Exposure of Toxocara canis eggs to Purpureocillium lilacinum as a biocontrol strategy: an experimental model evaluation.

Purpureocillium lilacinum is a nematophagous fungus used in biological control against some parasites, including Toxocara canis. This study researched the infectivity of embryonated T. canis eggs after exposure to the fungus P. lilacinum. T. canis eggs were exposed to P. lilacinum for 15 or 30 days and subsequently administered to Swiss mice (n=20). Control group consisted of mice who received T. canis embryonated eggs without fungal exposure. Forty-eight hours after infection, heart, lung, and liver from animals of each group were collected to assess larval recovery. The organs of mice that received embryonated eggs exposed to the fungus showed a lower average larval recovery (P<0.05) suggesting that exposure of T. canis eggs to P. lilacinum was able to reduce experimental infection. Under the evaluated conditions, the interaction time between the fungus and the parasite eggs was not a significant factor in larvae recovery. P. lilacinum may be considered a promising T. canis biological control agent. However, further studies are needed to determine a protocol for the use of this fungus as a biological control agent.

Exposure of Toxocara canis eggs to Purpureocillium lilacinum as a biocontrol strategy: an experimental model evaluation / Exposição de ovos de Toxocara canis a Purpureocillium lilacinum como estratégia de biocontrole: uma avaliação em modelo experimental

Abstract Purpureocillium lilacinum is a nematophagous fungus used in biological control against some parasites, including Toxocara canis. This study researched the infectivity of embryonated T. canis eggs after exposure to the fungus P. lilacinum. T. canis eggs were exposed to P. lilacinum for 15 or 30 days and subsequently administered to Swiss mice (n=20). Control group consisted of mice who received T. canis embryonated eggs without fungal exposure. Forty-eight hours after infection, heart, lung, and liver from animals of each group were collected to assess larval recovery. The organs of mice that received embryonated eggs exposed to the fungus showed a lower average larval recovery (P<0.05) suggesting that exposure of T. canis eggs to P. lilacinum was able to reduce experimental infection. Under the evaluated conditions, the interaction time between the fungus and the parasite eggs was not a significant factor in larvae recovery. P. lilacinum may be considered a promising T. canis biological control agent. However, further studies are needed to determine a protocol for the use of this fungus as a biological control agent.

Resumo Purpureocillium lilacinum é um fungo nematófago com potencial para uso no controle biológico de parasitos, incluindo Toxocara canis. Este estudo pesquisou a infectividade de ovos de T. canis embrionados após exposição ao fungo P. lilacinum . Ovos de T. canis foram expostos ao fungo por 15 ou 30 dias e subsequentemente administrados a camundongos Swiss (n=20). O grupo controle consistiu de camundongos que receberam ovos embrionados do parasita sem exposição ao fungo. Quarenta e oito horas após a infecção, coração, pulmão e fígado dos camundongos foram coletados para avaliar a recuperação larval. Os órgãos dos animais que receberam ovos embrionados expostos ao fungo apresentaram menor média de recuperação larval (P<0,05) do que os infectados com ovos sem exposição ao fungo, sugerindo que a exposição dos ovos de T. canis a P. lilacinum foi capaz de reduzir a infecção experimental. Nas condições avaliadas, o tempo de interação entre o fungo e os ovos do parasito não foi um fator significativo na recuperação das larvas. P. lilacinum pode ser considerado um promissor agente de controle biológico de T. canis, no entanto, mais estudos são necessários para avaliar o emprego deste fungo como um agente de controle biológico.

Survival of Pochonia chlamydosporia in the gastrointestinal tract of experimentally treated dogs.

The predatory capacity of the nematophagous fungus Pochonia chlamydosporia (isolate VC4) after passage through the gastrointestinal tract of dogs was assessed in vivo against Toxocara canis eggs. Twelve dogs previously wormed were divided into two groups of six animals and caged. The treatments consisted of a fungus-treated group (VC4) and a control group without fungus. Each dog of the fungus-treated group received a single 4 g dose of mycelial mass of P. chlamydosporia (VC4). Fecal samples from animals of both groups (treated and control) were collected at five different times (6, 12, 24, 36, and 48 h) after fungal administration, and placed in Petri dishes. Each Petri dish of both groups for each studied time interval received approximately 1000 T. canis eggs. Thirty days after the fecal samples were collected, approximately one hundred eggs were removed from each Petri dish of each studied time interval and evaluated by light microscopy (LM) and scanning electron microscopy (SEM). Microscopy examination of plates inoculated with the fungus showed that the isolate VC4 was able to destroy the T. canis eggs with destruction percentages of 28.6% (6 h), 29.1% (12 h), 32.0% (24 h), 31.7% (36 h), and 37.2% (48 h). These results suggest that P. chlamydosporia can be used as a tool for the biological control of T. canis eggs in feces of contaminated dogs.

[Toxocariasis]. / Toxocariasis.

The clinical presentation of toxocariasis, a zoonotic parasitosis transmitted from dogs and cats to humans, can be very diverse, which is one of the reasons why Toxocara-related disease may go unnoticed. This paper gives a brief summary of the various clinical presentations (covert/common toxocariasis, visceral larva migrans, ocular toxocariasis and neurotoxocariasis), diagnostic and differential-diagnostic considerations as well as treatment and prevention. In brief, the diagnosis of human toxocariasis relies mainly on patient data, anamnestic information, symptoms, eosinophil count and total-IgE levels.

Comparative in vitro effect of artemether and albendazole on adult Toxocara canis.

Since the integrity of Toxocara canis cuticle is essential for the nutritive and protective functions, light and scanning electron microscopic studies were undertaken to assess, for the first time, whether artemether had any effect on the cuticle including lips and sensory organs following 24 and 48 h incubation in vitro. The results were compared with those observed in the worm cuticle following incubation in albendazole, as it was 100% effective against adult nematode. The body cover changes seen after in vitro administration of artemether were similar to that induced by albendazole sulfoxide, active form, (ABZ-SO). However, the earlier onset of those changes was recorded with the former. The swollen appearance of the anterior end of T. canis, including the lips, distortion of some sensory papillae, and appearance of a number of lesions on the lips, were observed during in vitro action of artemether. Cuticular disruption had also been observed in adult T canis exposed to ABZ-SO. The surface changes were more severe than those observed following incubation in artemether, with which limited loss of the cuticle occurred in the lips of nematode but was not as widespread as that seen with ABZ-SO. However, the cuticular swelling of the anterior end of T canis was more pronounced than that with ABZ-SO. In the present study, artemether presented itself as a drug that might become important in nematode chemotherapy, besides its broad spectrum of activity against various trematodes.

Ultrastructural localization of Toxocara canis larval antigen reacted with a seropositive human serum.

Excretory-secretory products of Toxocara canis larvae have been considered as a major functional antigen in immune responses against toxocariasis. We studied ultrastructural localization of T. canis second-stage larval antigen using a seropositive human serum under immunogold electron microscopy. High-density gold particles were observed in the secretory cells, excretory duct, intestinal epithelium, and cuticle of the larval worm sections. The distribution of the positive reactions in the larval worms suggests that the nature of the antigen is excretory-secretory antigen including waste metabolites and secretory enzymes.

Synergistic effects of pyrantel and the febantel metabolite fenbendazole on adult Toxocara canis.

Pyrantel embonate and febantel are both constituents of Drontal Plus and Drontal Puppy broad spectrum anthelmintics for dogs. The effects of pyrantel and the febantel metabolite fenbendazole were investigated against Toxocara canis in-vitro by studying changes in worm motility and tissue damage. Pyrantel and fenbendazole were added to worms incubated in media for 8 h at the following concentrations: pyrantel: 12.2 microg, 25 microg, or 50 microg; fenbendazole: 50 microg, 100 microg or 200 microg; mixture of pyrantel and fenbendazole: 12.2 microg p + 50 microg f, 25 microg p + 100 microg f, 50 microg p + 200 microg f. Following this 8 h incubation period, one group of the worms was immediately fixed and studied by light- and electron microscopical examination. Other groups have been observed for further 8 h periods up to 56 hours and then studied in the same way.

A light and electron microscopic study on the synergistic effect of pyrantel and the febantel metabolite febendazole on adult Toxocara canis in vitro.

In the present study, we investigated the in vitro effects on the motility and morphology of tissues and organs of Toxocara canis of the two drug components of Drontal Plus and Welpan, pyrantel and fenbendazole (the active metabolite of the prodrug febantel), both alone and in combination. Although there was no significant difference observable between the effects of the single drugs and the drug combination on worm motility, the synergistic effect of pyrantel and fenbendazole was manifested by morphological alterations seen by light and electron microscopy. Thus, an earlier onset of damage to worm tissues and organs could be observed compared to the application of the individual drugs. In addition, a higher degree of damage and an increased number of vital organs were involved. There was dramatic, significantly greater and irreversible damage to the hypodermis, longitudinal muscle, intestine, nerve cords and genital organs induced by the pyrantel/fenbendazole combination. We hypothesise that these synergistic effects will also take place when dogs are treated either with Drontal Plus or Welpan in which lower dosages will be sufficient to destroy the worms.

In vitro studies on the effects of flubendazole against Toxocara canis and Ascaris suum.

Adult Toxocara canis and Ascaris suum were incubated in vitro in media containing 0.1, 1, 10 or 100 micro g/ml flubendazole in order to study drug-derived effects. This incubation was done for 8 h and repeated (in some groups) after 24 h for another 8 h. The onset and intensity of flubendazole-derived effects were dosage-dependent and time-dependent, i.e. the same grade of damage was reached when incubating for a longer period at a low dosage or for a shorter period in medium containing a high amount (10 or 100 micro g/ml) of flubendazole. A repeated incubation in drug-containing medium was superior to a single exposure. Flubendazole is apparently able to penetrate into the worm's interior via the cuticle. This became evident in worms with sealed orifices, which showed identical damage to worms which were not sealed. The type of tissue damage due to flubendazole was identical in both worm species when exposed to any of the drug dosages used. The principal mode of action of flubendazole was based on the complete reduction of microtubuli-polymerisation inside the parasite's cells. This apparently led to the complete destruction of the hypodermis, muscle layer and intestine. Flubendazole also stopped the formation of gametes. Summarising, even low concentrations of flubendazole (0.1 micro g/ml) led to significant and irreversible damage in all worms studied.

Biological interaction between soil fungi and Toxocara canis eggs.

Fungi from the soil of public areas in La Plata, Argentina were isolated and evaluated for their biological interaction with Toxocara canis eggs in vitro. We isolated and identified two fungal species: Fusarium pallidoroseum and Mucor hiemalis. Each species was co-cultured with T. canis eggs in sterile distilled water. The samples were observed by light and scanning electron microscopy (SEM) at days 4, 7 and 14 post-inoculation. Under the conditions of our experiments, F. pallidoroseum exhibited a high ovicidal activity on T. canis eggs, whereas M. hiemalis exhibited no such effects.

Toxocara canis (Nematoda:Ascaridae): the fine structure of the oviduct, oviduct-uterine junction and uterus.

The fine structure of the oviduct, oviduct-uterine junction and uterus of the nematode Toxocara canis (Werner, 1782) is described. Columnar-type epithelioid cells with numerous microvilli at the apical membrane border the oviduct lumen. Many electron dense secretory products are present in these cells. The cells lining the oviduct-uterine junction have no microvilli. They are coated with an electron-dense layer and contain numerous membrane-bound dense material containing bodies. Externally, the cells are surrounded by a basal lamina and muscle cells. The epithelial cells lining the greater part of the paired uteri appear to be rather flat. The oocytes inside the oviduct are covered with a dense thick plasma membrane and contain lipid droplets, dense granules and glycogen. The morphology of the oocytes before the fertilization inside the oviduct-uterine junction resembles that of the oocyte in the oviduct. After the fertilization the egg shell formation takes place. The egg shell of T.canis is composed of four layers: uterine, vitelline, middle chitinous and inner layer. The differences between the fine structure of the egg shell of T. canis and other related nematodes are discussed.

Toxocara canis: ultrastructural aspects of larval moulting in the maturing eggs.

The morphology of the surface of Toxocara canis larvae, developing in the eggs to reach infectivity, has been studied for the first time at an electron microscopical level. In most 11-day and some 15-day eggs, the larvae are surrounded by two shed cuticles. The outer first shed cuticle is composed of two layers. the inner second shed cuticle is much thicker than the outer one. The presence of both shed cuticles indicates that the larva has undergone two developmental stages in the maturing egg. The larvae in most 15-day eggs are surrounded by one shed cuticle composed of outer electron-dense and inner layers. This cuticular sheath is identical with the described inner second shed cuticle, except for its apparently reduced thickness. The infective larvae inside the 30-day eggs are enveloped by one cuticular sheath, derived from the second moulted cuticle, and consisting only of a single layer. The findings are discussed with respect to data concerning the moulting process in other nematode species.

Experimental murine toxocariasis histopathological study of chronic renal infection, transplacental transmission and ultrastructural study of egg shell.

Histopathological examination of kidney of swiss albino mice, 2 1/2 months after infection with Toxocara canis eggs revealed various pictures of pathological affection. There was diffuse mesangioproliferative glomerulonephritis with mesangeal expansion of most of glomeruli. Some glomeruli showed severe hyalinosis with marked adhesions to the bowman's capsule. The lumina of glomerular capillaries were markedly obliterated. There was also cystic dilatation of tubules with proteinaceous casts. The intersitial tissue showed mild inflammatory oedema. Chronic inflammatory cellular infiltration to form granulomatous inflammation was noticed. Transplacental transmission of Toxocara larvae was assessed in embryo of pregnant female swiss albino mice which were infected with Toxocara eggs in the 1st week of pregnancy. The larvae were detected in the liver of embryo. Ultrastructural study of the egg shell revealed its formation of 4 layers; a thin uterine membrane with occassional small bulges, vitelline layer, thick homogenous layer, and fibrous lamellar and lipid layer.

Toxocara canis (Nematoda, Ascarididae): ultrastructure of the rachis and the ovarian wall.

The oogonia and oocytes in the ovaries of Toxocara canis are joined to a cytoplasmic process called the rachis. The rachis is a much branched cytoplasmic mass without cell components in the germinal zone. At the end of the germinal zone and in the growth zone the cytoplasmic mass is formed into a central axial cylinder, containing small dense granules, lipid drops and glycogen. Throughout the growth zone shell granules similar to those present in the oocytes are also present in the rachis. Anterior to the opening of the ovaries into the oviduct the rachis disappears. The ovarian wall is composed of epithelial cells, adjoining the basal lamina. They are characterized by the presence of large numbers of mitochondria, especially in the germinal zone. The epithelial cells in the growth zone also contain rough endoplasmic reticulum, ribosomes and bundles of microfibrils. A dense tubular material occurs between the basal membrane of the epithelial cells and the basal lamina as well as in the wall intercellular spaces in the ovarian growth zone. Multivesicular labyrinth-like formations can also be observed in the epithelial intercellular spaces in the central portion of the T. canis ovary.

Effects of pyrantel pamoate on adult and preadult Toxocara canis worms: an electron microscope and autoradiography study.

Adult as well as preadult Toxocara canis isolated from the intestine of a beagle were incubated for 2, 4, and 14 h in medium containing either different concentrations of pyrantel pamoate (23.6, 236, and 2360 micrograms/ml medium) or tritiated pyrantel pamoate (2.36 micrograms/ml medium). These incubations were performed to study the effects of pyrantel pamoate on the morphology of the parasitic nematodes and to obtain information concerning the mode of uptake, the distribution, and the total amount of pyrantel pamoate ingested by T. canis. The results of the ultrastructure studies indicate that the intestine, hypodermis, and muscle cells are the organs that are predominantly affected by the drug. Additionally, it turned out that the duration of the treatment, i.e., the incubation time, was more important in determining the efficacy of pyrantel pamoate against T. canis than was the concentration itself. Autoradiography studies revealed that the adult worms ingest the drug orally, whereas preadults absorb pyrantel pamoate mainly through the whole body surface. Finally, measurements of the total amount of pyrantel pamoate taken up by T. canis indicated that adult worms can limit or even reduce the ingestion of pyrantel for more than 4 h, but then ingest large amounts of the drug. Preadult worms, however, absorb the drug more or less continuously during the first 14 h through the cuticula, albeit in lower concentrations than the adults. The different experiments elucidate differences in the uptake of pyrantel pamoate as well as in the total amount of drug ingested or absorbed by adult or preadult worms, leading to the assumption that repeated treatment with lower concentrations will be more effective than high concentrations given only once.

Lectin binding to secretory structures, the cuticle and the surface coat of Toxocara canis infective larvae.

Toxocara canis infective larvae are known to produce abundant glycosylated molecules which may be found associated with the surface or secreted into their environment. Using a range of fluorescein-conjugated and gold-conjugated lectins, the localization of particular carbohydrates was defined on the surface of live parasites, and internally at the ultrastructural level. Surface exposure of N-acetyl galactosamine and N-acetyl glucosamine was deduced by binding of FITC-conjugated Helix pomatia (HPA) and wheat-germ agglutinins (WGA). These sugars appear to be associated with a densely staining surface coat as conventional immuno-electron microscopy procedures dissipate this coat and reveal no surface binding site for these lectins. However, by using cryo-immuno-electron microscopical (C-IEM) techniques, the surface coat is retained and can be shown to bind WGA. The fluorescent lectins also revealed strong WGA binding to the secretory and amphidial pores, while the buccal opening and the cuticular alae bound HPA. Corresponding results were obtained at the ultra-structural level. Thus, HPA bound to the electron-dense area of the cuticle, areas of local cuticular thickening such as the alae and buccal labia, as well as to the oesophageal lumen. WGA also bound to the thickened cuticle of the alae and the buccal opening, but showed no reaction to either the electron-dense layer of the cuticle or the oesophageal lumen. Unlike HPA, WGA did bind specifically to the secretory column contents and the electron-dense regions of the lips associated with the chemosensory amphids. The compartmentalization of the sugars N-acetyl galactosamine and N-acetyl glucosamine, their sources and routes of surface expression and the possible association with the TES glycoprotein antigens are discussed.