °C. A saturated solution of sodium chloride (specific gravity [SpG] 1.20) was prepared both under field and laboratory conditions. Within 3 days of collection, 2-4 g of feces were tested for parasites using a flotation method by suspending each fecal sample in a saline solution. Feces were mixed with the flotation solution and crushed using a metal spoon.

Once a uniform consistency was achieved, the fecal sample was poured into a new plastic cup using a sieve and allowed to sit for 10-15 min. Three drops from the top of the mixture were collected using a sterilized inoculating loop. The slides were placed on the microscope and parasites were identified by trained parasitologists. Fecal samples from 1,055 dogs were screened microscopically. The number of positive samples for Ancylostoma spp. was 833 (78.95% frequency) and 222 (21.04%) for T. canis. These findings illustrate the importance of identifying zoonotic helminths in dogs living in urban and rural areas in Mexico using a coproparasitoscopic technique in the laboratory and under field conditions.

Introduction

Gastrointestinal parasites are one of the most common health problems that affect dogs¹. Estimates suggest that there are ~700 million domestic dogs worldwide, and approximately 175 million may be categorized as free-roaming². More than 60 parasite species are shared between dogs and humans, suggesting that dogs could be a source of infection for humans with these parasites³. Toxocara canis and Ancylostoma caninum are two parasitic species that infect dogs and, accidentally, human hosts. Currently, there are several studies about the locations where these helminths are able to survive and reproduce in Mexico. The prevalence of Toxocara in dogs varies from 0% to over 87% across the United States, Mexico, Central America, and the Caribbean⁴. Toxocara canis and Ancylostoma spp., as well as other parasitic species in dogs, have been previously reported in Mexico^{5,6,7,8,9,10,11,12,13} (Table 1).

Parasitic species	Region	Prevalence (%)	Reference
Ancylostoma caninum	Querétaro	42.90	5
	Tabasco	15.90	6
	Campeche	35.7 – 42.9	7
	Yucatán	73.8	8
Babesia	Morelos	13.60	9
	Veracruz	10.00
Coccidial oocysts	Yucatán	2.30	8
Ctenocephalides	Morelos	30.3	10
Dipylidium caninum	Yucatán	2.30	8
Dirofilaria	Yucatán	7.0 – 8.3	11
Giardia	Tabasco	3.00	6
	Yucatán	18.8	8
Leishmania	Chiapas	19.00	12
Tapeworms	Baja California	6.79	13
Toxocara canis	Querétaro	22.10	5
	Yucatán	6.20	8
Trichuris vulpis	Yucatán	25.40	8
Trypanosoma	Jalisco	8.10	9
	Campeche	7.60
	Chiapas	4.5 – 42.8
	Quintana Roo	20.1 – 21.3
	Toluca	17.50
	Yucatán	9.8 – 34

Table 1: Regional prevalence (%) of dog parasites in Mexico from 2001 to 2020. Findings from previous investigations conducted from 2001 to 2020 have enabled the identification of canine parasite distribution across several urban and rural settings in Mexico. These studies provide a deep understanding of epidemiological elements conducive to the persistence of canine parasites in different ecosystems, contributing to a comprehensive assessment of the zoonotic impact of some parasite species.

Life cycle stages of intestinal parasites, such as eggs, cysts, oocysts, or larvae can be found in stool samples. Thus, examination of fecal material provides valuable information about the parasites of an animal. The need for a method to detect Ancylostomidae eggs in human feces led to the use of the simple fecal smear in 1878, which was used for many years to detect gastrointestinal parasites but was considered not very sensitive. Thus, the need arose to develop better copromicroscopic methods¹⁴. More than 100 years have passed since the flotation technique for recovering and counting parasite eggs in fecal samples was first described¹⁵. Since then, several methods and variants of the flotation technique have been considered a standard for the detection of some parasites in their hosts.

For example, Lane described a method in 1924 involving the direct centrifugal flotation technique, which integrates centrifugation followed by floating the sediment in a saturated sodium chloride solution with SpG 1.2 in 1 g (Lane) or 10 g (Stoll's modification). The flotation technique was subsequently modified by using solutions with different SpG¹⁴. In 1939, Gordon and Whitlock reported the disadvantages of Stoll's technique due to interference from detritus in visualizing parasite eggs and developed the quantitative method known as McMaster¹⁶. In 1979, O'Grady and Slocombe demonstrated that the specific gravity of the solution, timing, and mesh sizes of strainers affect the accuracy of egg detection using the flotation technique¹⁷. During the last decades, because several modifications have been made to the flotation technique, there is an urgent need for standardization of flotation methods. Currently, detection of canine helminth infections in the context of prevention of zoonotic parasites is required to apply appropriate anthelmintic treatments to limit environmental contamination with infectious stages of zoonotic nematodes¹⁸.

Among qualitative methods, the fecal flotation technique is widely used and accepted because it does not require much equipment, is simple, inexpensive, and reproducible; yet it has a major drawback in that it lacks sensitivity when the intensity of the infection is low¹⁹. The ability to reveal the presence of a greater number of parasitic elements such as eggs, oocysts, cysts, or nematode larvae is usually determined by the density of the solution²⁰.

Previous reports have compared coproparasitological techniques for the detection of canine nematode eggs. With regard to the detection of motile protozoa, direct fecal smears are used; whereas sedimentation methods are useful for diagnosing heavy eggs of parasites such as trematodes²¹. One of the most widely used field-based diagnostic tests is the fecal smear method. However, the low level of sensitivity of this technique may be attributed to the fact that it does contain debris that interferes with the detection of parasite eggs. By incorporating a sieving step along with solutions that provide the proper SpG, the flotation method offers a clearer and less cluttered observation of ascarid and hookworm eggs. This leads to a more precise and efficient process for microscopic screening²². Likewise, simple flotation and direct centrifugal flotation techniques are very commonly utilized to recover parasite eggs and oocysts¹⁴. The classic flotation methods can be considered qualitative or quantitative depending on the use of a counting chamber such as the McMaster method¹⁵. Nonetheless, as the flotation technique has low sensitivity and focuses on the detection of parasites in the patent period, negative results should not be considered conclusive. However, the accuracy not only depends on the preservation procedure of fecal samples or the SpG of flotation solutions but also depends on the technical proficiency and experience in conducting fecal examinations of the user.

Consequently, other methods have been explored for the detection of canine parasites in feces. It has been generally recognized that one of the most widely used approaches for the diagnosis of intestinal helminth infections in dogs is the FLOTAC technique, a multivalent, sensitive, and accurate method that yields accurate and reliable results for the diagnosis of A. caninum in dogs when compared to a flotation protocol in a tube and the McMaster technique^19,23. Sedimentation methods are useful for recovering fluke eggs, embryonated nematode eggs, and most tapeworm eggs, which cannot be recovered on the surface of a flotation solution because these structures do not float²⁴. One method that has been proven to be superior to flotation/sedimentation techniques is the Modified Double Centrifugal Flotation method, as it enables the detection of cestode eggs in feces, is less time-consuming, separates Anoplocephala eggs from fecal debris, and decreases crystallization²⁵. Moreover, this technique has been successfully used to detect ascarid eggs with high sensitivity²⁶. Yet, some of these aforementioned techniques and centrifugal methods such as as the Ovassay, as opposed to the flotation protocol we propose in this study, require sample preservation in reagents such as formalin, commercial kits, sample processing under laboratory conditions, and the use of reagents such as zinc sulfate²⁷ which are expensive and require special disposal procedures to avoid environmental toxicity.

The use of techniques that increase the sensitivity of the flotation method by adding solutions with high SpG has been favored recently. However, it must be considered that the disadvantage of these solutions is the increase in debris in the final preparation and hence, the inaccurate detection of parasite eggs. In addition, the commercial availability of materials, reagents, cost, environmental impact issues, and difficulty of use of centrifugal methods affect the selection of a flotation technique¹⁴, which can be challenging in field conditions in contrast to the protocol that we present in this work. The preparation of the flotation solutions with table salt is advantageous over the utilization of sugar because under field conditions, sugar attracts insects such as wasps and bees and preparations become sticky. Further, solutions such as phenol, which is added to sugar solutions to avoid stickiness, or ZnSO₄are complex to properly discard according to environmental protection guidelines and cannot be disposed of in the field; unlike a table salt solution.

The goal of this manuscript is to demonstrate the steps to detect T. canis and Ancylostoma spp. eggs in fecal samples using an adaptation of the simple flotation technique under field and laboratory conditions. Following the protocol herewith described and using a microscope with a backup battery, the diagnosis of these canine zoonotic parasites in rural and suburban areas is possible when no laboratory equipment and infrastructure are available. The simple flotation method described in this work can provide quick results and is a non-invasive and cost-effective technique for routine screening.

Protocol

The use and care of dogs was approved by the National and Autonomous University of Mexico.

1. Collection of fecal samples

NOTE: Handle the dog with the help of a veterinarian or the owner of the animal.

1. In the case of feral dogs (Figure 1A) or nervous animals, collect samples from the ground right after defecation or no more than 10 min later.
2. Lubricate surgical gloves or thin-walled polyethylene bags with water or Vaseline. To collect fecal samples from the rectum, wear surgical gloves or thin-walled polyethylene bags.
3. Collect at least 2 g of feces from each dog (Figure 1B).
4. Identify individual fecal samples as follows: date, location (Global Positioning System [GPS] coordinates using Google Maps), assign an identification number for each animal, approximate age of the dog, gender of the dog, breed, indoor or outdoor (feral) dog.
5. Close the bags containing the fecal sample with a tight knot. Keep the bags refrigerated (4-8 °C) if stool samples are not analyzed within 3-4 h after collection.

2. Preparation of a saturated salt solution for field diagnosis

NOTE: If access to a balance, measuring material, stoves, or gas to boil water is absent or limited, the saturated saline solution can be easily prepared using water, common table salt, a plastic 12 oz (355 mL) cup, and a 1 L soda plastic empty bottle.

1. Wash thoroughly an empty 1 L soda bottle. Fill the bottle with 1 L of water.
2. Fill a 12 oz plastic cup with common table salt.
3. Transfer the salt to the soda bottle.
4. Close tightly the soda bottle with the screw cap. Shake the solution vigorously until the salt no longer dissolves.
   NOTE: Shaking the solution in the soda bottle until complete dissolution of salt can take up to 90 min.

3. Preparation of a saturated salt solution for laboratory diagnosis

1. Weigh 420 g of common table salt.
2. Dissolve 420 g of salt in 1 L of water.
3. Boil the solution until no more salt dissolves.
4. Filter the solution to discard the undissolved salt.
5. Check the concentration of the solution using a heavy fluid hydrometer or densitometer.
   NOTE: The ideal concentration has an SpG of 1.20 to achieve better results (Figure 2A). The floating capability of an egg is influenced by its interaction with the solution, contributing to the varying ability of eggs to float in solutions with the same specific gravity. Hence, to achieve optimal egg recovery, it is essential to consider the upper range of specific gravity in the flotation solution, ensuring it surpasses that of the targeted parasite elements⁶.

4. Flotation method

NOTE: If fecal samples are too dry or hard, macerate them in a mortar.

1. Using a spoon, place approximately 3 g of feces in one plastic cup (~8.5 cm in height and ~5.5 cm in diameter).
2. Add 1 mL of the saturated salt solution until a paste is obtained.
3. Stir for 1 min and add 100 mL of saturated salt solution.
4. Pass this suspension through a plastic strainer into a second plastic cup to avoid coarse particles (Figure 2B).
5. Let the suspension stand for 15-20 min.
6. Place an inoculation loop in a flame for 1 s to ensure it is free of eggs, cysts, or oocysts (Figure 2C).
7. Wait for 5 s for the inoculation loop to cool down (Figure 2D).
8. Take three drops from the surface of the suspension with the inoculation loop. Place each one of the three drops separately on one glass slide (Figure 2E-G)
   NOTE: Ensure that the drops are not in contact with each other (Figure 2H).
9. Observe under the microscope with a 10x objective (Figure 2I). Place a coverslip if magnification is increased to 40x.
10. When a positive result is observed in the droplet, assign a cross (+) in the laboratory logbook to record the presence of parasites in the patent period in random sites of the fecal suspension surface.

5. Interpretation of the flotation method

NOTE: Negative results are inconclusive.

1. Perform a series of three tests with samples from 3 consecutive days to increase the sensitivity of the test.
   NOTE: Positive results indicate the presence of parasites in the patent period.

Results

In this work, collection and coproparasitoscopic procedures for the identification of T. canis and Ancylostoma spp. are described. The rationale behind the adaptation of the simple fecal flotation method to detect canine helminth eggs is that this technique is cost-effective as the solutions, equipment, and materials are inexpensive. Hence, the method has a high sample handling capacity as multiple samples can be processed in a short period. Moreover, the simple fecal flotation method is easy to perform...

Discussion

Nematodes such as T. canis and Ancylostoma spp. can inhabit the small intestine of dogs and have the potential to be transmitted to humans. Clinical signs caused by T. canis are serious in young dogs, manifesting as poor growth, respiratory issues, or digestive tract lesions²⁸. In adult dogs, the infection typically tends to be mild. Diagnosis relies on identifying characteristic eggs in a fecal sample. This condition is a frequent cause for prescribing anthelmintic trea...

Materials

Name	Company	Catalog Number	Comments
3 x 1.2 V AA rechargeable batteries	Energizer		Sold in retail stores
Bunsen burner	Viresa	FER-M224
Disposable 12-oz glass cup	Uline Mexico	S-22275	Sold in retail stores
Glass slides	Velab, Mexico	VEP-P20
Inoculating loop	VelaQuin, Mexico	CRM-5010PH
Light Microscope	VelaQuin, Mexico	VE-B2
Lighter	Bic	J25	Sold in retail stores
One plastic cup (12 oz)	Amazon	ASIN B08C2CRHSH	Can be any kitchen plastic reuseable cup
Plastic cups  (size of a dice or urine sample cup) diameter 5.5 cm and height 8.5 cm, two cups	Amazon	Layhit-Containers-ZYHD192919	Can be any kitchen plastic reuseable cup
Plastic strainer 10 cm	Ecko	ASIN B00TUAAVWI	Can be any kitchen plastic strainer
Soda bottle	Coca-Cola	1-liter	Sold in retail stores
Spoon	Amazon Basics	ASIN B00TUAAVWI	Can be any kitchen spoon
Table salt	La Fina		Sold in retail stores