Combined effects of Limonene and Ivermectin on P-glycoprotein-9 gene expression of lambs Infected with Haemonchus contortus.

Although ivermectin (IVM) has a wide spectrum and long half-life, its frequent use as an anthelmintic for the last 42 years led to its worldwide tolerance by Haemonchus contortus. We evaluated the combination of limonene (LIM), a P-glycoprotein (Pgp) modulator, with IVM in lambs infected with a multidrug-resistant H. contortus. Twenty-four male Dorper lambs were artificially infected with two doses (seven days apart) of 8000 infective larvae of a multidrug-resistant isolate of H. contortus. The infection was patent 25 days later. Fifteen days before treatment with IVM (DAY -15), animals were divided into 4 groups: Infected-untreated control (CTL), IVM, LIM, and LIM+IVM. From DAY -15 to DAY + 14, groups LIM and LIM+IVM received 200 mg/kg of body weight/day of LIM via oral. On DAY 0, a single dose of IVM at 200 µg/kg of body weight was administered orally to groups IVM and LIM+IVM. On DAY + 7 and DAY + 14, fecal egg counts (FEC) were performed and on DAY + 14 animals were euthanized for total worm count (TWC), worm length, fecundity of females, and Pgp-9 gene expression. On DAY + 7, group LIM+IVM had 96.29% efficacy based on Fecal Egg Count Reduction TEST (FECRT) and a highly significant reduction in FEC (P = 0.0005) when compared to CTL. On DAY + 14, the efficacy of LIM+IVM was 82.87% on FECRT, although no differences were found among groups for FEC, TWC, worm length, or Pgp-9 gene expression. Female worms from the CTL group had higher egg counts in their uterus when compared to LIM. No differences were found for hematological or biochemical parameters, body weight, or weight gain among groups. Thus, LIM given daily at 200 mg/kg was safe for animals and, when combined with IVM, decreased egg shedding and could reduce pasture contamination, although it was unable to kill multidrug-resistant H. contortus.

Evaluation of encapsulated anethole and carvone in lambs artificially- and naturally-infected with Haemonchus contortus.

Molecules from natural sources, such as essential oils, have shown activity against parasites in vitro, but have not yet been explored extensively in vivo. Anethole and carvone (10% each), encapsulated with 80% of a solid matrix, referred to as EO (encapsulated oils), were tested in vivo in 2 experiments. In Experiment 1: Lambs were artificially infected with multidrug resistant Haemonchus contortus, or left uninfected, and treated (or not) with 50 mg/kg bw (body weight) of EO in a controlled environment. Thirty-two male lambs were kept in individual cages for a period of 45 days, after which animals were evaluated for parasitological, hematological, toxicological, and nutritional parameters. After 45 days of treatment, EO at 50 mg/kg bw provided a significant (P ≤ 0.05) reduction in fecal egg count (FEC). Although FEC was reduced, animals from both treatments had similar counts of total adult worms. The low FEC was caused probably by a significant reduction (P ≤ 0.05) in both male worm size and female fecundity. Dry matter intake of uninfected controls was significantly (P ≤ 0.05) reduced, although no toxicity was observed in treated animals. Thus, in Experiment 2, conducted for five months we used an EO dose of 20 mg/kg bw. Thirty-four weaned lambs, free of parasites, were divided in two groups and kept in collective pens. One group received EO at 20 mg/kg bw mixed with concentrate for 5 months and the other was kept as a control group (CTL). Parasitological and hematological parameters as well as body weight were evaluated. In the first 2.5 months, CTL and EO groups were confined, and both presented similar clinical parameters. Then, animals were allotted to graze on contaminated pastures to acquire natural infection for the next 2.5 months. The infection was patent after 25 days and both groups had similar decreases in weight gain, increases in FEC, and decreases in blood parameters. Coprocultures from CTL and EO groups established that parasite population was 90% Haemonchus sp. We concluded that the technology of encapsulation is safe and practical to deliver to lambs at the farm level and anethole and carvone at 50 mg/kg bw caused a significant decrease in FEC and, consequently, in pasture contamination by free living stages of H. contortus. However, EO at 20 mg/kg bw was not effective to prevent or treat sheep naturally-infected with gastrointestinal nematodes.

Inclusion complex and nanoclusters of cyclodextrin to increase the solubility and efficacy of albendazole.

Albendazole (ABZ), a benzimidazole widely used to control gastrointestinal parasites, is poorly soluble in water, resulting in variable and incomplete bioavailability. This has favored the appearance ABZ-resistant nematodes and, consequently, an increase in its clinical ineffectiveness. Among the pharmaceutical techniques developed to increase drug efficacy, cyclodextrins (CDs) and other polymers have been extensively used with water-insoluble pharmaceutical drugs to increase their solubility and availability. Our objective was to prepare ABZ formulations, including ß-cyclodextrin (ßCD) or hydroxypropyl-ß-cyclodextrin (HPßCD), associated or not to the water-soluble polymer polyvinylpyrrolidone (PVP). These formulations had their solubility and anthelmintic effect both evaluated in vitro. Also, their anthelmintic efficacy was evaluated in lambs naturally infected with gastrointestinal nematodes (GIN) through the fecal egg count (FEC) reduction test. In vitro, the complex ABZ/HPßCD had higher solubility than ABZ/ßCD. The addition of PVP to the complexes increased solubility and dissolution rates more effectively for ABZ/HPßCD than for ABZ/ßCD. In vivo, 48 lambs naturally infected with GIN were divided into six experimental groups: control, ABZ, ABZ/ßCD, ABZ/ßCD-PVP, ABZ/HPßCD, and ABZ/HPßCD-PVP. Each treated animal received 10 mg/kg of body weight (based on the ABZ dose) for three consecutive days. After 10 days of the last administered dose, treatment efficacy was calculated. The efficacy values were as follows: ABZ (70.33%), ABZ/ßCD (85.33%), ABZ/ßCD-PVP (82.86%), ABZ/HPßCD (78.37%), and ABZ/HPßCD-PVP (43.79%). In vitro, ABZ/HPßCD and ABZ/HPßCD-PVP had high solubility and dissolution rates. In vivo, although the efficacies of ABZ/ßCD, ABZ/ßCD-PVP, and ABZ/HPßCD increased slightly when compared to pure ABZ, this increase was not significant (P > 0.05).

Terminalia catappa: Chemical composition, in vitro and in vivo effects on Haemonchus contortus.

Haemonchus contortus is the most important nematode in small ruminant systems, and has developed tolerance to all commercial anthelmintics in several countries. In vitro (egg hatch assay) and in vivo tests were performed with a multidrug strain of Haemonchus contortus using Terminalia catappa leaf, fruit pulp, and seed extracts (in vitro), or pulp and seed powder in lambs experimentally infected with H. contortus. Crude extracts from leaves, fruit pulp and seeds obtained with 70% acetone were lyophilized until used. In vitro, the extracts had LC50=2.48µg/mL (seeds), LC50=4.62µg/mL (pulp), and LC50=20µg/mL (leaves). In vitro, seed and pulp extracts had LC50 similar to Thiabendazole (LC50=1.31µg/mL). Condensed tannins were more concentrated in pulp extract (183.92g of leucocyanidin/kg dry matter) than in either leaf (4.6g) or seed (35.13g) extracts. Phytochemical tests established that all extracts contained alkaloids, flavonoids, saponins, phenols, and terpenoids. Based on these results, in vivo tests were performed to evaluate the anthelmintic activity of T. catappa whole fruit (pulp+seed) powder. Male Santa Ines lambs were artificially infected with multidrug-resistant H. contortus and divided, according to similar fecal egg count (FEC) and weight, into two groups: Control (infected/untreated) and treated (infected/treated with whole fruit powder). Whole fruit powder was mixed with concentrate and provided at 2g/kg of body weight (BW) for five days. After treatment, parasitological analysis (FEC and egg hatch assay), renal profile (urea and creatinine), liver profile (aspartate aminotransferase) and BW were determined. In vitro (based on LC50), seed/pulp extracts had ovicidal effect similar to Thiabendazole but whole fruit powder had no anthelmintic effect on adult nematodes in the abomasum. We discuss the plausible causes of the lack of in vivo activity.

Synergistic interaction of ten essential oils against Haemonchus contortus in vitro.

Anthelmintic resistance in sheep gastrointestinal nematodes is a worldwide problem. Multi-drug resistant haemonchosis is the most serious impediment for small ruminant systems, and there are no new drug candidates currently under development. Molecules from natural sources have demonstrated anthelmintic activity against parasites. In this work, the monoterpenoids carvacrol, carvone, cineole, linalool, limonene, and thymol and the phenylpropanoids cinnamaldehyde, anethole, vanillin, and eugenol were assessed individually or in mixtures of ten binary, three ternary, and three quaternary combinations using the in vitro egg hatch assay with eggs of a multi-drug resistant strain of Haemonchus contortus. The main objective of this study was to identify the most effective interaction among essential oils with the greatest individual anthelmintic efficacy and to determine the most powerful combinations. The essential oils were ranked by their 50% lethal concentration (LC50) as follows (mg/mL): cinamaldehyde (0.018), anethole (0.070), carvone (0.085), carvacrol (0.11), thymol (0.13), linalool (0.29), vanillin (0.57), eugenol (0.57), cineole (4.74), and limonene (207.5). Quantification of synergism, additive effect, and antagonism were calculated for binary, ternary, and quaternary combinations. The best anthelmintic effect resulting from synergistic activity among 16 different combinations was for cinnamaldehyde:carvacrol (CL50 0.012mg/mL) and anethole:carvone (CL50 0.013mg/mL). These results indicate that these binary combinations would be promising to be tested in sheep infected with H. contortus.

Alternativas para o controle de nematoides gastrintestinais de pequenos ruminantes / Alternative techniques for the control of gastrointestinal nematodes in small ruminants

Sheep and goat farming requires an efficient management program, due to losses caused by parasites in susceptible animals. Many factors may collaborate to improve infection tolerance in the herd, such as: genetics, nutrition, physiological status, and age. The problem caused by resistance to antihelmintic agents has led to the spread of alternative techniques for parasite controls. The latest strategies include selective treatment with the FAMACHA method, phytotherapy, biological control with predatory fungi, and strategies that still await scientific confirmation, such as homeopathy, the dilution of resistance with the introduction of susceptible parasites, and the combination of drugs without antihelmintic effect. The main objective of these methods is to reduce the usage of antiparasitic agents, thus slowing the development of resistance and promoting the better use of effective products and newly released products. The objective of this article is to describe techniques for controlling nematodes in small ruminants, and it is aimed at technicians interested in increasing their knowledge about the mechanisms of resistance to antihelmintic agents as well as alternatives to the use of these products.

A criação de ovinos e caprinos depende de um manejo eficiente, devido, principalmente, ao prejuízo causado por parasitos em animais susceptíveis. Vários fatores colaboram para que os animais tolerem as infecções parasitárias, como: genética, nutrição, estado fisiológico e idade. O grave problema causado pela resistência anti-helmíntica promoveu a difusão de técnicas alternativas de controle parasitário. Dentre essas novas estratégias incluem-se a adoção do tratamento parcial seletivo com o método FAMACHA, fitoterapia, controle biológico com o uso de fungos nematófagos e estratégias que ainda necessitam de mais dados como a homeopatia, a diluição da resistência com a introdução de parasitos sensíveis e a combinação de produtos químicos sem efeito antiparasitário. O objetivo central destes métodos é reduzir o uso de antiparasitários, retardando o desenvolvimento da resistência, e promover melhor utilização de produtos ainda eficazes na propriedade e de novos produtos que venham a ser lançados. Este artigo abordará as técnicas descritas acima para o controle de nematoides de pequenos ruminantes e é direcionado aos técnicos interessados em ampliar seu conhecimento sobre os mecanismos de resistência dos parasitos aos anti-helmínticos e de alternativas ao uso desses produtos.

Prejuízos causados pela mastite em ovelhas Santa Inês / Damages caused by mastitis in Santa Inês Brazilian sheep breed

A mastite tem causado grandes prejuízos em criações de ovinos para corte no Brasil, onde o número de cordeiros nascidos e desmamados é de enorme importância econômica para o sistema de produção. O objetivo deste trabalho foi quantificar esses prejuízos em uma propriedade com ovinos da raça Santa Inês, em sistema semi-intensivo de produção, no Estado de São Paulo. Foram feitos exames clínicos, o Califórnia Mastitis Test (CMT), e as colheitas de leite para exame microbiológico das ovelhas na segunda semana de parição e no desmame. No mesmo dia das colheitas de amostras de leite, os cordeiros foram pesados, bem como no dia do nascimento. Quarenta e uma ovelhas pariram 55 cordeiros (taxa de natalidade 1,34) na estação de parição de 23/10/2008 a 4/1/2009. Destes, 20 (36%) morreram, 14 (70%) nas primeiras duas semanas de vida, e seis (30%) antes do desmame, que ocorreu em torno de 70 dias. 70% (14) das mortes foram atribuídas à condição de mastite da ovelha, e 30% (6) a outras causas, principalmente pneumonia. O ganho de peso diário do nascimento ao desmame de cordeiros filhos de ovelhas com mastite, em pelo menos uma glândula mamária, foi inferior (P < 0,10) ao de cordeiros filhos de ovelhas sadias. Das amostras positivas no exame microbiológico, 85,8% estavam positivas para bactérias do gênero Staphylococcus.

In meat sheep herds in Brazil, mastitis has provoked many damages, causing mortality and decreasing the numbers of lambs born and weaned, posing heavy economic impacts for the production system. The objective of this study was to quantify the damages caused by mastitis in a Santa Inês breed herd, reared in a semi-intensive production system, in São Paulo State, Brazil. Clinical exams were conducted and the California mastitis test was carried out, and milk samples were taken from the ewes for microbiological assay. The lambs were weighed at birth and on the days when the milk samples were taken. Forty-one ewes gave birth to 55 lambs (birth rate of 1.34) during the parturition season from 23 October 2008 to 4 January 2009. Twenty (36%) of them died, 14 (70%) in the first 2 weeks following birth, and 6 (30%) before weaning, which occurs at about 70 days. A total of 70% (14) of the deaths were imputed to the occurrence of mastitis in the respective ewe, and 30% (6) to other causes, mainly pneumonia. Mastitis decreased the average daily gain of the lambs from birth to weaning (P < 0.10). Most of the positive microbiological samples (85.8%) where positive for Staphylococcus spp.

Aspectos produtivos e sanitários de vacas mestiças leiteiras tratadas com produtos homeopáticos / Productive and sanitary aspects of crossbred milking cows receiving homeopathic products

Nos dias de hoje, a população mundial exige o consumo de alimentos de qualidade, sem resíduos de produtos químicos. Desempenho (produção e qualidade do leite, peso e condição corporal) e sanidade (mastite e infestação por ecto e endoparasitas) foram acompanhados em oito vacas leiteiras mestiças de um rebanho de 40 (20%), que recebeu diariamente produtos homeopáticos comerciais no concentrado para o controle de endo e ectoparasitas e mastite. No período de nove meses, que correspondeu a toda uma lactação, não houve necessidade de medicar com produtos alopáticos nenhuma vaca do rebanho, o que demonstrou ser possível criar vacas leiteiras mestiças não utilizando produtos químicos para o combate a parasitas (carrapato, mosca-do-chifre e verminose), ou antibióticos para o controle de mastite, e sem interferir na produtividade dos animais.

Nowadays the world population demands food of good quality, without any residues from chemical products. Eight cows from a herd of 40, which were consuming commercial homeopathic medicines at the daily concentration for control of endoand ectoparasites, were monitored in terms of their performance (production and quality of milk, body score and body weight) and sanitary aspects (parasites infestation and mastitis) during the lactation period. It was not necessary to give any allopathic (conventional) medicines to control mastitis or parasites (ticks, horn-fly, verminosis) in any cow of the herd during the nine months of the lactation period. The results showed that it is possible to produce milk without using chemical products, and not interfering in the productivity of the crossbred animals.

Performance and nematode infection of ewe lambs on intensive rotational grazing with two different cultivars of Panicum maximum.

The daily live weight gain (DLWG), faecal nematode egg counts (FEC), and packed cell volume (PCV) of Suffolk, Ile de France and Santa Inês ewe lambs were evaluated fortnightly for 56 days in the dry season (winter) and 64 days in the rainy season (summer) of 2001-2002. The animals were distributed in two similar groups, one located on Aruana and the other on Tanzania grass (Panicum maximum), in rotational grazing system at the Instituto de Zootecnia, in Nova Odessa city (SP), Brazil. In the dry season, 24 one-year-old ewe lambs were used, eight of each breed, and there was no difference (p > 0.05) between grasses for DLWG (100 g/day), although the Suffolk had higher values (p < 0.05) than the other breeds. In the rainy season, with 33 six-month-old ewe lambs, nine Suffolk, eight Ile de France and 16 Santa Inês, the DLWG was not affected by breed, but it was twice as great (71 g/day, p < 0.05) on Aruana as on Tanzânia grass (30 g/day). The Santa Inês ewe lambs had the lowest FEC (p < 0.05) and the highest PCV (p < 0.05), confirming their higher resistance to Haemonchus contortus, the prevalent nematode in the rainy season. It was concluded that the best performance of ewe lambs on Aruana pastures in the rainy season is probably explained by their lower nematode infection owing to the better protein content of this grass (mean contents 11.2% crude protein in Aruana grass and 8.7% in Tanzania grass, p < 0.05) which may have improved the immunological system with the consequence that the highest PCV (p < 0.05) observed in those animals.