Dipylidium caninum (D. caninum) is the causal agent of dipilidiasis, a parasitical disease caused by the adult cestode in dogs, cats, foxes, coyotes, and other wild carnivores^1-7 and well known as a zoonotic disease affecting humans^8,9. The control of cestodes with potential zoonotic risk for pet animals is of great importance for public health worldwide^2,10,11 given the close contact both dogs and cats have with humans¹², with the risk of infection even higher in children^8,9. The adult D. caninum cestode grows to a length of 50 cm, while its macroscopic structure has the appearance of rosary beads or a grouping of cucumber seeds¹³. Unlike nematodes, cestode parasites do not have a digestive system and grow from proliferating cells in the neck, producing several hundred segments known as proglottids and obtaining food via the tegument or body wall¹³. Located in the small intestine of the host, D. caninum absorbs digested material and obtains the nutrients necessary for survival, reproducing without the difficulty undergone by other parasites, because, as a hermaphrodite parasite, it forms gravid proglottids full of infective eggs via continuous differentiation^9,14,15.

Pyrantel (E-1,4,5,6-tetrahydro-1-methyl-2-pyrimidine) is an imidazothiazole anti-helminthic derived from the tetrahydropyrimidines and used widely by veterinarians on small species (dogs and cats), given its wide spectrum of action against mature and immature gastrointestinal parasites that infect domestic animals¹⁶. Oxantel (1 methyl-2-(3-hydroxyphenyl-ethenyl) 1,4,5,6-tetrahydropyrimidine) is an m-oxyphenol derivative of pyrantel¹⁷ that activates the N-subtype, a nicotine and methyridine-sensitive subtype. Pyrantel activates the L-subtype, a levamisole- and pyrantel-sensitive subtype, which explains why differences in their target nematodes have been reported¹⁶.

Tetrahydropyrimidines comprise a wide variety of pyrantel, morantel, and oxantel salts, all of which have nicotinic agonist effects which alter the parasite’s neuromuscular system, affecting muscular contraction and causing tonic paralysis. The nicotinic acetylcholine receptors (nAchRs) are essential to the parasite’s nervous function, presenting a different distribution and physiology than that found in mammals⁹. Nicotinic agonist anti-helminthics act on the nicotinic acetylcholine in the parasite’s neuromuscular junction, causing neuromuscular depolarization and spastic paralysis^18,19. Parasite nAchRs have five glycoprotein subunits located around a central ion channel²⁰, comprising a pentameral ligand-gated ion channels, which is a structure with significant pharmacological effects¹⁶. Moreover, nAchRs are functionally diverse due to their extensive gene families encoding subunits and three pharmacological sub-populations of receptors¹⁶. The activation of the acetylcholine receptor in nematodes has been divided into three pharmacological subtypes according to the degree of nicotinic affinity^21,22, characterized and defined as follows: the N-subtype, with significant sensitivity to nicotine, methyridine, and oxantel; the L-subtype, with affinity to levamisole and pyrantel; and, the B-subtype, with a greater sensitivity to bephenium.

Pyrantel’s mechanism of action functions by blocking muscular excitation via the activation of an agonist of the nAchR^18,19, altering the neuromuscular system and, thus, provoking muscular contraction and paralysis, which result in the death of the parasite⁹. Oxantel (m-oxyphenol) has been shown to be effective against gastrointestinal nematodes of great impact on both animal and public health^23,24. While the effect of pyrantel-oxantel (P-O), as a combined treatment, on nematode parasites has been reported previously²⁵, there are no scientific reports on the effects of a P-O combination, either in vivo or in vitro, on motility, tegument thickness, or other anatomical structures in tapeworms. Therefore, the present study aimed to evaluate the in vitro cestocidal effect of P-O on the motility and tegument thickness of adult Dipylidium caninum cestodes and to describe the histological changes in the organisms’ gravid proglottids.

All the euthanasia procedures applied in the present study were performed following the guidelines recommended and approved by the Ethics Committee for Animal Experimentation of the Faculty of Agricultural and Livestock Sciences at the Autonomous University of the State of Morelos and the Code of Ethics of the World Medical Association (Declaration of Helsinki).

Two hundred and sixty-six (266) naturally infected stray dogs were sampled at the Canine and Feline Control Center in the municipality of Tlahuac, Mexico City, after capture by the municipal animal control brigade. The animals were euthanized by authorized personnel via an anesthetic overdose, with the small intestine of each subject then obtained via a transversal incision on their abdomen. The small intestines obtained, still connected via the gastroduodenal and ileocecal valves, were stored in plastic bags, labelled with progressive numbers, and transported to the Animal Production Laboratory at the Faculty of Agricultural and Livestock Sciences at the Autonomous University of the State of Morelos, located in the city of Cuernavaca²⁶.

Each intestine sample was individually dissected via longitudinal incision and examined for the presence of D. caninum cestodes, which were identified via the macroscopic appearance of the proglottids and allocated using an optical microscope to verify their morphology²⁷. Direct observation was carried out under a microscope to determine the viability of the parasites at 40X objective, with cestodes presenting full motility for a one-minute period considered viable for subsequent experimentation^28-30.

The cestocidal effect of P-O on adult parasites was assessed (treated group (P-O), n= 21; control group (CG), n= 21), with the individuals placed in 90x60 mm Petri dishes containing 10 mL of RPMI (Roswell Park Memorial Institute) 1640 and incubated at 37 °C for 10 h. Distilled water and PMSF (phenylmethane sulfonyl fluoride) were used as a protease inhibitor vehicle for the control group, in order to maintain the structural anatomy and physiology of the cestodes. For the treated group, a commercial deworming drug (75 mg Pyrantel Pamoate and 75 mg Oxantel Pamoate (Vermiplex), Holland Animal Health Laboratories, Jiutepec, Morelos, Mexico) was used, wherein macerated tablets were added to distilled water in the Petri dishes.

After incubation, direct observation was carried out in a stereoscopic microscope every hour to determine the cestocidal effects. The motility test was conducted in triplicate²⁶, wherein cestode motility was evaluated on scale of 0 to 5, as follows: 0 indicated completely motionless tapeworms that did not respond to manual stimulation; 1 indicated movement only when prodded; 2 indicated spontaneous activity, but solely at either end of the organism, namely the scolex and the end of strobila; 3 indicated slow and spontaneous activity throughout the assessment; 4 indicated that the subject was more active; and, 5 indicated that the subject was highly active^28-30.

Adult parasite segments (gravid proglottids) were obtained and fixed in 10% paraformaldehyde and kept under refrigeration at 4 °C until the histological procedure was carried out. In order to observe the histological structure and quantify the height of the secretory epithelium and the thickness of the lamina propria, tissue sections were obtained and placed on glass slides previously treated with poly-l-lysine. The paraffin was removed from the sections with xylol, hydrated with alcohols of different concentrations, and made permeable with 0.1% triton X-100 in sodium citrate for 20 min, while endogenous peroxidase activity was then inhibited by incubating the tissue for 25 min in a 0.3% H₂O₂ solution at an ambient temperature. The preparations were washed with a phosphate buffer solution (PBS1X) and marked with a hydrophobic pencil around the tissue^31-33. D. caninum cestode segments were processed, obtaining 5-µm semi-serial sections. Hematoxylin and eosin staining was used to evaluate the histological structure and tegument thickness²⁷. For each histological section, six microscope fields were observed using a 40X objective, while six tegument thickness measurements were carried out using the Motic 2.0 image analyzer and then photographed^26,27.

The experimental data corresponding to in vitro motility observations were analyzing using a Z-test, while the tegument thickness measurements were compared via a Student’s t-test³⁴. Differences were considered statistically significant when P<0.05, while the in vitro motility results are expressed as a percentage and the histology results are expressed as both mean and standard error.

Results for the efficacy of P-O in terms of the motility of D. caninum parasites are available in Table 1. At the beginning (Hour 0) of the experiment, both groups presented total motion (P=0.07). The P-O showed progressive motility reduction (from 72.0 to 4.8 %) from the first to the fifth hour post-incubation, thus achieving a mean hourly reduction of 19 %, while, from hours six to ten, 0 % motility was observed. In the control group (CG), 100 % motility was observed for the first 2 h post-incubation, while motility reduced from 96.2 to 4.2 % from the third hour to the end of the experiment, giving a mean reduction of 10 % per hour.

A (P=0.0001) difference was observed between the treated and CG groups between the first hour and the end of the experiment. In the first hour post-incubation, the motility of the treated group was 72.0 %, while this was 100 % for the CG (P=0.0001). Motility of 55.7, 41.5, 31.5, 20.0, and 4.2 % was observed at hours 6, 7, 8, 9 and 10 post-incubation, while 0.0 % motility was observed in the treated group from the sixth hour onward post-incubation (P=0.0001).

The effects of P-O on the tegument thickness (P=0.001) of adult D. caninum cestodes are shown in Figures 1 and 2, wherein P-O reduced tegument thickness by 42.5 % (10.24 ± 0.21 µm) (Figure 2b; grey arrowhead), while this was 17.81 ± 0.33 µm for the CG (Figure 2a; grey arrowhead).

**Significant differences among groups (P=0.001).

Figure 1 Effect of Pyrantel-Oxantel on tegument thickness (mean ± SEM) in Dipylidium caninum adult cestodes 

A) Intact tegument thickness (grey arrowhead), immature calcareous corpuscles along the surface and near the tegument (black arrowheads), intact embryophore and vitelline layer (purple arrowhead), and the embryo (red arrowheads). B) Reduction in tegument thickness (grey arrowhead), reduction in numbers of immature calcareous corpuscles (black arrowhead), the appearance of mature calcareous corpuscles (blue arrowhead), morphology and distribution of egg sacs (purple arrowhead), embryophore, vitelline layer, embryo, and distended structures (purple and red arrowheads).

Figure 2 Histological sections of Dipylidium caninum tapeworms. Tegument thickness obtained in untreated cestodes (A) and those treated with Pyrantel-Oxantel (B) 

The effects of P-O on the general structure of D. caninum, as compared to the structure presented by the CG, are shown in Figures 2a and b. A significant finding regarding the histology of the gravid proglottids observed was the concentration of immature calcareous corpuscles (CCs) (black arrowheads) along the surface and near the tegument (Figure 2a, b). In the CG, the immature CCs concentration was higher than that observed in the proglottids treated with P-O (Figure 2a; black arrowhead), while mature CCs were only observed in the P-O group (Figure 2b; blue arrowhead), in which an alteration of the D. caninum eggs was also observed. In the group treated with P-O, the morphology and distribution of egg sacs was found to have been altered, while, in the CG, the egg sacs presented a normal morphology and distribution (Figure 2b; purple arrowhead). Finally, the embryophore, vitelline layer, and embryo remained almost completely intact in the CG, while, in the P-O group, such structures were distended (Figure 2a; red arrowhead).

The present study shows the effects of P-O on the motility and tegument thickness of adult D. caninum cestodes, as well as other histological structures such as calcareous corpuscles, egg sacs, embryophore, the vitelline layer, and the embryo. The motility results obtained in the present study show that P-O has a direct effect on motility, causing 100 % motility inhibition 6 h post-incubation. Some drugs have a rapid effect on the neuromuscular transmission of some parasites, with, for example, pyrantel and oxantel acting as agonists on the synaptic and extra-synaptic nAchRs in the nematode muscle cells, producing contraction and spastic paralysis³⁵. Early studies evaluated the effects of pyrantel pamoate against Caenorhabditis elegans³⁶, while pamoate pyrantel and pamoate oxantel, used in combination, have been found to be effective against Trichuris muris³⁷Ascaris lumbricoides³⁸, although this treatment has been found to be ineffective against Ancylostoma ceylanicum and Necator americanus³⁷. The effects of pyrantel pamoate and oxantel pamoate, 24 h after exposure, include muscle contraction, motility inhibition, and a reduction in the size of the parasite^36,37, in both infective larvae and adult organisms. While the effects found in these studies concur with the findings of the present study, the P-O treatment applied caused 100% mortality in D. caninum tapeworms from the sixth hour post-exposure onward.

The effects of pyrantel pamoate (in paste form) were evaluated in the common horse tapeworm A. perfoliata^39-41, with a 92-98 % reduction in motility obtained in adult tapeworms after 7 to 16 d of treatment in naturally-infected horses examined at necropsy⁴⁰. Said results concur with the 100% mortality observed in vitro in the present study 6 h post-incubation. As pyrantel pamoate salt is practically insoluble in water, it is absorbed at a reduced rate in the gastrointestinal tract, thus enabling it to reach microenvironmental sites on the target parasites more easily than other pyrantel salts such as tartrate, which is more soluble in water and more rapidly absorbed via the gastrointesinal tract, to be then metabolized and excreted in both urine and, in small quantities, in feces⁴⁰. Therefore, the present study shows, in vivo, the advantage of the P-O treatment applied in the present study in dogs naturally infected with D. caninum tapeworm, as, at least, a lethal effect was observed in vitro.

The combination of pyrantel with other drugs has been found to have a limited anthelmintic potential, as observed in A. ceylanicum and N. americanus³⁷in vitro, which presented antagonistic and non-lethal effects. However, the present study found a synergetic effect and an increased potency via the combination of pamoate pyrantel and pamoate oxantel, obtaining 100 % mortality in the D. caninum adult cestode. A similar effect was observed in an earlier study using a combination of embonate pyrantel, embonate oxantel, and praziquantel, obtaining 100 % mortality in vitro eight hours post-incubation³³. While the foregoing results concur with those obtained by the present study, these results show 100 % mortality in vitro 6 h post-incubation. The effect of P-O observed in the present study is due to the capacity of the drug to remain in microsites on the parasites, thus increasing its absorption by the cestode along the length of the tegument and shortening the time in which it takes effect, thereby increasing mortality.

The tegument is one of the major structures in a cestode, which requires this anatomical feature both for absorbing semi-digested material from the small intestine of the host and improving the cestode’s physiological function and reproduction, given that, unlike nematodes, cestodes have no digestive tract. Therefore, the absorption capacity of the tegument in cestodes is higher than that found in nematodes. In relation to the foregoing, during their establishment, helminth parasites produce increased levels of pro-inflammatory cytokines in the host⁴², producing, in consequence, cachexia.

The present study found that P-O substantially reduced tegument thickness in the D. caninum tapeworm. There are reports in the literature of this treatment affecting the tegument to different degrees, causing changes and irreversible morphological damage to the tegument and parenchyma, alterations in muscular organization, the absence of tegumentary microvilli, the loss of membrane cells in subtegumental tissue, the development of a dense granular tegument, and large vacuoles generating a patchy and porous appearance, in the following organisms: the rodent tapeworms Hymenolepis nana^43,44, Hymenolepis microstoma⁴⁴, Taenia taeniformis^44,45, Echinococcus multilocularis^44,45, and Hymenolepis diminuta^29,44,45; Taenia solium in experimentally-infected hamsters⁴⁶; Taenia crassiceps^47-49; Mesocestoides corti⁵⁰; Raillietina echinobothrida^51-54 in domestic fowl; Anoplocephala perfoliata³⁰ in horses; D. caninum³³ in dogs, cats, and humans; the trematode Fasciola hepatica in rats⁵⁵; Artyfechinostomum sufrartyfex⁵⁶ in humans; Fasciolopsis buski^53,56; the gastrointestinal swine nematode Ascaris suum⁵³; the gastrointestinal canine hookworm nematode Ancylostoma ceylanicum⁵⁷; the gastrointestinal rodent nematodes Rodentolepis microstoma⁵⁷, Trichuris muris⁵⁸, and Heligmosomoides polygyrus⁵⁹; and the parasitic plant nematodes of the genera Meloidogyne and Globodera⁶⁰. The results of the present study concur with the results mentioned above, such as the reduction (thinning) of the tegument by 42.5 % in D. caninum parasites treated with P-O after 6 h of in vitro incubation. The effect of this treatment on tegument thickness may be more pronounced in parasites incubated for more than six hours, as shown by the histological results; however, histological results were not obtained from the treated subjects more than 6 h post-incubation.

The present study showed the presence of CCs in gravid segments in both the CG or P-O groups; however, in the P-O group, the number of CCs was lower than that observed in the CG. Biomineralization is a widespread phenomenon in invertebrates, with calcium carbonate one of the most abundant biominerals involved in said process⁶¹. In cestodes, minerals produce CCs, the function of which has been the subject of scientific speculation, with some hypotheses proposed, one of which positing that the CCs represent approximately 10 % of the parasites’ body weight and that they are to be commonly found in the parenchyma of many metacestodes and adult cestodes^61,62. Another hypotheses is that CCs play an important role in detoxification⁶³, as, because they are mainly produced in the absence of oxygen, they are thought to anaerobically buffer acids and serve as a reservoir for inorganic ions⁶⁴. It should be noted that CCs are, in part, an excretory product that serves to remove metabolic waste from the body by passing through the tegument⁶². Calcareous corpuscles are composed of an organic base coupled with inorganic substances, such as potassium, sodium, magnesium, silicate, calcium, phosphate⁶¹, and sulfate in different cysticercus larvae and adult cestodes⁶⁴. Their organic base includes DNA, RNA⁶⁵, proteins^66,67, and glycogen^46,68. Therefore, in the present study, the decreased number of immature CCs distributed near the tegument in the P-O group was due to both a high level of absorption via the tegument of the D. caninum cestodes and the length of in vitro incubation. Moreover, the 100 % mortality obtained by the present study is also due to both a loss of the protein and glycogen required by the metabolic process in the cells and a probable disintegration of the organism’s DNA.

As few studies have been conducted on the density and location of CCs in different parts of the strobila of D. caninum cestodes, Khalifa et al²⁷ conducted a comparative histochemical and ultrastructural study to ascertain the differences in the location, distribution, composition, and functions of the CCs of D. caninum and T. taeniaeformis. The results of the present study in relation to CCs concur with those obtained by Khalifa et al²⁷ as the distribution of the CCs was concentrated on the lateral sides of the gravid segments of the D. caninum cestode and were affected by the P-O treatment.

Hematoxilin and eosin staining of the gravid segments showed that, in the CG, the D. caninum eggs were grouped in sacs as observed by Khalifa et al²⁷ while the P-O group presented signs of morphological alterations, such as distention of the eggs, embryophore, embryo, and vitelline layer. While few studies have observed the structure of the gravid segments of D. caninum, Peña et al³³ observed the effects of the toxins of B. thuringiensis on D. caninum, with their results concurring with those of the present study in showing effects on motility, tegument thickness, and the eggs, effects which reduce the percentage of motility, thin the tegument, and damage the organism’s eggs. However, Peña et al³³ used a commercial drug containing pyrantel embonate, oxantel embonate and praziquantel as a positive control.

Other strategies have been used to decrease the infectivity of D. caninum eggs, with a study conducted in Brazil by Araujo et al⁶⁹ evaluating the effect of the nematophagous fungi Poconia chlamydospora, Duddingtonia flagrans, and Monacrosporium thaumasium on egg capsules. Their results showed that Poconia chlamydospora isolates had an ovicidal activity (type 2 and 3) for between 5 and 15 d after in vitro incubation. However, to date, the activity of a synthetized drug is found to have faster and more pronounced effects, as observed with the P-O treatment used in the present study.

Based on the results of the present study, P-O has in vitro cestocidal effects against the D. caninum tapeworm, showing a lethal effect and decreasing motility by 100 % within the first 6 h after in vitro incubation. Moreover, P-O has a direct effect on tegument thickness, reducing it by 42 %. The present study is the first conducted on the in vitro effects of P-O on mortality, the reduction of tegument thickness, and alterations in histological structures, such as the eggs, embryophore, embryo, and vitelline layer of the D. caninum tapeworm. The use of a P-O combination is an optional drug therapy for the control of D. caninum in naturally-infected dogs and cats.