Effect of oral administration of Bacillus thuringiensis var. oswaldocruzi to sheep on the development of larvae in fecal cultures.

Haemonchus contortus is one of the most important gastrointestinal nematodes infecting sheep, being a production-limiting factor in sheep herds. Biological control has been used for many years either in combination with traditional parasiticides or as an alternative treatment itself. Bacillus thuringiensis can be a promising tool for an integrate control of H. contortus in sheep herds by disrupting nematode's life cycle, thus decontaminating pasture. This study aims to evaluate the larvicidal efficacy of Bacillus thuringiensis var. oswaldocruzi (Bto) on the development of H. contortus larvae in fecal sheep cultures. Bto concentrations (~1010 colony forming units, CFU / mL) were orally administered to thirty-six ewes naturally infected with H. contortus in three different pharmaceutical forms: hard gelatin capsule, mucoadhesive gel or suspension. The different treatments were carried out in a single oral administration (SOA). All formulations were effective in inhibiting larvae development compared with control groups in all time points studied (p < 0.05). The mucoadhesive gel showed ~90% of efficacy on inhibiting larvae development during all the time points post SOA. The suspension had 85% efficacy on inhibiting larvae development at 24-48 h and 96% at 72-96 h. The hard gelatin capsule was 57%, 62%, 73% and 96% effective inhibiting larvae development at 24, 48, 72 and 96 h, respectively. This study highlights that disrupting the infective larval stage in the environment Bacillus thuringiensis var. oswaldocruzi is a promising prophylactic alternative in the H. contortus control.

Eimeria bateri Natural Infection: Oocysts Reductions in Grey Quail (Coturnix coturnix) Treated with Bacillus thuringensis var. israelensis

Abstract Coccidiosis, a disease caused by the parasitic Eimeria spp., affects birds of all ages, particularly young birds more intensely. Infected poultry presents significant economic losses. Bacillus thuringiensis var israelensis (Bti) is a Gram-positive, spore-forming bacterium that produces proteins with high specific parasiticidal activity against various orders of parasites. Thus, the aim of the present study was to evaluate the parasiticidal potential of Bti in quails that were naturally infected with Eimeria bateri. Twenty 12-week-old male quails (Coturnix coturnix coturnix), naturally infected with Eimeria bateri, were randomly divided into two groups of 10 birds: Bti treated and control. The treated group was supplemented with Bti (1×108 spores∙g-1) in the feed, while; the control group received the same feed without Bti. To evaluate the occurrence of oocysts, samples of feces were collected every week for four weeks. Significant (P < 0.05) oocysts reductions of 56.64% and 94.51% were noted in the Bti treated group at 2nd and 4th week of study, respectively. The Bti supplementation may contribute to the reduction of oocysts in quails and environmental contamination. Bacillus thuringiensis var israelensis appeared to be a promising complementary alternative in E. bateri control.

Purpureocillium lilacinum and Trichoderma virens for biological control of trichostrongylid parasites of sheep: an in vitro evaluation.

The study evaluated the ovicidal activity of enzymatic extracts of Purpureocillium lilacinum and Trichoderma virens against trichostrongylid eggs from sheep. Filtered extract (FE) and macerated crude extract (MCE) were prepared from fungal cultures in minimal broth. In the experiment, 100 trichostrongylid eggs, obtained from the feces of naturally infected sheep, were exposed to fungal extracts for 24 and 48 hours/25°C. In the control group, eggs were incubated in minimal broth. The number of L1 larvae was ascertained. Each treatment consisted of four repetitions and the experiment was repeated five times. It was observed that the effect of FE and MCE of P. lilacinum and T. virens on egg hatchability differed from that of the control group. MCE of T. virens and P. lilacinum showed higher ovicidal activity than FE over both periods and at 48 hours of exposure, respectively. From the percentage reductions in hatchability of the eggs, MCE was shown to be superior to FE for both fungi. This study demonstrated the ovicidal potential of these fungi against trichostrongylid eggs. However, further studies are needed in order to identify the molecules responsible for the ovicidal effects, and to evaluate the behavior of fungal extracts in biotic and abiotic interactions.

Ovicidal in vitro activity of the fixed oil of Helianthus annus L. and the essential oil of Cuminum cyminum L. against Fasciola hepatica (Linnaeus, 1758).

The fascioliasis is a parasitic disease of importance in veterinary medicine and public health. For this parasitosis, the treatment by synthetic fasciolicides is used and due to their intense use although they have been shown less effective because of the establishment of resistant Fasciola hepatica population to these drugs, with a global concern. The use of derived products of plants with biological activity has been shown promising in the control of parasites. In this context, we evaluated the chemical composition and action of ovicidal in vitro fixed oil of Helianthus annuus L. (FOH) and essential oil of Cuminum cyminum L. (EOC), as well as their combination (FOH + EOC) of F. hepatica. In the assay in vitro of F. hepatica were submitted to different concentrations of oils, such as FOH (2.3 mg/mL + 0,017 mg/mL); EOC (2.07 mg/mL + 0,004 mg/mL) and the combination of (1.15 mg/mL + 1.03 mg/mL to 0,0085 mg/mL + 0,008 mg/mL) as well as a positive control of thiabendazole (0.025 mg/mL) and a negative control with distilled water and tween. The identification of the majority chemical compounds was performed by gas chromatography. The -cell viability of the oils was tested in MDBK cellular line by the MTT method. The majority compounds in the FOH were the linoleic (53.6%) and oleic (35.85%) unsaturated fatty acids, and the majority phytochemicals compounds in the EOC were the Cumaldehyde (26.8%) and the 2-Caren 10-al (22.17%). The EOC and the combination presented effectiveness of 99% (±1) and of 94% (±1) in the concentration of 0.03 mg/mL and 0.035 mg/mL+0.03 mg/mL, respectively, and the FOH was insufficiently active as ovicidal. The cell viability at this concentration of EOC was 93%. From the results above we could infer that the EOC is promising as a new alternative for the fascioliasis control.

Ovicidal and larvicidal potential of Rosmarinus officinalis to control gastrointestinal nematodes of sheep / Potencial ovicida e larvicida de Rosmarinus officinalis para controle de nematódeos gastrintestinais de ovinos

Abstract Gastrointestinal Nematode Infection (GIN) are the main constraint to the production of small ruminants. Studies of medicinal plants have been an important alternative in the effort to control these parasites. Therefore, the purpose of this study was to evaluate the in vitro ovicidal and larvicidal activity of essential oil of Rosmarinus officinalis. The oil was extracted, analyzed by gas chromatography and tested on GIN eggs and larvae in six concentrations, 227.5mg/mL, 113.7mg/mL, 56.8mg/mL, 28.4mg/mL, 14.2mg/mL and 7.1mg/mL. To determine the ovicidal activity, GIN eggs were recovered from sheep feces and incubated for 48h with different concentrations of the oil. For the evaluation of larval migration, third-stage larvae (L3) were obtained by fecal culture, and associated with the essential oil for 24h at the same concentrations, after which they were left for another 24 hours on microsieves, followed by the count of migrating and non-migrating larvae. The assays of R. officinalis oil showed a significant (p<0.05) 97.4% to 100% inhibition of egg hatching and a significant (p<0.05) 20% to 74% inhibition of larval migration. The main constituent revealed by gas chromatography was Eucalyptol. The results indicate that R. officinalis essential oil has ovicidal and larvicidal activity on sheep GINs.

Resumo As infecções por nematódeos gastrintestinais (ING) constituem a maior limitação à produção de pequenos ruminantes. Na busca do controle desses parasitos, estudos com plantas medicinais têm sido uma importante alternativa. Visto isto, o estudo desenvolvido teve como objetivo avaliar a ação ovicida e larvicida in vitro do óleo essencial de Rosmarinus officinalis. O óleo foi extraído, analisado por cromatografia gasosa e testado sobre ovos e larvas de ING em seis concentrações, 227,5mg/mL; 113,7mg/mL; 56,8mg/mL; 28,4mg/mL; 14,2mg/mL; 7,1mg/mL. Para determinar a ação ovicida, ovos de ING foram recuperados de fezes de ovinos e incubados por 48h com as diferentes concentrações do óleo. Na avaliação da migração das larvas, as larvas de terceiro estágio (L3) foram obtidas por coprocultura, e associadas ao óleo essencial por 24h nas mesmas concentrações, permanecendo por mais 24h em microtamises, seguindo-se a contagem de larvas que migraram e que não migraram. Os testes in vitro com o óleo de R. officinalis mostraram o nível de significância (p<0.05) 97,4% a 100% na inibição da eclodibilidade e 20% a 74% na inibição da migração das larvas. Na análise por cromatografia gasosa o constituinte majoritário foi o eucaliptol. Os resultados apresentados mostram que o óleo essencial de R. officinalis possui ação ovicida e larvicida sobre ING de ovinos.

Ovicidal and larvicidal potential of Rosmarinus officinalis to control gastrointestinal nematodes of sheep.

Gastrointestinal Nematode Infection (GIN) are the main constraint to the production of small ruminants. Studies of medicinal plants have been an important alternative in the effort to control these parasites. Therefore, the purpose of this study was to evaluate the in vitro ovicidal and larvicidal activity of essential oil of Rosmarinus officinalis. The oil was extracted, analyzed by gas chromatography and tested on GIN eggs and larvae in six concentrations, 227.5mg/mL, 113.7mg/mL, 56.8mg/mL, 28.4mg/mL, 14.2mg/mL and 7.1mg/mL. To determine the ovicidal activity, GIN eggs were recovered from sheep feces and incubated for 48h with different concentrations of the oil. For the evaluation of larval migration, third-stage larvae (L3) were obtained by fecal culture, and associated with the essential oil for 24h at the same concentrations, after which they were left for another 24 hours on microsieves, followed by the count of migrating and non-migrating larvae. The assays of R. officinalis oil showed a significant (p<0.05) 97.4% to 100% inhibition of egg hatching and a significant (p<0.05) 20% to 74% inhibition of larval migration. The main constituent revealed by gas chromatography was Eucalyptol. The results indicate that R. officinalis essential oil has ovicidal and larvicidal activity on sheep GINs.

Detection of Toxocara canis DNA in tissues of experimentally infected mice.

The main etiological agent of toxocariasis is the helminth Toxocara canis. Several difficulties are found in the diagnosis of this disease, because of nonspecific clinical signs and possible cross-reactions that may occur in the available test, the indirect ELISA. Therefore, molecular diagnosis has been indicated as an alternative to conventional diagnosis. The purpose of this study was to evaluate the polymerase chain reaction (PCR) technique for the identification of T. canis in tissues of experimentally infected mice. To this end, nine mice were inoculated with 1500 embryonated eggs and were divided into two groups, the first euthanized 48 h (G1) and the other 30 days post inoculation (G2). Lungs, brain, liver and blood were collected from all the animals for DNA Extraction and tissue digestion, also was collected blood samples for DNA extraction and ELISA test (serum). Toxocara canis DNA was identified in all the inoculated animals using the ITS-2 target gene. The PCR test successfully identified the parasite in the brain, lung and liver of the animals euthanized 48 h PI and 30 days PI. This technique yielded good results in the identification of the parasite in the brain, being more sensitive than the method for the recovery of larvae, in the group with acute infection (48 h PI). The infection was confirmed by PCR within 48 h after infection, while the ELISA indicated serological conversion occurred only 14 days after inoculation. This study demonstrates the ability of PCR to identify T. canis in the liver, lungs and brain during acute and chronic infection.

Environmental contamination by parasitic forms in a socially vulnerable community in southern Rio Grande do Sul state: a serious public health problem / Contaminação ambiental por formas parasitárias em comunidade em vulnerabilidade social no sul do estado do Rio Grande do Sul: um grave problema de saúde pública

In Brazil, a significant percentage of the population lives without basic sanitation, experiencing so-called social vulnerability. The fact that these people share the environment with animals promotes the establishment of zoonotic parasitic infections, as well as the resultant parasitic cycles. Thus, parasites present in the environment must be identified, so that control measures can be recommended. In this context, this study's objective was to evaluate environmental contamination by parasitic forms in a socially vulnerable community in southern Rio Grande do Sul. A total of 100 soil samples collected from the community were processed by a sodium dichromate centrifuge-flotation technique and analyzed by a compound microscope (40X objective) for the identification of parasite eggs, oocysts and cysts. All points were positive for two or more parasites, with the identification of 33.59% non-identified coccidian oocysts, Strongylida (25.4%), Ascaridida (21.31%), Trichuris spp. (8.19%), Toxocara spp. (3.27%), Amoebas (4.08%), Dioctophyma renale (2.45%), and Giardia spp.(1.63%). The presence of parasitic forms in all points analyzed surpasses other studies of environmental contamination carried out in the southern region of Brazil. In addition, the identification of several parasitic forms with zoonotic potential is concerning, since it shows the possibility of parasitic transmission to humans and other animals. In view of the results, the conclusion is that the environment analyzed is contaminated by parasitic forms, constituting a serious public health problem. Therefore, implementing educational and preventive measures in the community to control parasites is of crucial importance.(AU)

No Brasil, uma parcela significativa da população não possui saneamento básico e vive em situação de vulnerabilidade social, compartilhando o ambiente com animais, possibilitando o estabelecimento de infecções parasitárias zoonóticas e a manutenção do ciclo dos parasitos. Assim, para que medidas de controle sejam preconizadas, torna-se necessário a identificação dos parasitos presentes no ambiente. Neste contexto, este trabalho avaliou a contaminação ambiental por formas parasitárias em comunidade de vulnerabilidade social no sul do Rio Grande do Sul. Foram coletadas cem amostras de solo da comunidade, que foram processadas pela técnica de centrifugo-flutuação em solução de dicromato de sódio e analisadas em microscópio composto (objetiva 40X) para a identificação dos ovos, oocistos e cistos de parasitos. Todos os pontos de coleta foram positivos para dois ou mais parasitos, sendo diagnosticados oocistos de coccídios não-identificados (33,59%), Strongylida (25,4%), Ascaridida (21,31%), Trichuris spp. (8,19%), Toxocara spp. (3,27%), Amebas (4,08%), Dioctophyma renale (2,45%), Giardia spp. (1,63%). A quantidade de formas parasitárias em todos os pontos analisados supera a contida em outros estudos de contaminação ambiental já realizados na região sul do Brasil. Além disso, a identificação de diversas formas parasitárias com potencial zoonótico é preocupante, pois evidencia a possibilidade de transmissão de parasitoses ao homem e a outros animais. Diante dos resultados, conclui-se que o ambiente em questão está contaminado por formas parasitárias, constituindo um sério problema de saúde pública. Ressalta-se a importância da implantação de medidas educativas e preventivas com a comunidade para o controle dos parasitos.(AU)

Controlling gastrointestinal nematodes in cattle by Bacillus species.

In this study, we tested the in vitro and in vivo larvicidal activity of Bacillus species against gastrointestinal nematodes in cattle, and their viability in the presence of anthelmintics. For in vitro tests, cattle feces naturally infected with trichostrongylides were incubated with spore suspensions of Bacillus circulans (Bcir), B. thuringiensis var. osvaldocruzi (Bto), B. thuringiensis var. israelensis (Bti) or B. thuringiensis var. kurstaki (Btk). Subsequently, residual larvae were counted and identified. All of the Bacillus species showed 60% or more larvicidal effects. Bcir and Bti were selected to be incubated with anthelmintics (moxidectin, nitroxynil and levamisole), and after 24, 72, and 144h, their viability was evaluated. Bti showed highest drug resistance, maintaining a concentration of 1×107CFU/mL. Based on this result, Bti was selected for in vivo tests on calves naturally infected with gastrointestinal nematodes. The calves were dived into four groups: Group 1, Bti suspension of ∼1×109CFU orally administered; Group 2, Bti suspension of ∼1×109CFU orally administered with levamisole (subcutaneously, 150mg); Group 3, only levamisole (subcutaneously, 150mg), and Group 4 untreated. Then 24 and 48h after treatment, larvae numbers were counted. We observed a reduction of 84%, 100%, and 100% after 48h of treatment, respectively, for Groups 1, 2 and 3 treatments in comparison with the untreated. The tested Bacillus species showed larvicidal activity against bovine trichostrongylides, and its association with anthelmintics. It may serve as a promising integrated alternative for control of gastrointestinal nematodes in cattle.

First isolation of Dioctophyme renale eggs from an urban environment and identification of those from animal urine / Primeiro isolamento de ovos de Dioctophyme renale no ambiente urbano e identificação destes na urina de animais

Abstract Dioctophyme renale is a zoonotic parasite with worldwide distribution, although its occurrence is little known. The objective here was to evaluate the presence of parasite eggs in the environment and in the urine of dogs and cats in an urban area. Soil samples and urine were evaluated respectively by means of the Caldwell-Caldwell technique and urinalysis. Out of the 100 soil samples, 3% presented D. renale eggs, and out of the 43 urine samples, 18.6% were positive, including the feline samples. Thus, D. renale eggs are present in the urban environment, and dogs and cats are parasitized by this nematode, which therefore represents a risk to public health.

Resumo Dioctophyme renale é um parasito zoonótico com distribuição cosmopolita, entretanto sua ocorrência é pouco conhecida. Assim, o objetivo foi avaliar a presença de ovos do parasito no ambiente e na urina de cães e gatos de zona urbana. As amostras de solo e urina foram avaliadas, respectivamente, pela técnica de Caldwell e Caldwell e por urinálise. Das 100 amostras de solo, 3% apresentaram ovos de D. renale, e das 43 amostras de urina, 18,6% foram positivas, incluindo a de felino. Conclui-se que ovos de D. renale estão presentes no ambiente urbano, assim como os caninos e felinos apresentam-se parasitados, representando um risco para a saúde pública.

First isolation of Dioctophyme renale eggs from an urban environment and identification of those from animal urine.

Dioctophyme renale is a zoonotic parasite with worldwide distribution, although its occurrence is little known. The objective here was to evaluate the presence of parasite eggs in the environment and in the urine of dogs and cats in an urban area. Soil samples and urine were evaluated respectively by means of the Caldwell-Caldwell technique and urinalysis. Out of the 100 soil samples, 3% presented D. renale eggs, and out of the 43 urine samples, 18.6% were positive, including the feline samples. Thus, D. renale eggs are present in the urban environment, and dogs and cats are parasitized by this nematode, which therefore represents a risk to public health.