Carvacryl acetate nanoencapsulated with chitosan/chichá gum exhibits reduced toxicity in mice and decreases the fecal egg count of sheep infected with gastrointestinal nematodes.

The nanoencapsulation of biocomposites with anthelmintic action has been proposed as an alternative for improving their efficiency. Thus, the current study aimed to evaluate the efficacy of carvacryl acetate nanoencapsulated with biopolymers (nCVA) in the control of sheep gastrointestinal nematodes. CVA was nanoencapsulated with chitosan/chichá gum and characterized in terms of its efficacy of encapsulation (EE), yield and zeta potential. The acute toxicity of nCVA was evaluated in mice. For the fecal egg count reduction test, 40 animals were divided into four groups (n = 10) and orally administered the following treatments: G1, 250 mg kg−1 CVA; G2, 250 mg kg−1 nCVA; G3, chitosan/chichá gum (negative control) and G4, 2.5 mg kg−1 monepantel (positive control). Feces were collected on days 0 and 16 posttreatment to determine the eggs per gram of feces (epg). The EE and yield of nCVA were 72.8 and 57.5%, respectively. The nanoparticles showed a size of 764.5 ± 302.5 nm, and the zeta potential at pH 3.2 was +22.0 mV. nCVA presented a 50% lethal dose (LD50) of 2609 mg kg−1. By 16 days posttreatment, CVA, nCVA and monepantel reduced the epg by 52.9.7, 71.5 and 98.7%, respectively, and the epg of sheep treated with nCVA differed from that of the negative control (P > 0.05) but did not differ from that of sheep treated with CVA. In conclusion, the nanoencapsulation of CVA reduced its toxicity, and nCVA showed anthelmintic activity.

Chemical composition and in vitro activity of Calotropis procera (Ait.) latex on Haemonchus contortus.

Calotropis procera is among the species of medicinal plants that have traditionally been used for the treatment of parasites in small ruminants, stimulating the scientific validation of anthelmintic effects. This study aimed to investigate the chemical composition of ethyl acetate extract of Calotropis procera latex (EAECPL), assess the in vitro effect against Haemonchus contortus and the structural changes caused in the adult worm. The latex was collected, lyophilized and subjected to washing with the ethyl acetate solvent to obtain EAECPL. The constituents of the extract were isolated by column chromatography and identified by (13)C and (1)H nuclear magnetic resonance spectroscopy. The egg hatching test (EHT), larval development test (LDT) and adult worms motility test (WMT) were conducted to evaluate the effectiveness of EAECPL on eggs, larvae and adult of H. contortus, respectively. The worms obtained from the WMT, after 24h exposure to EAECPL or controls were observed on a scanning electron microscope (SEM). The results were analysed by variance analysis and compared with Tukey's test (P<0.05). Three compounds were isolated from EAECPL and identified as urs-19(29)-en-3-yl acetate, (3ß)-Urs-19(29)-en-3-ol, and 1-(2',5'-dimethoxyphenyl)-glycerol. In the EHT, EAECPL inhibited larval hatching by 91.8% at dose of 4mg/ml. In the LDT 1mg/ml inhibited 99.8% larval development. In the WMT, EAECPL in the concentration of 100µg/ml inhibited 100% motility of worms, 12h post-exposition. In the SEM, obvious differences were not detected between the negative control worms and the worms treated with EAECPL. In this study, EAECPL showed an effect on inhibition egg hatching, larval development and motility of the adult worms of H. contortus. This should be related both to the identified compounds, as well as the other compounds present in the EAECPL, acting alone or synergistically.

Comparative efficacy and toxic effects of carvacryl acetate and carvacrol on sheep gastrointestinal nematodes and mice.

Carvacrol is a compound isolated from some essential oils. It has been reported to possess anthelmintic activity. Acetylation of this monoterpene has been proposed as a potential way to reduce the toxicity and enhance the pharmacological effects of carvacrol. This study aimed to evaluate the effect of carvacryl acetate (CA) using in vitro and in vivo assays with gastrointestinal nematodes of small ruminants. The egg hatching test (EHT), larval development test (LDT) and adult worm motility (AWM) assessment were conducted to evaluate the effect of the acetylated product and pure carvacrol on Haemonchus contortus eggs, larvae and adults. The structural changes induced in adult H. contortus were assessed using scanning electron microscopy (SEM). CA and carvacrol acute toxicity was evaluated in mice. Finally, the efficacy of 250 mg/kg CA and 2.5mg/kg monepantel (positive control) were evaluated in 30 sheep naturally infected with gastrointestinal nematodes by the fecal egg count reduction test (FECRT). In vitro tests were analyzed by analysis of variance (ANOVA) followed by comparison with Tukey's test. The efficacy was calculated by the Boot Street program using the arithmetic average. The number of eggs in feces (epg) of the groups were transformed to log (x+1) and subjected to ANOVA to compare differences among the groups by Tukey's test. The level of significance was P<0.05. CA and carvacrol inhibited larval hatching by 89.3 and 97.7% at doses of 8.0 and 1.0mg/ml, respectively. At the concentration of 2mg/ml, CA and carvacrol inhibited 100% of larval development. At a concentration of 200 µg/ml, CA and carvacrol inhibited the motility of adult worms by 100% and 58.3% at 24h post-exposure, respectively. CA caused cuticle and vulvar flap wrinkling and bubbles to emerge from the tegument. Carvacrol caused more discreet effects on the cuticle and vulvar flap. The LD10 and LD50 of CA were 566.7 mg/kg and 1544.5mg/kg, respectively. The LD10 and LD50 of carvacrol were 546.8 mg/kg and 919 mg/kg, respectively. CA and monepantel reduced the epg of sheep by 65.9 and 96.4%, respectively, at 16 days post-treatment. CA showed in vitro and in vivo anthelmintic activity and was less toxic than carvacrol.

In vitro activity of Lantana camara, Alpinia zerumbet, Mentha villosa and Tagetes minuta decoctions on Haemonchus contortus eggs and larvae.

The resistance of gastrointestinal nematodes to anthelmintics has increased the need to evaluate natural products that can replace or assist current strategies to control gastrointestinal nematodes. The objective of this study was to evaluate the effect of decoctions of Lantana camara (DLc), Alpinia zerumbet (DAz), Mentha villosa (DMv) and Tagetes minuta (DTm) on Haemonchus contortus by two in vitro tests. The effects of increasing concentrations of lyophilized decoctions (0.31 to 10mg/ml) were assessed using the egg hatch test (EHT). The decoctions were then tested in the larval artificial exsheathment assay. H. contortus third stage larvae (L3) were exposed to 0.31 mg/ml A. zerumbet and M. villosa decoctions and 0.62 mg/ml T. minuta and L. camara decoctions for 3h and then exsheathment procedure at 10 min intervals. An inhibitor of tannins, polyvinyl polypyrrolidone (PVPP), was used to study if tannins were responsible for the inhibitory effect on hatching and exsheathment of larvae. A. zerumbet, M. villosa and T. minuta showed a dose-dependent effect in the EHT, which did not disappear after the addition of PVPP. No effect was observed for L. camara in the EHT. However, the decoctions inhibited the process of larval exsheathment, which may be related to tannin action because the addition of PVPP reversed the inhibitory effect. A. zerumbet, M. villosa and T. minuta decoctions showed inhibitory activity on H. contortus larvae hatching and exsheathing. The decoctions of these plants could be used to control gastrointestinal nematodes following confirmation of their anthelmintic activity in vivo.

The use of Eucalyptus staigeriana nanoemulsion for control of sheep haemonchosis / Uso da nanoemulsão de Eucalyptus staigeriana no controle da hemoncose em ovinos

Sustainable control of gastrointestinal nematodes (GIN) in small ruminants has been based on the use of alternative methods, including targeted selective treatment, such as FAMACHA. Another GIN control alternative is the use of herbal medicines, although in many cases their use is based on empirical knowledge. Biopolymer nanoformulations has been investigated to maximize the essential oil effects against sheep gastrointestinal nematodes. The aim of the present study was to combine a Eucalyptus staigeriana essential oil nanoemulsion (EsNano) with FAMACHA as an alternative control for sheep haemonchosis. The study was performed over six months at a commercial sheep farm located in a semiarid region of Northeast Brazil. Initially, a fecal egg count reduction test (FECRT) in sheep with levamisole, ivermectin and oxfendazole in sheep was performed used to determine the most effective anthelmintic to use as the positive control. Levamisole has been selected because it showed efficacy superior to 95%. EsNano was obtained and then its physicochemical properties were characterized. The average (±SE) size of the particles in the nanoemulsion was 276.8 (±12.3) nm with bimodal distribution and polydispersity. Nine visits were performed, from April to September 2013, with an interval of 17 days. One hundred sixty-two male and female sheep were divided into three groups (n=54 each) and were treated when FAMACHA score was 3, 4, or 5: G-EsNano 250mg kg-1 EsNano; G-Lev 7.5mg kg-1 levamisole (positive control), and G-Neg was not treated (negative control). Feces from sheep were collected to quantify the number of eggs per gram of feces (epg) and to identify nematode genera. Sheep weight gain was monitored. The epg data for each group and the average sheep weight gains were analyzed by variance analysis and compared with the Tukey's test (P<0.05). Significant difference between the number of animals treated with EsNano and levamisole was not observed in any visit (P>0.05). The epg variation was similar in the G-EsNano and G-Lev groups on visits (P>0.05), except the second and fifth evaluation in the epg groups were significantly different (P<0.05). Haemonchus spp. was the most prevalent nematode. There was no significant weight gain in any of the treated groups (P<0.05). The combination of phytotherapy and FAMACHA can be an alternative to minimize the use of synthetic anthelmintics to control resistant GIN populations of small ruminants.(AU)

O controle sustentável de nematoides gastrintestinais (NGI) em pequenos ruminantes tem sido baseado na utilização de métodos alternativos, incluindo o tratamento alvo-seletivo, tal como o FAMACHA. Outra alternativa de controle de NGI é o uso de plantas medicinais, embora, em muitos casos, a sua utilização seja baseada no conhecimento empírico. Nanoformulações biopoliméricas tem sido investigadas para maximizar os efeitos de óleos essenciais sobre nematoides gastrointestinais em ovinos. O objetivo do presente estudo foi combinar a nanoemulsão do óleo essencial de Eucalyptus staigeriana (EsNano) com o método FAMACHA como uma alternativa para o controle da hemoncose em ovinos. Este estudo foi realizado ao longo de seis meses em uma fazenda comercial de ovinos localizada em uma região semiárida do Nordeste do Brasil. Inicialmente, um teste de redução da contagem de ovos nas fezes (FECRT) em ovinos com levamisol, ivermectina e oxfendazole foi realizado para determinar o anti-helmíntico mais eficaz, para posterior uso como controle positivo. Levamisol foi selecionado porque mostrou eficácia superior a 95%. EsNano foi obtido e, em seguida, as suas propriedades físico-químicas foram caracterizadas. O tamanho médio (±SE) das partículas na nanoemulsão foi 276,8 (±12,3) nm, com distribuição bimodal e polidispersividade. Foram realizadas nove visitas, de abril a setembro de 2013, com um intervalo de 17 dias. Cento e sessenta e dois ovinos machos e fêmeas foram divididos em três grupos (n=54 cada) e foram tratados quando o FAMACHA foi 3, 4 ou 5: G-EsNano 250 mg kg-1 EsNano; G-Lev 7,5mg kg-1 de levamisol (controlo positivo), e G-Neg não foi tratado (controle negativo). As fezes dos ovinos foram coletadas para quantificar o número de ovos por grama de fezes (opg) e identificar os gêneros de nematóides. O ganho de peso de ovinos foi monitorado. Os dados do opg de cada grupo e os ganhos de peso médio dos ovinos foram analisados por análise de variância e comparadas com o teste de Tukey (P<0,05). Diferença significativa entre o número de animais tratados com EsNano e levamisol não foi observada em nenhuma visita (P>0,05). A variação opg foi semelhante para os grupos G-EsNano e G-Lev nas vistas (P>0,05), excetuando a segunda e quinta avaliação em que os opg dos grupos foram significativamente diferentes (P>0,05). O nematoide Haemonchus spp. foi o mais prevalente. Não houve aumento significativo de peso em qualquer um dos grupos tratados (P>0,05). Assim, a combinação de fitoterapia e FAMACHA pode ser uma alternativa para minimizar o uso anti-helmínticos sintéticos para controlar populações resistentes NGI em pequenos ruminantes.(AU)

Anthelmintic activity of Jatropha curcas L. seeds on Haemonchus contortus.

The aim of this study was to evaluate the anthelmintic activity of hexane (HE), ethyl acetate (EA) and ethanol (EE) extracts obtained from the seeds of Jatropha curcas using the egg hatch inhibition assay (EHA) and the artificial larval exsheathment inhibition assay (LEIA). For the egg hatch assay, HE, EA and EE were used in concentrations of 3.12, 6.25, 12.5, 25 and 50 mg ml(-1), accompanied by a negative control (5% Tween 80) and a positive control (0.025 g ml(-1) thiabendazole). In LEIA, the extracts were tested at a concentration of 1000 µg ml(-1), accompanied by a negative control (PBS). To evaluate the effect of tannins, the extract with the greatest effect was incubated with polyvinyl polypyrrolidone (PVPP). The EE (50 mg ml(-1)) inhibited 99.8% of egg hatching. After the addition of PVPP, the ovicidal effectiveness of EE was reduced to 91.9%. Using the HE and EA, inhibition of egg hatching was 15.3% and 32.2%, respectively. In the LEIA, 18.9% of L3 incubated with EE were exsheathed (p<0.01). The addition of PVPP to EE reversed the inhibitory effect on larval exsheathment. The percentage of exsheathment of L3 incubated with HE (99.6%) and EA (97.8%) did not differ from the control group (p>0.05). The results show that the effects of EE on eggs are not solely due to the tannins. However, these secondary metabolites are implicated in blocking the larval exsheathment.