Evaluation of different fungal enzyme extract of eggs of ANCYLOSTOMA spp

The role of companion animals or pets as zoonotic disease reservoirs has been recognized as a significant public health hazard throughout the world. Ancylostoma caninum and A. braziliense are zoonotic parasites which are transmitted to man through contact with soil contaminated with both eggs and larvae of these ancylostomids. The considerable prevalence of environmental contamination by Ancylostoma spp eggs, together with the difficulties of applying control measures and soil disinfection, as well as the development of resistance to anthelmintic therapy, highlights the need for alternative methods to help control these helminths. Taking into account that these nematodes spend part of their life cycle in the soil and that nematophagous fungi usually found in this ecosystem establish parasitic or predatory relationships with these parasites, thus playing an important role as natural enemies, the use of these agents in the effective control of nematodes is viable and desirable. Thus, nematophageous fungi can be used when the environment is already contaminated. This study aimed to determine helminth prevalence in dog feces collected on streets and parks in Pelotas County, Rio Grande do Sul State, Brazil, as well as to evaluate the in vitro ovicidal activity of different crude enzymatic extract preparations of CG193 and MICLAB 009 Paecilomyces lilacinus, MICLAB 008 Trichoderma virens and CG502 Trichoderma harzianum fungal isolates on Ancylostoma spp. eggs. Fifteen random feces samples were collected from 14 different places in town between January and March 2012, totaling 210 samples, which were submitted to coproparasitological tests. The fungal isolates evaluated were grown in minimum liquid medium under agitation at 28ºC for five days. Fungal preparations consisted of supernatant liquid medium without filtration (crude extract), filtered (filtered extract), macerated mycelium (crude macerate), and macerated mycelium submitted to filtration (filtered macerate). Ancylostoma spp. eggs were obtained from the feces of naturally parasitized dogs. In vitro assays consisted of four treatments and one control group. 4mL of each fungal extract was poured into Petri dishes added with a 1 mL suspension containing approximately 103 Ancylostoma spp. eggs. The control group consisted of 1mL suspension containing 1000 Ancylostoma spp. eggs added with 4mL sterile distilled water. All dishes were incubated at 25º C for 24 hours. Each assay had five replicates. Following, the total number of larvae present in each treatment and in the control group was counted. The results obtained showed that the overall prevalence of environmental contamination on public streets and parks was 57,6%, including both mono (67,8%) and multi (32,2%) infections. Ancylostoma spp. was the most frequent occurrence (88,4%), followed by Trichuris vulpis. (38,8%). When evaluating the in vitro ovicidal activity, the different fungal formulations tested for each fungus were found to differ (p<0,05) from the control group, showing a relevant ovicidal effect. When the egg hatching reduction percentage was calculated, the highest reduction occurred when the crude macerate preparation was used, showing 68,43% and 47,05% MICLAB 009 and CG193 P. lilacinus and 56,43% T. harzianum reduction percentages, respectively. The crude macerate reduction percentage for the T. virens isolate (52,25%) was slightly lower than that for the filtered macerate (53,64%). The finding that 88,4% of the feces were positive for Ancylostoma spp. eggs not only reveals the high environmental contamination rates in the municipality of Pelotas, but also warns of the urgent need to implement health education and responsible dog ownership programs, as well as the need to adopt additional control measures. The evaluation of the ovicidal activity showed that, regardless of the fungal extract tested, the fungus species evaluated were effective in reducing Ancylostoma spp. egg hatchability, and thus are potential candidates for the biological control of this nematodeThe role of companion animals as reservoirs of zoonotic diseases has been recognized as significant public health problem worldwide.O papel dos animais de companhia como reservatórios de doenças zoonóticas tem sido reconhecido como significativo problema de saúde pública em todo o mundo. Ancylostoma caninum e A. braziliense são parasitos zoonóticos transmitidos ao homem pelo contato com solos contaminados com ovos e larvas destes ancilostomídeos. As consideráveis prevalências de contaminação ambiental por ovos de Ancylostoma spp., aliada as dificuldades de medidas de controle e desinfecção do solo, assim como o surgimento de resistência a terapia anti-helmíntica, evidenciam a necessidade de métodos alternativos que auxiliem no controle destes helmintos. Considerando-se o fato que os nematoides que infectam animais gastam parte do seu ciclo de vida no solo e que os fungos nematófagos, comumente encontrados nesse ecossistema, desenvolvem relações parasíticas ou predatórias com esses parasitos, desempenhando importante papel como inimigos naturais, torna-se viável e desejável o emprego destes agentes no controle eficaz de nematoides. Desta forma, os fungos nematófagos podem ser utilizados em situações em que o ambiente já está contaminado. Os objetivos do presente estudo foram verificar a prevalência de helmintos em fezes de cães coletadas em vias públicas do município de Pelotas, RS, Brasil, e avaliar a atividade ovicida in vitro de diferentes preparações de extratos brutos enzimáticos dos isolados fúngicos Paecilomyces lilacinus CG193, P. lilacinus MICLAB 009, Trichoderma virens MICLAB 008 e Trichoderma harzianum CG502 sobre ovos de Ancylostoma spp. No período de janeiro a março de 2012 foram coletadas aleatoriamente 15 amostras de fezes em 14 pontos do município, totalizando 210 amostras, as quais foram submetidas a exames coproparasitológicos. Os isolados fúngicos avaliados foram cultivados em meio mínimo líquido, em agitação, durante cinco dias a 28ºC. As preparações fúngicas consistiram do meio líquido sobrenadante sem filtração (extrato bruto) e filtrado (extrato filtrado), do micélio macerado (macerado bruto) e micélio macerado submetido a filtração (macerado filtrado). Os ovos de Ancylostoma spp. foram obtidos a partir de fezes de cães naturalmente parasitados. Os ensaios in vitro consistiram em quatro tratamentos e um grupo controle. Em placas de Petri foram vertidos 4mL de cada extrato fúngico e a esse volume foi acrescido 1mL de uma suspensão contendo aproximadamente 103 ovos de Ancylostoma spp. O grupo controle consistiu de 1mL de suspensão contendo 1000 ovos de Ancylostoma spp. acrescido de 4mL de água destilada estéril. Todas as placas foram incubadas a 25ºC, durante 24 horas. Cada ensaio foi constituído de cinco repetições. Após esse período, o número total de larvas presente em cada tratamento e no grupo controle foi contato. Os resultados obtidos demonstram que a prevalência geral de contaminação ambiental em vias públicas foi de 57,6% tanto em mono (67,8%) como em multi-infecções (32,2%). Ancylostoma spp. ocorreu em maior frequência (88,4%), seguido de Trichuris vulpis. (38,8%). Na avaliação da atividade ovicida in vitro foi observado que as diferentes formulações fúngicas testadas em cada fungo diferiram (p