Protective effect of the probiotic Lactobacillus acidophilus ATCC 4356 in BALB/c mice infected with Toxocara canis.

Human toxocariasis consists of chronic tissue parasitosis that is difficult to treat and control. This study aimed to evaluate the action of the probiotic Lactobacillus acidophilus ATCC 4356 on larvae of Toxocara canis and the effect of IFN-γ cytokine on parasite-host in vivo (1.109 CFU) and in vitro (1.106, 1.107, 1.108, 1.109 CFU) interactions. Four groups of six BALB/c mice were formed: G1 - L. acidophilus supplementation and T. canis infection; G2 - T. canis infection; G3 - L. acidophilus supplementation; and G4 - PBS administration. Mice were intragastrically suplemented with probiotics for 15 days before inoculation and 48 h after inoculation with 100 T. canis eggs. The inoculation of T. canis was also perfomed intragastrically. The recovery of larvae took place through digestion of liver and lung tissues; the evaluation of IFN-γ gene transcription in leukocytes was performed by qPCR. The in vitro test consisted of incubating the probiotic with T. canis larvae. The supplementation of probiotics produced a reduction of 57.7% (p = 0.025) in the intensity of infection of T. canis larvae in mice, whereas in the in vitro test, there was no larvicidal effect. In addition, a decrease in the IFN-γ gene transcription was observed in both, T. canis-infected and uninfected mice, regardless of whether or not they received supplementation. The probiotic L. acidophilus ATCC 4356 reduced T. canis infection intensity in mice, however, the probiotic did not have a direct effect on larvae, demonstrating the need of interaction with the host for the beneficial effect of the probiotic to occur. Yet, the proinflammatory cytokine IFN-γ did not apparently contributed to the observed beneficial effect of probiotics.

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.

In vitro effect of Chrysosporium indicum and Chrysosporium keratinophylum on Toxocara canis eggs / Efecto in vitro de Chrysosporium indicum y Chrysosporium keratinophylum sobre huevos de Toxocara canis

The degree of antagonism exercised by fungi on geohelminth development varies according to the morphological alterations caused by different fungal species. Saprophytic fungi may exert ovicidal or ovistatic effects. The aim of this study was to apply scanning electron microscopy (SEM) to observe the action of two soil saprophytic species of Chrysosporium (C. indicum and C. keratinophylum) on Toxocara canis eggs. The fungal strains to be tested were incubated for 28 days at 28 °C in 2% water agar with a suspension of unembryonated T. canis eggs. A suspension of T. canis eggs in 2% water agar was used as control group. The assay was done in triplicate for each fungus and the control group. SEM observations were performed on the 4th, 7th, 14th, 21st, and 28th day after inoculation. The effect of the fungi on eggs was evaluated in accordance with the alterations observed on the surface and the changes in the normal characteristics of the eggs. Hyphae around the eggs, appresoria penetrating the shell and changes in the typical egg membrane were observed in this assay. Type 3 effect (alterations that occur both in the embryo and the shell, and hyphal penetration of the eggs) was the prevalent effect. SEM allowed us to observe clearly the morphological alterations in T. canis eggs due to the effect of C. indicum and C. keratinophylum. Both saprophytic species of Chrysosporium alter the egg structure and alterations increase as exposure increases.

El grado de antagonismo ejercido por los hongos sobre el desarrollo de los geohelmintos depende de la especie fúngica y las alteraciones morfológicas que causan. Los hongos saprofitos pueden tener efecto ovicida u ovistático sobre los huevos. El objetivo fue aplicar la microscopía electrónica de barrido (MEB) para observar la acción de 2 especies de Chrysosporium (C. indicum y C. keratinophylum) saprofitas de suelos, sobre huevos de Toxocara canis.Las especies a ensayar se sembraron en agar agua al 2% con una suspensión de huevos no embri-onados de T. canis y se incubaron 28 días a 28 °C. Como grupo control se utilizó una suspensión de huevos de T. canis en agar agua al 2%. El ensayo se realizó por triplicado para cada hongo y el grupo control. Las observaciones con MEB se realizaron a los 4, 7, 14, 21 y 28 días de incubación. La acción de los hongos se evaluó según las alteraciones en la superficie y los cambios en las características normales de los huevos. En este ensayo se observaron: hifas rodeando los huevos, appresorios penetrando la cubierta y cambios en la membrana característica del huevo, prevaleciendo el efecto tipo 3 (alteraciones que se producen tanto en el embrión como en la cubierta y penetración de hifas al interior de los huevos). La aplicación de la MEB permitió observar claramente que las 2 especies de Chrysosporium saprofitas de suelos, afectan el normal desarrollo de los huevos de T. canis, alteran su estructura y las alteraciones aumentan con el tiempo de exposición.

Lactobacillus rhamnosus reduces parasite load on Toxocara canis experimental infection in mice, but has no effect on the parasite in vitro.

Human toxocariasis is a neglected global parasitic zoonosis. The efficacy of drug treatment for this disease has been hindered by the biological complexity of the main etiological agent, the nematode Toxocara canis. Experimental studies have shown the potential of probiotics to promote a reduction in the parasite load of T. canis larvae. This study aimed to evaluate the effect of probiotic Lactobacillus rhamnosus ATCC 7469 on the parasite load of BALB/c mice with acute toxocariasis and evaluate the direct effect of this probiotic on T. canis larvae in vitro. In vivo administration of probiotics reduced the parasite load of T. canis larvae by 53.3% (p = 0.0018) during the early stage of infection in mice. However, when analyzed in vitro, it was observed that the probiotic did not present a deleterious effect on the larvae, as approximately 90% of these remained viable. These results demonstrate the potential of the probiotic L. rhamnosus in the reduction of T. canis larvae in BALB/c mice and suggest it could be used as an alternative means for the controlling of visceral toxocariasis. However, further studies are required to elucidate the mechanisms of action promoted by this probiotic.

In vitro effect of Chrysosporium indicum and Chrysosporium keratinophylum on Toxocara canis eggs.

The degree of antagonism exercised by fungi on geohelminth development varies according to the morphological alterations caused by different fungal species. Saprophytic fungi may exert ovicidal or ovistatic effects. The aim of this study was to apply scanning electron microscopy (SEM) to observe the action of two soil saprophytic species of Chrysosporium (C. indicum and C. keratinophylum) on Toxocara canis eggs. The fungal strains to be tested were incubated for 28 days at 28°C in 2% water agar with a suspension of unembryonated T. canis eggs. A suspension of T. canis eggs in 2% water agar was used as control group. The assay was done in triplicate for each fungus and the control group. SEM observations were performed on the 4th, 7th, 14th, 21st, and 28th day after inoculation. The effect of the fungi on eggs was evaluated in accordance with the alterations observed on the surface and the changes in the normal characteristics of the eggs. Hyphae around the eggs, appresoria penetrating the shell and changes in the typical egg membrane were observed in this assay. Type 3 effect (alterations that occur both in the embryo and the shell, and hyphal penetration of the eggs) was the prevalent effect. SEM allowed us to observe clearly the morphological alterations in T. canis eggs due to the effect of C. indicum and C. keratinophylum. Both saprophytic species of Chrysosporium alter the egg structure and alterations increase as exposure increases.

Trichoderma virens as a biocontrol of Toxocara canis: In vivo evaluation.

BACKGROUND: Microorganisms have been widely studied as biological control agents of parasites of medical and veterinary importance. Coprophagous arthropods, bacteria and fungi are among the different organisms evaluated as potential biological control agents. Nematophagous fungi capture and digest the free forms of nematodes in the soil. Due to its zoonotic potential, Toxocara canis have been brought to the attention of researchers. AIMS: The aim of the present study was to determine whether the administration of embryonated T. canis eggs exposed to the nematophagous fungus Trichoderma virens reduces parasite infection in experimental animals. METHODS: Embryonated T. canis eggs were exposed to T. virens mycelium for 15 days at 25°C. Subsequently, 100 fungus-exposed eggs were orally administered to 20 Swiss mice. As a positive control, another 20 mice received 100 embryonated eggs that were not exposed to the fungus. After 48h, the animals were killed, and heart, lungs and liver were harvested for the recovery of larvae. RESULTS: The organs of the animals that received embryonated T. canis eggs exposed to the fungus showed a lower mean larval recovery when compared with the animals that received embryonated eggs without fungus exposure (p<0.05). CONCLUSIONS: The exposure of T. canis eggs to T. virens reduces the experimental infection, demonstrating the potential of this nematophagous fungus as a biocontrol agent.

Fungi predatory activity on embryonated Toxocara canis eggs inoculated in domestic chickens (Gallus gallus domesticus) and destruction of second stage larvae.

The objective of this study was to evaluate the infectivity of Toxocara canis eggs after interacting with isolated nematophagous fungi of the species Duddingtonia flagrans (AC001) and Pochonia chlamydosporia (VC4), and test the predatory activity of the isolated AC001 on T. canis second stage larvae after 7 days of interaction. In assay A, 5000 embryonated T. canis eggs previously in contact with the AC001 and VC4 isolated for 10 days were inoculated into domestic chickens (Gallus gallus domesticus), and then these animals were necropsied to collect material (digested liver, intestine, muscles and lungs) at 3-, 7-, 14-, and 21-day intervals after inoculation. In assay A, the results demonstrated that the prior interaction of the eggs with isolated AC001 and VC4 decreases the amount of larvae found in the collected organs. Difference (p < 0.01) was observed in the medium larvae counts recovered from liver, lung, intestine, and muscle of animals in the treated groups when compared to the animals in the control group. At the end of assay A, a percentage reduction of 87.1 % (AC001) and 84.5 % (VC4) respectively was recorded. In the result of assay B, the isolated AC001 showed differences (p < 0.01) compared to the control group, with a reduction of 53.4 % in the recovery of L2. Through these results, it is justified to mention that prior interaction of embryonated T. canis eggs with the tested fungal isolates were efficient in reducing the development and migration of this parasite, in addition to the first report of proven predatory activity on L2.

Fungal ovicidal activity on Toxocara canis eggs.

BACKGROUND: Visceral toxocariasis is a parasitic zoonosis caused by Toxocara canis. The prevalence of this parasite in dogs, soil contamination and the resistance of eggs increase human exposure to the disease. Moreover, the difficulties of the control measures justify the need for alternative ones. AIMS: The objective of this study was to evaluate the in vitro ovicidal activity of fungi isolated from soils from public places in the city of Pelotas, Rio Grande do Sul, Brazil, on Toxocara canis. METHODS: Samples of soil from ten localities were inoculated onto Petri dishes with 2% water-agar (WA) that contained antibiotics, and incubated at 25°C/21 days. Isolated fungi were tested in vitro for ovicidal activity, with five replicates. One mL of an embryonated Toxocara canis egg suspension (10(3) eggs) was poured over the fungal cultures after 10 days of growth. At intervals of 7, 14 and 21 days, 100 eggs were removed from each plaque and evaluated by optical microscopy. RESULTS: Acremonium, Aspergillus, Bipolaris, Fusarium, Gliocladium, Mucor and Trichoderma were isolated from the soil. A significant ovicidal type 3 effect was observed in Trichoderma, Fusarium solani complex and Acremonium. Those isolates from the genus Trichoderma showed their ovicidal effect on the 14th day of fungus-egg interaction. The other fungal genera tested showed a type 2 effect. CONCLUSIONS: These results suggest that the use of Trichoderma and Fusarium solani complex in biological control of T. canis is promising; however, further studies should be performed.

Antagonistic activity of the fungus Pochonia chlamydosporia on mature and immature Toxocara canis eggs.

In vitro tests were performed to evaluate the ability of 6 isolates of the nematophagous fungus Pochonia chlamydosporia to infect immature and mature Toxocara canis eggs on cellulose dialysis membrane. There was a direct relationship between the number of eggs colonized and the increase in the days of interaction, as well as between the number of eggs colonized and the increase in the concentration of chlamydospores (P<0.05). Immature eggs were more susceptible to infection than mature eggs. The isolate Pc-04 was the most efficient egg parasite until the 7th day, and showed no difference in capacity to infect mature and immature eggs in comparison to Pc-07 at 14 and 21 days of interaction, respectively. Isolate Pc-04 was the most infective on the two evolutionary phases of the eggs at most concentrations, but its ability to infect immature eggs did not differ from that presented by the isolates Pc-07 and Pc-10 at the inoculum level of 5000 chlamydospores. Colonization of infective larvae inside or outside the egg was observed in treatments with the isolates Pc-03, Pc-04, Pc-07 and Pc-10. The isolate Pc-04 of P. chlamydosporia has great biological capacity to destroy immature and mature T. canis eggs in laboratory conditions.

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.

[Destruction of Toxocara canis eggs by the nematophagous fungus Pochonia chlamydosporia]. / Destruição de ovos de Toxocara canis pelo fungo nematófago Pochonia chlamydosporia.

INTRODUCTION: Toxocara canis is an ascarid parasite of the small intestine of dogs that causes visceral larva migrans in humans. METHODS: With the aim of demonstrating the effectiveness of the fungus Pochonia chlamydosporia on Toxocara canis eggs under laboratory conditions, a trial was set up in Petri dishes with 2% agar-water. RESULTS: There was ovicidal activity of 43.8% (p < 0.01) in the treated group in relation to the control group over the periods studied. CONCLUSIONS: The results from the present study suggest that Pochonia chlamydosporia can potentially be used as an alternative biological control for embryonated Toxocara canis eggs.

Destruição de ovos de Toxocara canis pelo fungo nematófago Pochonia chlamydosporia / Destruction of Toxocara canis eggs by the nematophagous fungus Pochonia chlamydosporia

INTRODUÇÃO: Toxocara canis é um ascarídeo parasita do intestino delgado de cães, causador da larva migrans visceral em seres humanos. MÉTODOS: Com o objetivo de demonstrar a eficácia do fungo Pochonia chlamydosporia sobre ovos de Toxocara canis em condições laboratoriais, foi montado ensaio experimental em placas de Petri com ágar-água 2 por cento. RESULTADOS: Houve atividade ovicida de 43,8 por cento (p<0,01) do grupo tratado em relação ao grupo controle durante os intervalos estudados. CONCLUSÕES: Os resultados demonstrados no presente trabalho sugerem a empregabilidade de Pochonia chlamydosporia como uma alternativa de controle biológico dos ovos embrionados de Toxocara canis.

INTRODUCTION: Toxocara canis is an ascarid parasite of the small intestine of dogs that causes visceral larva migrans in humans. METHODS: With the aim of demonstrating the effectiveness of the fungus Pochonia chlamydosporia on Toxocara canis eggs under laboratory conditions, a trial was set up in Petri dishes with 2 percent agar-water. RESULTS: There was ovicidal activity of 43.8 percent (p < 0.01) in the treated group in relation to the control group over the periods studied. CONCLUSIONS: The results from the present study suggest that Pochonia chlamydosporia can potentially be used as an alternative biological control for embryonated Toxocara canis eggs.

Ovicidal activity of Pochonia chlamydosporia and Paecilomyces lilacinus on Toxocara canis eggs.

An assessment was made of the ovicidal activity of egg-parasitizing fungi Pochonia chlamydosporia (isolates VC1 and VC4) and Paecilomyces lilacinus on Toxocara canis eggs in vitro. The fungal isolates were inoculated onto Petri dishes with 2% water-agar (2% WA) and stored at 25 degrees C for 10 days in an incubator, in the dark. The control group was comprised of Petri dishes without fungi, containing the 2%WA medium only. Later, 1000 embryonated eggs were placed on the surface of the plates with fungal isolates and also on the control plates, and were then incubated at 25 degrees C for 7, 14 and 21 days. At these intervals, the eggs were retrieved and underwent percentage assessment according to the following parameters: no changes; type 1 effect, physiological and biochemical effect without morphological damage to eggshell, with visualization of hyphae adhered to eggshell; type 2 effect, lytic effect with morphological changes in embryo and eggshell, without hyphal penetration through the eggshell; type 3 effect, lytic effect with morphological changes in embryo and eggshell, with hyphal penetration and internal egg colonization. All the fungal isolates showed ovicidal activity (type 3 effect) on T. canis eggs, with 13.8%, 20.5% and 20.3% of ovicidal activity using P. chlamydosporia isolate VC1 after 7, 14 and 21 days, whereas isolate VC4 showed 15.2%, 19.0% and 21.7% of ovicidal activity at the same time intervals. P. lilacinus showed ovicidal activity of 12.3%, 18.8% and 20.0% after 7, 14 and 21 days. P. chlamydosporia and P. lilacinus were effective in vitro on T. canis eggs and can be considered a potential candidate to biological controller of those nematodes.

[Nematophagous fungi of Toxocara canis eggs in a public park of La Plata, Argentina]. / Hongos nematófagos de huevos de Toxocara canis en un paseo público de La Plata, Argentina.

Fungi have showed a great potential for the biological control of nematodes. However, they have not been evaluated for the control of animal and/or human parasites transmitted by egg contaminated soils. Environmental contamination with Toxocara spp. eggs is a public health problem. Accidental swallowing of Toxocara canis eggs (a nematode of dogs) usually results on a zoonotic infection (toxocarosis). The objectives of this research were: 1) To test the presence of antagonistic fungi against T. canis in the soil in public places of La Plata city, Argentina, infected with eggs of this parasite, 2) To determine the possible association between biotic and abiotic factors of the soil with the presence of fungal parasites of egg nematodes. Soil samples were tested for: textural type, organic matter (%), pH, presence of egg-parasite fungi, of larvae and of nematode eggs, in particular of Toxocara spp. The studied area showed the following characteristics: pH: 6.6-8.0, organic matter: 1.2-70%, with a predominantly loam texture. The following antagonistic fungal genera were identified: Acremonium, Aspergillus, Chrysosporium, Fusarium, Humicola, Mortierella, Paecilomyces and Penicillium. A prevalence of 70% was detected for nematode eggs, of 33% for Toxocara spp. eggs and of 90% for larvae. No association between the presence of egg-parasite fungi and the considered factors was found. More studies are necessary to know the natural antagonism factors to T. canis eggs for its in situ biological control.

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.

Biological activity of Paecilomyces genus against Toxocara canis eggs.

Saprophytic soil fungi can exert ovicidal and ovistatic effects on helminths with differing degrees of efficiency. The representatives of such fungi from temperate regions, Paecilomyces lilacinus (Thom) Samson and P. marquandii (Masse) Hughes, exhibit recognized ovicidal activity on some nematodes. We evaluated the action in vitro of P. lilacinus and P. marquandii on the zoonotic canine roundworm eggs of Toxocara canis. Eggs exposed and unexposed to fungal samples were observed by both light and scanning electron microscopy on days 4, 7 and 14 post-inoculation. Ovicidal activity of P. lilacinus on T. canis eggs was considered to be high and that of P. marquandii to be intermediate.