Anthelmintic activity of Eucalyptus citriodora essential oil and its major component, citronellal, on sheep gastrointestinal nematodes / Atividade anti-helmíntica do óleo essencial de Eucalyptus citriodora e seu componente majoritário, citronelal, sobre nematoides gastrintestinais de ovinos

Abstract This study aimed to evaluate the anthelmintic activity of Eucalyptus citriodora essential oil and citronellal on sheep gastrointestinal nematodes. Essential oil composition was determined by gas chromatography mass spectrometry. The substances were evaluated in vitro using adult worm motility test (AWMT) and transmission electron microscopy (TEM). The acute toxicity test in mice and the fecal egg count reduction test (FECRT) in sheep were performed. Citronellal was confirmed as the essential oil major constituent (63.9%). According to the AWMT, 2 mg/mL of essential oil and citronellal completely inhibited Haemonchus contortus motility at 6 h post exposure. H. contortus exposed to essential oil and citronellal exhibited internal ultrastructural modifications. The lethal dose 50 values in mice were 5,000 and 2,609 mg/kg for essential oil and citronellal, respectively. E. citriodora essential oil reduced sheep epg at 14 days post treatment by 69.5% (P<0.05). No significant differences were observed in epg between the citronellal and negative control groups (P>0.05). The interaction between citronellal and other constituents in the essential oil may be relevant for its in vivo anthelmintic activity. Thus, E. citriodora essential oil and citronellal pharmacokinetic studies may help elucidate the anthelmintic activity of these compounds.

Resumo Este trabalho objetivou avaliar a atividade anti-helmíntica do óleo essencial de Eucalyptus citriodora e citronelal sobre nematoides gastrintestinais de ovinos. A composição do óleo essencial foi determinada por cromatografia gasosa acoplada à espectrometria de massas. As substâncias foram avaliadas in vitro utilizando-se teste de motilidade de vermes adultos (AWMT) e microscopia eletrônica de transmissão (TEM). Teste de toxicidade aguda em camundongos e teste de redução da contagem de ovos fecais (FECRT) em ovinos foram realizados. Citronelal foi confirmado como componente majoritário do óleo essencial (63,9%). No AWMT, 2 mg/mL de óleo essencial e citronelal inibiram completamente a motilidade de H. contortus 6 h pós-exposição. H. contortus expostos ao óleo essencial e citronelal exibiram modificações ultraestruturais internas. Os valores da dose letal 50 em camundongos foram 5.000 e 2.609 mg/kg para óleo essencial e citronelal, respectivamente. Óleo essencial de E. citriodora reduziu opg de ovinos 14 dias pós-tratamento em 69,5% (P<0,05). Não houve diferença significativa de opg entre grupo controle negativo e citronelal (P>0,05). A interação entre citronelal e outros constituintes do óleo essencial pode ser relevante na atividade anti-helmíntica in vivo. Portanto, avaliação farmacocinética do óleo essencial de E. citriodora e citronelal pode auxiliar a elucidar a atividade anti-helmíntica desses compostos.

Patrón espacio-temporal de larvas y huevecillos de nemátodos gastrointestinales en pastizales ganaderos de Veracruz, México / Spatio-temporal pattern of larvae and eggs of gastrointestinal nematodes in cattle pastures in Veracruz, Mexico

The spatial and temporal distribution of gastrointestinal nematodes of cattle has been little studied in Mexico. Previous studies have described periods of higher larval presence, vertical and horizontal migration in grasslands, and the frequency of adult nematodes; as well as the effect of pasture trichomes on the migration and survival of Haemonchus larvae. The aim of this study was to determine the time-space layout and spread of gastrointestinal nematode larvae on pasture, and to estimate the effect of ivermectin applied to cattle on the time-dependent abundance of their eggs in a ranch in Veracruz. To determine the spatio-temporal arrangement, monthly morning grass samples were obtained from 30 sampling points from July 2008 to June 2009. Third stage larvae (L3) from each point were counted, and aggregation patterns were estimated through variance/mean and negative binomial K indices. Additionally, the number of eggs per gram in cattle feces was determined, from samples with (CI) and without ivermectin (SI), using standard techniques. A total of 20 276L3 larvae were recovered in the pasture, of which an 80% corresponded to Haemonchus contortus. The highest nematode density with more than 5 000L3/kgDM was detected in October 2008, and the lowest in February and March 2009. The L3 showed an aggregated spatial pattern of varying intensity throughout the year. The number of eggs in the stool was not reduced with the ivermectin application to cattle, which suggested a failure of control. However, the highest parasite loads were observed from July to November 2008. We concluded that the application of ivermectin was not effective to control nematodes eggs, and that L3 populations fluctuated on pasture for ten months, providing an infection source to grazing animals afterwards. Rev. Biol. Trop. 61 (4): 1747-1758. Epub 2013 December 01.

El conocer la disposición espacio-temporal y diseminación de las larvas de nematodos gastrointestinales en los pastizales, y estimar el efecto de la ivermectina aplicada a bovinos sobre la abundancia de sus huevecillos, permite estimar dónde y cuándo se presentan las poblaciones más altas, que puede servir para establecer planes de muestreo y orientar medidas de control, así como para definir el riesgo de contaminación de los pastizales de manera diferencial. Para la determinación de la disposición espacio-temporal de L3 en el pastizal, se recolectaron, contaron e identificaron mensualmente las larvas en 30 puntos de muestreo, posteriormente se generaron mapas de disposición espacial con los datos obtenidos de los conteos de L3 en cada punto y mes de muestreo, y se calculó el patrón de disposición mediante los índices varianza/media y K binomial negativa. El número de huevecillos por gramo de heces de los bovinos con (CI) y sin ivermectina (SI), se calculó con la técnica de McMaster. En el pastizal se recuperaron 20 276L3, correspondiendo el 80% a H contortus. En octubre 2008 se detectó la más alta densidad de nematodos con más de 5 000L3/kgMS. Las L3 presentaron un patrón espacial agregado de intensidad variable durante todo el año. Las mayores densidades poblacionales de nematodos fueron en octubre 2008 y las menores en febrero y marzo 2009. La aplicación de ivermectina a los bovinos no redujo el número de huevecillos presentes en las heces, debido a que los tratamientos fueron estadísticamente iguales. De julio a noviembre 2008, se observaron las mayores cargas parasitarias.

Frequency of benzimidazole resistance in Haemonchus contortus populations isolated from buffalo, goat and sheep herds / Frequência da resistência ao benzimidazol em populações de Haemonchus contortus isoladas de rebanhos de bubalinos, caprinos e ovinos

Anthelmintic resistance is an increasing problem that threatens livestock production worldwide. Understanding of the genetic basis of benzimidazole resistance recently allowed the development of promising molecular diagnostic tools. In this study, isolates of Haemonchus contortus obtained from goats, sheep and buffaloes raised in Brazil were screened for presence of the polymorphism Phe200Tyr in the β-tubulin 1 gene, which confers resistance to benzimidazole. The allelic frequency of the mutation conferring resistance ranged from 7% to 43%, and indicated that resistance to benzimidazole could be found in nematodes isolated from all the ruminant species surveyed. Although significant variation in the frequency of the F200Y mutation was observed between different herds or host species, no significant variation could be found in populations isolated from animals within the same herd. These findings suggest that screening of samples from a few animals has the potential to provide information about the benzimidazole resistance status of the entire herd, which would enable a considerable reduction in the costs of diagnosis for the producer. Molecular diagnosis has practical advantages, since it can guide the choice of anthelmintic drug that will be used, before its application in the herd, thus reducing the economic losses driven by anthelmintic resistance.

A resistência aos anti-helmínticos é um problema crescente que ameaça a produção pecuária em todo o mundo. A compreensão da base genética da resistência ao benzimidazol permitiu, recentemente, o desenvolvimento de métodos diagnósticos moleculares promissores. Neste estudo, isolados de Haemonchus contortus obtidos a partir de rebanhos de caprinos, ovinos e bubalinos criados no Brasil foram avaliados quanto à presença do polimorfismo F200Y no gene da β-tubulina1, o qual confere resistência ao benzimidazol. A frequência alélica da mutação variou de 7% a 43%, indicando que a resistência ao benzimidazol pode ser encontrada em nematoides isolados a partir de todas as espécies de ruminantes pesquisadas. Embora tenha sido observada variação significativa das frequências de mutação F200Y entre rebanhos/espécies hospedeiros distintos, não foi encontrada variação significativa entre populações isoladas de animais dentro de um mesmo rebanho. Estes achados sugerem que a avaliação de amostras de alguns poucos animais tem o potencial de fornecer informações sobre o nível de resistência ao benzimidazol de todo o rebanho, possibilitando uma redução considerável dos custos de diagnóstico para o produtor. O diagnóstico molecular apresenta vantagens práticas, uma vez que pode guiar a escolha da base anti-helmíntica a ser utilizada antes da sua aplicação no rebanho, reduzindo, portanto, as perdas ocasionadas pela resistência aos fármacos anti-helmínticos.

Cloning and characterization of a selenium-independent glutathione peroxidase (HC29) from adult Haemonchus contortus

The complete coding sequence of Haemonchus (H.) contortus HC29 cDNA was generated by rapid amplification of cDNA ends in combination with PCR using primers targeting the 5'- and 3'-ends of the partial mRNA sequence. The cloned HC29 cDNA was shown to be 1,113 bp in size with an open reading frame of 507 bp, encoding a protein of 168 amino acid with a calculated molecular mass of 18.9 kDa. Amino acid sequence analysis revealed that the cloned HC29 cDNA contained the conserved catalytic triad and dimer interface of selenium-independent glutathione peroxidase (GPX). Alignment of the predicted amino acid sequences demonstrated that the protein shared 44.7~80.4% similarity with GPX homologues in the thioredoxin-like family. Phylogenetic analysis revealed close evolutionary proximity of the GPX sequence to the counterpart sequences. These results suggest that HC29 cDNA is a GPX, a member of the thioredoxin-like family. Alignment of the nucleic acid and amino acid sequences of HC29 with those of the reported selenium-independent GPX of H. contortus showed that HC29 contained different types of spliced leader sequences as well as dimer interface sites, although the active sites of both were identical. Enzymatic analysis of recombinant prokaryotic HC29 protein showed activity for the hydrolysis of H2O2. These findings indicate that HC29 is a selenium-independent GPX of H. contortus.

Haemonchus contortus-sheep relationship: a review

La hemoncosis, causada por el nematodo del abomaso de rumiantes Haemonchus contortus, constituye una de las enfermedades parasitarias más notables del ganado ovino en todo el mundo. Las infecciones provocan síndromes anémicos y de mala digestión/absorción que pueden causar la muerte en los casos agudos y disminución de la producción en las formas crónicas. Las claves principales para la aparición de esta enfermedad son el comportamiento biológico del helmito, su patogenia, además de la respuesta del hospedador. El conocimiento actualizado de estos aspectos permitirá una mayor eficaciencia de los métodos de diagnóstico y contro del proceso y, como consecuencia, la disminución de los riesgos de aparición de esta enfermedad. La presente revisión analiza y discute algunos aspectos de la relación ganado ovino-H. contortus de interés para disminuir el impacto de esta parasitosis sobre la salud y producción ganaderas.

Haemonchosis, caused by the abomasal nematode Haemonchus contortus, is among the most relevant parasitic diseases of small ruminants all over the world. The infections are responsible for anemic and bad digestion/absorption syndromes causing the death of severely infected animals in acute cases and the reduction of production scores in the chronic forms of the disease. The keys for the appearance of the disease include the biological behavior of the helminth and mechanisms of pathogenesis, besides the host’s response. The updated knowledge of these aspects would result in a higher efficiency of diagnostic and control methods, thus reducing the risks of appearance of the disease. In the present review, several aspects of the sheep - H. contortus relationship are analyzed with the aim of reducing the impact of this parasitosis on livestock health and productivity.

Characterization of HC58cDNA, a putative cysteine protease from the parasite Haemonchus contortus

Because of the complexity of the cathepsin B-like (CBL) family, an information on the biological and biochemical characteristics of individual CBL genes is lacking. In this study, we investigated the degradative effects of the recombinant HC58 protein isolated from Haemonchus contortus parasites on protein substrates over a broad pH range in vitro. This protein, which hydrolyzed the synthetic peptide substrates Z-FR-AMC and Z-RR-AMC, had characteristics of the cysteine protease class of proteins. In the acidic pH range, the isolated protein actively degraded hemoglobin (Hb), the heavy chain of goat immunoglobulin G, and azocasein. By contrast, it degraded fibrinogen in the alkaline pH range. These activities were strongly inhibited in the presence of the cysteine protease inhibitor E-64. While the protein digested Hb, it did not induce the agglutination of erythrocytes from its natural host. These results suggest that the HC58 protein may play a role in the nutrition of this parasite.

Parasitological characteristics and tissue response in the abomasum of sheep infected with Haemonchus spp

A histopathological study was conducted on the abomasal mucosa of 40 Corriedale sheep exposed to natural infection with Haemonchus spp. The sheep were allowed to graze on contaminated pastures for 14 days and, after being housed for 28 days, they were slaughtered. Fecal samples were collected for fecal egg counts (FEC) and abomasum samples were obtained for histopathological examination and eosinophil, mast cell and globule leucocyte counts. The number of Haemonchus spp. present in the abomasum was estimated from a 10 per cent aliquot of its content. The number of eggs per female was estimated for 10 Haemonchus spp. females collected from each animal. The following significant correlation coefficients were obtained between the characteristics analyzed: number of Haemonchus and FEC (r = 0.86), female length and number of eggs per female (r = 0.60), female length and FEC (r = 0.53), number of eosinophils and number of Haemonchus (r = 0.48), number of eosinophils and number of globule leucocytes (r = 0.54), number of mast cells and female length (r = -0.39), and number of mast cells and number of globule leucocyte (r = 0.34). The characteristics that showed the highest correlation with animal load was FEC

A possible role of caesium radiations in the control of propagation of hookworm.

Faeces charcoal mixtures containing eggs of sheep hookworm, Haemonchus contortus were exposed to caesium radiations (23 r to 1656 r) from 1st to 7th day of incubation. Each plate was exposed only once. A dose related inhibition of transformation of eggs to infective larvae was observed. The maximum effect was observed on 2nd day of incubation. The radiation has maximum effect on the 1st stage larvae followed by 2nd stage. The 3rd stage or ineffective larvae are relatively resistant to the effect of radiation.