Purpureocillium lilacinum and Trichoderma virens for biological control of trichostrongylid parasites of sheep: an in vitro evaluation / Purpureocillium lilacinum e Trichoderma virens no controle biológico de tricostrongilídeos de ovinos: uma avaliação in vitro

Abstract 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.

Resumo O estudo avaliou a atividade ovicida de extratos enzimáticos de Purpureocillium lilacinum e Trichoderma virens sobre ovos de tricostrongilídeos de ovinos. Extrato filtrado (EF) e extrato macerado bruto (EMB) foram preparados a partir de culturas fúngicas em caldo mínimo. No ensaio experimental, 100 ovos de tricostrongilídeos, obtidos de fezes de ovinos naturalmente infectados, foram expostos durante 24 e 48 horas/25ºC aos extratos dos fungos. No grupo controle, os ovos foram incubados em caldo mínimo. O número de larvas L1 foi determinado. Cada tratamento consistiu em quatro repetições e o experimento foi repetido cinco vezes. Observou-se que o efeito ovicida do EF e EMB de P. lilacinum e T. virens diferiu do grupo controle. O EMB de T. virens e P. lilacinum evidenciou atividade ovicida superior ao EF em ambos os períodos avaliados e em 48 horas de exposição, respectivamente. O percentual de redução de eclodibilidade evidenciou que o EMB foi superior ao EF em ambos os fungos. Este estudo demonstra o potencial ovicida desses fungos sobre ovos de tricostrongilídeos. No entanto, estudos adicionais são necessários para identificar as moléculas responsáveis pelo efeito ovicida, bem como avaliar o comportamento dos extratos fúngicos em interações bióticas e abióticas.

Proteomic analysis of Mecistocirrus digitatus and Haemonchus contortus intestinal protein extracts and subsequent efficacy testing in a vaccine trial.

BACKGROUND: Gastrointestinal nematode infections, such as Haemonchus contortus and Mecistocirrus digitatus, are ranked in the top twenty diseases affecting small-holder farmers' livestock, yet research into M. digitatus, which infects cattle and buffalo in Asia is limited. Intestine-derived native protein vaccines are effective against Haemonchus, yet the protective efficacy of intestine-derived M. digitatus proteins has yet to be determined. METHODOLOGY/PRINCIPAL FINDINGS: A simplified protein extraction protocol (A) is described and compared to an established method (B) for protein extraction from H. contortus. Proteomic analysis of the H. contortus and M. digitatus protein extracts identified putative vaccine antigens including aminopeptidases (H11), zinc metallopeptidases, glutamate dehydrogenase, and apical gut membrane polyproteins. A vaccine trial compared the ability of the M. digitatus extract and two different H. contortus extracts to protect sheep against H. contortus challenge. Both Haemonchus fractions (A and B) were highly effective, reducing cumulative Faecal Egg Counts (FEC) by 99.19% and 99.89% and total worm burdens by 87.28% and 93.64% respectively, compared to the unvaccinated controls. There was no effect on H. contortus worm burdens following vaccination with the M. digitatus extract and the 28.2% reduction in cumulative FEC was not statistically significant. However, FEC were consistently lower in the M. digitatus extract vaccinates compared to the un-vaccinated controls from 25 days post-infection. CONCLUSIONS/SIGNIFICANCE: Similar, antigenically cross-reactive proteins are found in H. contortus and M. digitatus; this is the first step towards developing a multivalent native vaccine against Haemonchus species and M. digitatus. The simplified protein extraction method could form the basis for a locally produced vaccine against H. contortus and, possibly M. digitatus, in regions where effective cold chains for vaccine distribution are limited. The application of such a vaccine in these regions would reduce the need for anthelmintic treatment and the resultant selection for anthelmintic resistant parasites.

Effectiveness of Asteraceae extracts on Trichostrongylidae eggs development in sheep / Eficácia de extratos da família Asteraceae no desenvolvimento dos ovos de Trichostrongilídeos de ovinos

Data on in vitro evaluation of extracts of three species of the Asteraceae family on the development of Trichostrongylidae eggs in sheep are presented. Egg hatchability was tested using herbal extracts prepared in a Soxhlet extractor, and using hydrolate prepared by means of hydrodistillation. The laboratory tests showed that the ethanol extract from flowers of the species Aster lanceolatus presented high activity against Trichostrongylidae eggs development in sheep, inhibiting larva formation by 91 percent within 48 hours, and maintaining similar rates after 72 hours.

Apresentam-se dados da avaliação in vitro de três espécies vegetais da família Asteraceae sobre o desenvolvimento dos ovos de Trichostrongilídeos de ovinos. Realizou-se o teste de eclodibilidade com extratos vegetais preparados por aparelho de Soxhlet e hidrolato preparado por hidrodestilação. Os testes laboratoriais evidenciaram que o extrato etanólico das flores da espécie Aster lanceolatus apresenta alta atividade sobre o desenvolvimento dos ovos de Trichostrongilídeos de ovinos, inibindo em 91 por cento a formação da larva em 48 horas, mantendo-se índices próximos em 72 horas.

Atividade in vitro do extrato hidroalcoólico de Lippia sidoides Cham sobre larvas de terceiro estádio de nematódeos gastrintestinais (família Trichostrongylidae) de caprinos / In vitro activity of the hydroalcoholic extract of Lippia sidoides Cham on buoyant larvae of third-stage gastrointestinal nematodes (family Trichostrongylidae) of goats

Avaliou-se in vitro a atividade larvicida do extrato hidroalcóolico de alecrim pimenta (Lippia sidoides Cham) sobre larvas de terceiro estádio de nematoides gastrintestinais de caprinos. Os resultados encontrados demonstraram que a concentração de 500 mg mL-1 apresentou atividade efetiva e com capacidade de ação de 95,89%, demonstrando desta forma a possibilidade do uso terapêutico anti-helmíntico para esse extrato.

The in vitro larvicidal activity of the hydroalcoholic extract of rosemary pepper (Lippia sidoides Cham) was evaluated on buoyant larvae of third-stage gastrointestinal gastrointestinal nematodes of goats. The results demonstrated that the concentration of 500 mg mL-1 presented effective activity, with a capacity of action of 95.89%, thus demonstrating the possibility of anthelmintic therapeutic use for this extract.

Vaccination of young lambs against infection with Nematodirus battus using gamma irradiated larvae.

Helminthologically naIve 6-week-old Suffolk lambs were given 1-3 doses of 20000 gamma-irradiated infective larvae (L3) of the nematode Nematodirus battus at weekly intervals. Following an anthelmintic drench they were challenged with 50000 viable L3 at 10 weeks of age. Nematode worm burdens 14 days post-challenge showed a significant (P<0.01) 66% reduction in the single vaccine dose group. The two and three dose groups had mean worm burdens which were 30 and 42% lower than controls, respectively, although these were not statistically significant. There was little measurable stimulation of the immune system in the vaccinated lambs, suggesting that the repeatedly dosed animals may have developed immunological unresponsiveness to the parasite.

Rejection of heterologous nematodes by sheep immunized with larval or adult Trichostrongylus colubriformis.

Sheep immunized by truncated larval infections or by the adoptive transfer of adult Trichostrongylus colubriformis were subsequently challenged with single infections of T. colubriformis, Nematodirus spathiger, Haemonchus contortus or Ostertagia circumcincta or combinations of the parasites. Sheep vaccinated with larval infections were > 90% protected by 4 days after challenge (DAC) against T. colubriformis L3 given in a single or combined infection, whereas no significant protection was exhibited against a single-species infection with the unrelated nematodes N. spathiger or O. circumcincta. Sheep challenged with T. colubriformis together with N. spathiger or O. circumcincta were equally protected against both intestinal nematodes, but O. circumcincta was not affected. Sheep immunized with adult T. colubriformis and challenged with a combined infection of T. colubriformis, N. spathiger and H. contortus expelled around 40 and 80% of the intestinal parasites by 4 and 11 DAC, respectively, but showed no protection against the abomasal parasite, H. contortus. The results confirm the previous findings on 'self-cure' and the non-specific rejection of unrelated parasites living in the same or downstream niches in the gut when the nematode used to induce immunity is included in the challenge infection. The results also indicate that even though L3 antigens effectively elicited the non-specific rejection, antigens produced by L4 and later stages could also induce rejection of unrelated worms in the second week after infection.