Ana Carolina de Souza Chagas

Challenges of nematode control in ruminants: Focus on Latin America

Gastrointestinal nematodes (GIN) of ruminants (cattle, sheep and goats) are ubiquitous and can cause severe injuries to infected animals and significant losses in farming revenues. GIN are able to survive severe environmental and host conditions, but mankind has developed a number of ingenious methods for parasite control. The commerce and use of modern anthelmintic drugs with a broad spectrum of activity has been a solid tool for nearly 40 years, however the continuous use of these drugs, has led to the selection of populations of drug-resistant worms worldwide. At present, the ever-growing agricultural systems in Latin America are facing many challenges and cannot rely on the far-reaching objective of parasitic elimination from the host or the environment. The lack of extensive programs for monitoring drug resistance exacerbates the negative consequences of reduced efficacy, which is evident in some areas with the increase in mortality rate even after treatment. Experts agree that new schemes of parasitic control are needed and should be based on the strategy of targeted selective treatment where affected hosts are identified and treated accordingly. In this article, we will focus our discussion on the challenges for the control of GIN in Latin America by 2020 imposed by reduced drug efficacy. We will evaluate phenotypic and molecular markers, methods for single-animal evaluation, and the implementation of schemes for anthelmintic treatment that address parasites in refugia.

In vitro efficacy of plant extracts and synthesized substances on Rhipicephalus (Boophilus) Microplus (Acari: Ixodidae)

Herbal drugs have been widely evaluated as an alternative method of parasite control, aiming to slow development of resistance and obtain low-cost biodegradable parasiticides. This study evaluated the in vitro efficacy on Rhipicephalus (Boophilus) microplus of extracts from Carapa guianensis seed oil, Cymbopogon martinii and Cymbopogon schoenanthus leaf essential oil, and Piper tuberculatum leaf crude extract and similar synthesized substances. In the immersion test, engorged females were evaluated in five dilutions ranging from 10% to 0.030625% concentration. In the larval test on impregnated filter paper, the concentration ranged from 10% to 0.02%. The treatments and controls were done in three replicates. Chemical analysis of the oils was performed by gas chromatography. The main compounds were oleic acid (46.8%) for C. guianensis and geraniol for C. martinii (81.4%), and C. schoenanthus (62.5%). The isolated and synthesized substances showed no significant effect on larvae and adult. C. martinii and P. tuberculatum showed the best efficacy on the engorged females. The LC50 and LC90 were 2.93% and 6.66% and 3.76% and 25.03%, respectively. In the larval test, the LC50 and LC90 obtained for C. martinii, P. tuberculatum, and C. schoenanthus were 0.47% and 0.63%, 0.41% and 0.79%, 0.57% and 0.96%, respectively. The fact that geraniol is present in greater quantities in C. martinii explains its higher activity in relation to C. shoenanthus. It is necessary to validate the in vivo use of safe and effective phytoparasiticidal substances. Efforts should be focused on developing formulations that enhance the efficacy in vivo and lengthen the residual period.

Anthelmintic activity of Cymbopogon martinii, Cymbopogon schoenanthus and Mentha piperita essential oils evaluated in four different in vitro tests

Anthelmintic resistance is a worldwide concern in small ruminant industry and new plant-derived compounds are being studied for their potential use against gastrointestinal nematodes. Mentha piperita, Cymbopogon martinii and Cymbopogon schoenanthus essential oils were evaluated against developmental stages of trichostrongylids from sheep naturally infected (95% Haemonchus contortus and 5% Trichostrogylus spp.) through the egg hatch assay (EHA), larval development assay (LDA), larval feeding inhibition assay (LFIA), and the larval exsheathment assay (LEA). The major constituent of the essential oils, quantified by gas chromatography for M. piperita oil was menthol (42.5%), while for C. martinii and C. schoenanthus the main component was geraniol (81.4% and 62.5%, respectively). In all in vitro tests C. schoenanthus essential oil had the best activity against ovine trichostrongylids followed by C. martini, while M. piperita presented the least activity. Cymbopogon schoenanthus essential oil had LC(50) value of 0.045 mg/ml in EHA, 0.063 mg/ml in LDA, 0.009 mg/ml in LFIA, and 24.66 mg/ml in LEA. The anthelmintic activity of essential oils followed the same pattern in all in vitro tests, suggesting C. schoenanthus essential oil could be an interesting candidate for nematode control, although in vivo studies are necessary to validate the anthelmintic properties of this oil.

In vitro anthelmintic activity of aqueous leaf extract of Annona muricata L. (Annonaceae) against Haemonchus contortus from sheep.

Despite the overall progress of sheep farming in Brazil, infections with the gastrointestinal parasite Haemonchus contortus represent one the most important problems in sheep production, aggravated by the increasing resistance of nematodes to traditional anthelmintic drugs caused by inadequate sheep flock management by breeders. Ethnopharmacological data indicate Annona muricata as a promising alternative for the control of gastrointestinal nematodes because of its general anthelmintic properties. The aim of this work was to evaluate the in vitro anthelmintic effects of A. muricata aqueous leaf extract against eggs, infective larvae and adult forms of parasitic nematode H. contortus. At higher doses, A. muricata extract showed 84.91% and 89.08% of efficacy in egg hatch test (EHT) and larval motility test (LMT), respectively. In the adult worm motility test, worms were completely immobilized within the first 6-8h of nematode exposition to different dilutions of extract. Phytochemical analysis indicated the presence of phenolic compounds in A. muricata aqueous leaf extract that may be responsible for the anthelmintic effects observed. Moreover those results validate the traditional use of A. muricata as a natural anthelmintic and then the pharmacological potential of its compounds for future in vivo investigations.

The anthelmintic effect of plant extracts on Haemonchus contortus and Strongyloides venezuelensis

The indiscriminate use of anthelmintics has resulted in the establishment of parasite resistance. Thus, this study aimed to evaluate the in vitro antiparasitic effect of plant extracts on Haemonchus contortus in sheep and the in vivo effect on Strongyloides venezuelensis in Rattus norvegicus. The plant extracts from Piper tuberculatum, Lippia sidoides, Mentha piperita, Hura crepitans and Carapa guianensis, produced at different research institutions, were chemically analyzed and evaluated through the egg hatch test (EHT) and larval development test (LDT) in H. contortus. P. tuberculatum (150 and 250 mg kg(-1) of body weight) was evaluated for its anthelmintic action on R. norvegicus experimentally infected with S. venezuelensis. In the EHT, the LC(50) and LC(90) of the extracts were respectively as follows: 0.031 and 0.09 mg mL(-1) for P. tuberculatum, 0.04 and 0.13 mg mL(-1) for L. sidoides, 0.037 and 0.10 mg mL(-1) for M. piperita, 2.16 and 17.13 mg mL(-1) for H. crepitans and 2.03 × 10(-6) and 1.22 × 10(-12) mg mL(-1) for C. guianensis. In the LDT, the LC(50) and LC(90) were respectively: 0.02 and 0.031 mg mL(-1) for P. tuberculatum, 0.002 and 0.04 mg mL(-1) for L. sidoides, 0.018 and 0.03 mg mL(-1) for M. piperita, 0.36 and 0.91 mg mL(-1) for H. crepitans and 17.65 and 1890 mg mL(-1) for C. guianensis. The extract of P. tuberculatum showed the following substances: piperamides as (Z)-piplartine, (E)-piplartine, 8,9-dihydropiplartine, piperine, 10,11-dihydropiperine, 5,6 dihydropiperlongumine and pellitorine. The major compounds of the oils were thymol (76.6%) for L. sidoides, menthol (27.5%) for M. piperita and oleic acid (46.8%) for C. guianensis. Regarding the in vivo test, neither dose of P. tuberculatum caused any significant reduction (P>0.05) in worm burden and fecal egg counts compared with the control group. We conclude that the extracts of P. tuberculatum, L. sidoides and M. piperita have effective activity when tested in vitro, but the doses of the extract of P. tuberculatum have no effect when employed in in vivo tests.

Anthelmintic effect of plant extracts containing condensed and hydrolyzable tannins on Caenorhabditis elegans, and their antioxidant capacity

Although tannin-rich forages are known to increase protein uptake and to reduce gastrointestinal nematode infections in grazing ruminants, most published research involves forages with condensed tannins (CT), while published literature lacks information on the anthelmintic capacity, nutritional benefits, and antioxidant capacity of alternative forages containing hydrolyzable tannins (HT). We evaluated the anthelmintic activity and the antioxidant capacity of plant extracts containing either mostly CT, mostly HT, or both CT and HT. Extracts were prepared with 70% acetone, lyophilized, redissolved to doses ranging from 1.0mg/mL to 25mg/mL, and tested against adult Caenorhabditis elegans as a test model. The extract concentrations that killed 50% (LC(50)) or 90% (LC(90)) of the nematodes in 24h were determined and compared to the veterinary anthelmintic levamisole (8 mg/mL). Extracts were quantified for CT by the acid butanol assay, for HT (based on gallic acid and ellagic acid) by high-performance liquid chromatography (HPLC) and total phenolics, and for their antioxidant activity by the oxygen radical absorbance capacity (ORAC) assay. Extracts with mostly CT were Lespedeza cuneata, Salix X sepulcralis, and Robinia pseudoacacia. Extracts rich in HT were Acer rubrum, Rosa multiflora, and Quercus alba, while Rhus typhina had both HT and CT. The extracts with the lowest LC(50) and LC(90) concentrations, respectively, in the C. elegans assay were Q. alba (0.75 and 1.06 mg/mL), R. typhina collected in 2007 (0.65 and 2.74 mg/mL), A. rubrum (1.03 and 5.54 mg/mL), and R. multiflora (2.14 and 8.70 mg/mL). At the doses of 20 and 25mg/mL, HT-rich, or both CT- and HT-rich, extracts were significantly more lethal to adult C. elegans than extracts containing only CT. All extracts were high in antioxidant capacity, with ORAC values ranging from 1800 μmoles to 4651 μmoles of trolox equivalents/g, but ORAC did not correlate with anthelmintic activity. The total phenolics test had a positive and highly significant (r=0.826, p ≤ 0.01) correlation with total hydrolyzable tannins. Plants used in this research are naturalized to the Appalachian edaphoclimatic conditions, but occur in temperate climate areas worldwide. They represent a rich, renewable, and unexplored source of tannins and antioxidants for grazing ruminants, whereas conventional CT-rich forages, such as L. cuneata, may be hard to establish and adapt to areas with temperate climate. Due to their high in vitro anthelmintic activity, antioxidant capacity, and their adaptability to non-arable lands, Q. alba, R. typhina, A. rubrum, and R. multiflora have a high potential to improve the health of grazing animals and must have their anthelmintic effects confirmed in vivo in both sheep and goats.

Caenorhabditis elegans as a model to screen plant extracts and compounds as natural anthelmintics for veterinary use.

The most challenging obstacles to testing products for their anthelmintic activity are: (1) establishing a suitable nematode in vitro assay that can evaluate potential product use against a parasitic nematode of interest and (2) preparation of extracts that can be redissolved in solvents that are miscible in the test medium and are at concentrations well tolerated by the nematode system used for screening. The use of parasitic nematodes as a screening system is hindered by the difficulty of keeping them alive for long periods outside their host and by the need to keep infected animals as sources of eggs or adults when needed. This method uses the free-living soil nematode Caenorhabditis elegans as a system to screen products for their potential anthelmintic effect against small ruminant gastrointestinal nematodes, including Haemonchus contortus. This modified method uses only liquid axenic medium, instead of agar plates inoculated with Escherichia coli, and two selective sieves to obtain adult nematodes. During screening, the use of either balanced salt solution (M-9) or distilled water resulted in averages of 99.7 (± 0.73)% and 96.36 (± 2.37)% motile adults, respectively. Adult worms tolerated DMSO, ethanol, methanol, and Tween 80 at 1% and 2%, while Labrasol (a bioenhancer with low toxicity to mammals) and Tween 20 were toxic to C. elegans at 1% and were avoided as solvents. The high availability, ease of culture, and rapid proliferation of C. elegans make it a useful screening system to test plant extracts and other phytochemical compounds to investigate their potential anthelmintic activity against parasitic nematodes.

Haemonchus contortus: A multiple-resistant Brazilian isolate and the costs for its characterization and maintenance for research use

The aim of this work was to determine the resistance level of Haemonchus contortus isolated from the Santa Inês flock of the Embrapa (Brazilian governments Agricultural Research Company), Southeast Livestock Unit (CPPSE), as well as to determine costs of characterizing and maintaining this isolate in host donors. Forty-two male Santa Inês lambs were experimentally infected with 4000 H. contortus infective larvae of the field isolate of CPPSE, called Embrapa2010, and divided into six treatment groups, which received triclorfon, albendazol plus cobalt sulfate, ivermectin, moxidectin, closantel and levamisole phosphate, as well as a negative control group (water). Egg per gram (EPG) counts were performed at 0, 3, 7, 10 and 14 days post treatment when the animals were slaughtered for parasite count. The data were analyzed using the RESO statistical program, considering anthelmintic resistance under 95% of efficacy. EPG and worm count presented a linear and significant relation with 94% determination coefficient. The susceptibility results obtained by RESO through both criteria (EPG and worm count) were equal, except for closantel, showing that the isolate Embrapa2010 is resistant to benzimidazoles, macrocyclic lactones and imidazothiazoles. The need of a control group did not appear to be essential since the result for susceptibility in the analyses with or without this group was the same. Suppression in egg production after treatment did not occur in the ivermectin and moxidectin groups. In the control group, the establishment percentage was just 12.5 because of the low number of third-stage larvae, resistance (innate and infection immunity) of the animals studied plus good nutrition. Drug classes presented similar efficacy between adults and immature stages. The costs for isolate characterization were calculated for 42 animals during 60 days. The total cost based on local market rates was approximately US

Gastrointestinal nematode infection in beef cattle of different genetic groups in Brazil.

8000. The precise identification of Brazilian isolates and their establishment in host donors would be useful for laboratorial anthelmintic resistance diagnoses through in vitro tests, which has an annual cost of approximately US

In vitro anthelmintic effect of Melia azedarach L. and Trichilia claussenii C. against sheep gastrointestinal nematodes.

2500 for maintenance in host donors.