Acacia farnesiana pods (plant: Fabaceae) possesses anti-parasitic compounds against Haemonchus contortus in female lambs.

Acacia farnesiana pods are rich in secondary metabolites and their biological activities have been recorded as antibacterial, antioxidant and anthelmintic. Previously, an in vitro bioguided study showed the important ovicidal and larvicidal effects of an organic fraction (EtOAc-F) from a hydroalcoholic extract of A. farnesiana pods against Haemonchus contortus. The present study aimed to assess the in vivo anthelmintic effect of EtOAc-F from A. farnesiana pods on the H. contortus faecal egg elimination in female lambs and on the infective larvae (L3) population reduction in coprocultures. The EtOAc-F was obtained from a hydroalcoholic extract from A. farnesiana pods through chromatographic procedures; additionally, some secondary compounds were identified using high-performance liquid chromatography (HPLC). Twenty-one 'Katahdin' crossbred female lambs ranging from three to four months of age, with body weights 21.9 ± 0.39 kg were used. Animals were orally infected with H. contortus (L3) by a single dose of 350 L3/kg BW. Three experimental groups (n = 7) were assigned as follows: 1) Control (untreated), 2) Albendazole, as a positive control (at 7.5 mg/kg BW, unique dose) and 3) EtOAc-F (at 100 mg/kg BW, once every third day, with three applications in total). Individual faecal samples were collected once a week for 5 weeks (at days 38, 45, 52, 59 and 66) post-treatment, to measure the faecal egg counts (FEC) and to obtain the H. contortus (L3) population from faecal cultures. The highest FEC reduction caused by EtOAc-F was 67.7%; meanwhile, albendazole showed a total FEC reduction after the second week post-treatment (day 45). On the other hand, the fraction caused an important reduction in the larval population in coprocultures (54.3-68.5%). The phytochemical analysis revealed the presence of galloyl derivatives and flavonoids as major compounds. The A. farnesiana pods could serve as a natural anthelmintic for the control of H. contortus, and perhaps for controlling other parasites of veterinary importance.

Anthelmintic-like activity of polyphenolic compounds and their interactions against the cattle nematode Cooperia punctata.

Polyphenolic compounds (PCs) have been proposed as one of the most bioactive group of secondary metabolites occurring in nature and have been associated to anthelmintic (AH)-like activity of plants against cattle nematodes. However, little is known regarding their synergetic / antagonistic interactions. This study assessed the in vitro AH-like activity of commercial PCs: quercetin, caffeic acid, rutin and coumarin, and their combinations against the egg hatching and larval exsheathment of Cooperia punctata; one of the most prevalent nematodes affecting grazing cattle in tropical regions. The molecules selected for the in vitro analysis were identified as bioactive phytochemicals of plants through bio-guided fractionation in previous studies. To estimate mean effective concentrations (EC50) five increasing concentrations were used for both Egg hatching inhibition assay (EHIA) and larval exsheathment inhibition assay (LEIA) (0.6-9.8 mg mL-1 and 0.15-2.4 mg mL-1, respectively). From the four molecules, only rutin did not affect egg hatching; while quercetin, showed no bioactivity against eggs or larvae (P > 0.766 and P > 0.621, respectively). Best-fit EC50 estimated through the EHIA was considered for PCs classification as bioactive (coumarin and caffeic acid) and non-bioactive (quercetin and rutin). Phytochemical interactions were subsequently assessed combining bioactive:non-bioactive PCs (8:2 ratio), and the nature of their interaction was classified using the fractional inhibitory concentration index (FICindex). Combinations had a highly synergistic interaction against larval exsheathment (FICindex < 0.5) except for coumarin:rutin against egg hatching (FICindex> 0.5). Quercetin and rutin acted as PCs AH-like activity enhancers, reducing EC50 of bioactive molecules in a range of 43%-64% and 68%-83% for EHIA and LEIA, respectively. A linear relationship between low molecular weight of molecules and ovicidal activity was observed; where, molecules with lower molecular weight displayed better-fit EC50 for ovicidal activity. Furthermore, coumarin and caffeic acid bioactivity against free-living stages of C. punctata makes them suitable candidates as markers for anthelmintic-like activity in bioactive forages. Combinations used through this investigation showed a potent anthelmintic-like activity against free-living forms of C. punctata, representing a first step towards the identification of promising alternatives for nematode control.

In vitro ovicidal activity of Baccharis conferta Kunth against Haemonchus contortus.

The indiscriminate use of chemical drugs to deworm livestock tends to trigger an anthelmintic resistance problem. In this context, the use of plant extracts rich in secondary metabolites could be an alternative method for the control of gastrointestinal nematodes. Baccharis conferta Kunth is a native plant species from Mexico that is widely used by several ethnic groups as forage for farm animals and medicinally to treat gastrointestinal diseases such as acute stomach ache, dysentery, diarrhoea, vomiting, indigestion, colic, intestinal spasms, urinary problems, and cramps. The aim of the present study was to isolate and characterise the ovicidal constituents of B. conferta and to determine a possible mode of action against Haemonchus contortus. The ovicidal activity was determined using the egg hatching inhibition test (EHI) to assess the methanol extract obtained from B. conferta foliage. The dry extract was partitioned (water/ethyl acetate) to obtain an ethyl acetate (BcEtOAc-F) and aqueous fraction. BcEtOAc-F showed an ovicidal activity of 72.32% EHI at 1 mg/mL. The chromatographic fractionation of BcEtOAc-F resulted in three active sub-fractions with higher ovicidal activity: BcC1R4 (99.15% EHI at 1.0 mg/mL); BcC1R5 (92.51% EHI at 0.75 mg/mL); and BcC1R8 (96.8% EHI at 3.0 mg/mL). Chemical analysis of the BcC1R4 fraction allowed the identification of the major active compound, isokaempferide (1, 98.06% EHI at 1 mg/mL). While, 4,5-di-O-acid caffeoylquinic (3; 96.8% EHI at 3 mg/mL) and an inactive flavone (vicenin-2, 2) were identified as the main compounds in BcC1R8. Chemical characterisation of the isolated compounds was performed via spectroscopic (NMR) and spectrometric (UPLC-MS) analyses. Additionally, the environmental and confocal scanning microscopy analyses revealed that isokaempferide was able to cross the eggshell layer without breaking it and attach itself to the embryo, causing its death. The flavonol, isokaempferide, and the hydroxycinamic acid, 4,5-di-O-caffeoylquinic, displayed powerful ovicidal effects, proving to be a potential alternative for the development of a phytodrug for the control of haemonchosis.

In vitro nematicidal effect of Chenopodium ambrosioides and Castela tortuosa n-hexane extracts against Haemonchus contortus (Nematoda) and their anthelmintic effect in gerbils.

The in vitro nematicidal effect of Chenopodium ambrosioides and Castela tortuosa n-hexane extracts (E-Cham and E-Cato, respectively) on Haemonchus contortus infective larvae (L3) and the anthelmintic effect of these extracts against the pre-adult stage of the parasite in gerbils were evaluated using both individual and combined extracts. The in vitro confrontation between larvae and extracts was performed in 24-well micro-titration plates. The results were considered 24 and 72 h post confrontation. The in vivo nematicidal effect was examined using gerbils as a study model. The extracts from the two assessed plants were obtained through maceration using n-hexane as an organic agent. Gerbils artificially infected with H. contortus L3 were treated intraperitoneally with the corresponding extract either individually or in combination. The results showed that the highest individual lethal in vitro effect (96.3%) was obtained with the E-Cham extract at 72 h post confrontation at 40 mg/ml, followed by E-Cato (78.9%) at 20 mg/ml after 72 h. The highest combined effect (98.7%) was obtained after 72 h at 40 mg/ml. The in vivo assay showed that the individual administration of the E-Cato and E-Cham extracts reduced the parasitic burden in gerbils by 27.1% and 45.8%, respectively. Furthermore, the anthelmintic efficacy increased to 57.3% when both extracts were administered in combination. The results of the present study show an important combined nematicidal effect of the two plant extracts assessed against L3 in gerbils.

In Vitro Anthelmintic Activity of Methanolic Extract from Caesalpinia coriaria J. Willd Fruits against Haemonchus contortus Eggs and Infective Larvae.

The aim of this study was to evaluate the in vitro lethal effect of a methanolic extract (ME) from Caesalpinia coriaria fruits against Haemonchus contortus eggs and infective larvae. The anthelmintic activity was assessed using the egg hatching inhibition assay (EHI) and the mortality test. The ME was assessed using five concentrations as follows: 6.15, 3.12, 1.56, and 0.78 mg/mL to eggs and 150, 100, 75, and 50 mg/mL to larvae, respectively. Ivermectin (5 mg/mL) was used as positive control and 4% methanol and distilled water were used as negative controls. The data of ovicidal and larvicidal effect were analyzed with a completely randomized design through ANOVA analysis using the general linear model (GLM) and lethal concentrations (LC50 and LC90) were estimated through a Probit analysis using the SAS program. A clear ME increased concentration dependence effect was observed in the EHI and mortality tests. The highest activity of the methanolic extract was observed at the highest concentration (P < 0.05) to obtain a similar effect to the positive control (ivermectin), with LC50 = 78.38 and 0.00064 mg/mL and LC90 =235.63 and 0.024 mg/mL, respectively, for larvae and eggs. The results indicate that the C. coriaria fruit ME possesses in vitro ovicidal and larvicidal properties (gallotannins: methyl gallate) against H. contortus that needs to be investigated more in vivo for the control of gastroenteric nematodes in ruminants.

Age of Haemonchus contortus third stage infective larvae is a factor influencing the in vitro assessment of anthelmintic properties of tannin containing plant extracts.

The larval exsheathment inhibition assay (LEIA) of infective larvae (L3) is an in vitro method used to evaluate the anthelmintic (AH) activity of tannin-containing plant extracts against different species of gastrointestinal nematodes, including Haemonchus contortus. Some conditions remain to be defined in order to standardize the LEIA, i.e. the optimal age of larvae produced from donor animals to use in the assays. Therefore, this study aimed at identifying the effect of age and age-related vitality of H. contortus infective larvae produced under tropical conditions, on the in vitro AH activity measured with the LEIA. The same acetone:water (70:30) extract from Acacia pennatula leaves was used to perform respective LEIA tests with H. contortus L3 of different ages (1-7 weeks). Each week, the L3 were tested against different concentrations of extract (1200, 600, 400, 200, 100, 40µg/mL of extract) plus a PBS control. Bioassays were performed with a benzimidazole (Bz) resistant H. contortus (Paraíso) strain. In order to identify changes in L3 vitality on different weeks (1-7), two assays testing larval motility were included only with PBS: the larval migration assay (LMA) and the larval motility observation assay (LMOA). Mean effective concentrations causing 50% and 90% exsheathment inhibition (EC50, EC90) were obtained for every week using respective Probit analyses. On the first week, the larvae had lowest EC50 and EC90 (39.4 and 65.6µg/mL) compared to older larvae (P<0.05). The EC50 and EC90 for weeks 2-5 were similar (P>0.05), while older larvae tended to show higher EC50 and EC90 (P<0.05). Motility showed strong negative correlations with age of larvae (r≥-0.83; P <0.05) and EC50 (r≥-0.80; P<0.05), suggesting that the lower extract efficacy could be associated with decaying vitality of larvae associated with age. More stable efficacy results were found between two to five weeks of age.

Caffeoyl and coumaroyl derivatives from Acacia cochliacantha exhibit ovicidal activity against Haemonchus contortus.

ETHNOPHARMACOLOGY RELEVANCE: Acacia cochliacantha is a small tree whose foliage is traditionally used in Mexico for treatment of kidney pain, gastrointestinal illnesses and to kill intestinal parasites. In recent decades, the study of vegetal extracts has offered other possible alternatives for the control of Haemonchus contortus. Considering that this nematode affects dramatically the health and productivity of small ruminants, the aim of this study was to identify the anthelmintic compounds from A. cochliacantha hydro-alcoholic extract (HA-E) through an ovicidal test. MATERIAL AND METHODS: In vitro egg hatch assay was conducted to determinate the anthelmintic effects of a HA-E (60g). Liquid-liquid ethyl acetate/water extraction gave two fractions (EtOAc-F, 1.92g; Aq-F; 58.1g). The less polar compounds from ethyl acetate fraction were extracted by addition of dichloromethane offering a precipitate phase (Mt-F, 1.25g) and a soluble mixture (DCMt-F 1.15g). All fractions were evaluated for ovicidal activity obtaining the egg hatching inhibition (EHI, 0.07-25mg/mL). Ivermectin (0.5mg/mL) was used as a reference drug (positive control), and distilled water, 2.5% DMSO and 2% methanol were used as negative controls. The isolated compounds from the most active fractions were subjected to spectroscopic (1H NMR) Spectrometric (MS) and UV HPLC analysis in order to identify the bioactive compounds. RESULTS: The less polar treatments (AcOEt-F, DCMt-F, DCMt-P) showed the highest ovicidal activities (98-100% EHI; at 0.62-1.56mg/mL) and the major compounds found in these fractions were identified as caffeoyl and coumaroyl derivatives, including caffeic acid (1), p-coumaric acid (2), ferulic acid (3), methyl caffeate (4), methyl-p-coumarate (5), methyl ferulate (6) and quercetin. In case of the less active fractions (Aq-F, Mt-F) were constituted principally by glycosylated flavonoids. CONCLUSION: These results show that caffeoyl and coumaroyl derivatives from Acacia cochliacantha leaves had promising anthelmintic activity against Haemonchus contortus. This leguminous may offer an alternative source for the control of gastrointestinal nematodes of small ruminants.

Relative expression analysis of IL-5 and IL-6 genes in tropical sheep breed Pelibuey infected with Haemonchus contortus.

Haemonchus contortus is a parasitic nematode of Pelibuey sheep, a meat breed used in tropical regions. Due to anthelmintic problems, the identification of hosts resistant to H. contortus is another option of control. The aim of this study was to analyse the relative expression of IL-5 and IL-6 genes in Pelibuey sheep after H. contortus infection. Nineteen lambs infected with H. contortus and three more lambs without infection were studied. The haemonchosis was determined by the number of eggs per gram of faeces (epg) and by the estimation of the percentage of the packed cell volume (%pcv). Peripheral blood mononuclear cells (PBMCs) were obtained to extract RNA at 0, 1, 2, 7, 14, 21 and 28 days after infection to quantify the relative expression of IL-5, IL-6 and GAPDH by real-time PCR. Five lambs were classified as low responders (lr) to haemonchosis with averages of 1519 ± 315·3 epg and 31·49 ± 5·13%pcv, and 14 lambs were identified as high responders (hr) with averages of 530 ± 132 epg and 34·88 ± 3·75%pcv. The expression ratio of IL-5 was significantly different compared with control lambs at 2, 7 and 14 days post-infection (PI), and IL-6 was significantly different after 14 days. The highest level of relative expression for IL-5 and IL-6 genes was 9·9-fold and 12-fold after 2 and 14 days for hr hosts (P < 0·05) compared with control group, respectively. In conclusion, the Pelibuey breed in grazing areas exhibited different expression of IL-5 and IL-6 obtained from PBMCs against H. contortus, suggesting the importance of these cytokines in regulating the nematode infection.

Benzimidazole-resistant gastrointestinal nematodes in indigenous Chiapas and Pelibuey sheep breeds from Chiapas, Mexico.

Because of the natural adaptation of Mexican sheep, the aim of the present study was to identify the presence or absence of gastrointestinal parasitic nematodes (GIN) resistant to benzimidazole (BZ) in both Chiapas and Pelibuey sheep breeds on local farms. Both male and female GIN-infected grazing sheep of the two breeds were selected. Sheep faecal samples were collected to obtain infective larvae (L3). This evolving stage of the parasite was used for taxonomic identification of the genus, based on its morphological characteristics. BZ anthelmintic resistance was evaluated using a nematode-compound in vitro interaction bioassay and the allele-specific polymerase chain reaction technique to detect mutations of residues 198 and 200 on isotype 1 of the ß-tubulin gene. Three BZ-based compounds (febendazole (FBZ), tiabendazole (TBZ) and albendazole (ABZ)) at concentrations of 1, 0.5, 0.25, 0.125, 0.062 and 0.03 mg/ml were used to estimate the anthelmintic efficacy and lethal dose (LD50, LD90 and LD99) of the drugs. Two parasitic nematodes, Haemonchus and Teladorsagia, were identified in both isolates. Also, the proportions of anthelmintic resistance identified in GIN of the two sheep breeds were 68% in isolates from the Chiapas breed and 71.8% in the Pelibuey breed. The specific lethal activity obtained with FBZ was higher than 90%. However, TBZ and ABZ showed a lethal activity lower than 50%. High variability in the discriminating dose values was found among the BZ drugs. For example, FBZ LD ranged from 0.01 to 1.20 mg/ml; on the other hand, TBZ and ABZ required a dose ranging from 0.178 to 759 mg/ml. In addition, amino acid changes of Phe (TTC) to Tyr (TAC) at codon 200 of the ß-tubulin gene, showing resistance to BZ, and no changes at codon 198 Glu (GAA) to Ala (GCA) were observed for both isolates. These results confirmed the presence of a genetic mutation associated with BZ in both Chiapas and Pelibuey nematode isolates.

Anthelmintic resistance in sheep farms: update of the situation in the American continent.

The present paper reviews the frequency of anthelmintic resistance in sheep farms in different countries of the American continent and describes some aspects that might influence the trend in sheep farms. The situation of anthelmintic resistance in sheep farms has been explored mainly in south of the continent (Argentina, Brazil and Uruguay) where sheep farming is an important industry. In those three countries, as well as in Paraguay, the first comprehensive surveys of anthelmintic resistance were performed among countries in the continent, which showed evidence of high frequency of sheep farms with anthelmintic resistance. Today, it is common to find sheep flocks with multiple-resistant worms. In North and Central America, a similar situation has been reported in sheep farms in the south of the United States of America, parts of Mexico and Costa Rica. On the other hand, other areas of the continent show low frequency of farms with anthelmintic resistance. From many areas no results have been published regarding situation on anthelmintic resistance or, alternatively, published results have received limited dissemination. Although the diagnosis of anthelmintic resistance is important for decision making of helminth management/control at the farm level, this is still an aspiration rather than a reality. For decades, researchers working on anthelmintic resistance in the American continent have expressed the need to change farmers' attitudes towards anthelmintic drugs. A common advice has been to check the anthelmintic drug efficacy regularly and reduce the dependence on these with alternative control measures. In spite of such advice, the challenge to stop/delay the advancement of anthelmintic resistance against the available anthelmintic drugs is still present. The evidence suggests that anthelmintic resistance is a growing phenomenon in the American continent. The situation described might be the tip of the iceberg, as anthelmintic resistance is still largely under-diagnosed. Hence, a different approach to tackle the advancement of anthelmintic resistance in sheep farms must be found. Awareness of farmers on the importance of diagnosis of anthelmintic resistance is not enough. Technical support schemes that provide the diagnostic service cheaply and timely must be implemented together with the research aiming at the adoption of control methods to reduce the dependence on conventional anthelmintic drugs. Unless these elements are readily available for producers, the negative consequences of anthelmintic resistance on sheep farming in America will continue to worsen with time.

Research and implementation of novel approaches for the control of nematode parasites in Latin America and the Caribbean: is there sufficient incentive for a greater extension effort?

The widespread presence of anthelmintic resistant gastrointestinal parasitic nematodes in outdoor ruminant production systems has driven the need to identify and develop novel approaches for the control of helminths with the intention to reduce the dependence on commercial anthelmintic drugs. This paper identifies what has been done in Latin America (LA) in terms of estimating the prevalence of anthelmintic resistance (AR) in ruminant production systems and the application of different novel approaches for the control of helminths in those systems, including research and extension activities. Firstly, the paucity of knowledge of AR is discussed in the context of different countries, as well as, the available economic resources for research, the technical infrastructure available and the practical difficulties of the production systems. It is then proposed that the search for novel approaches is not only driven by AR but also by the need for techniques that are feasible for application by resource-poor farmers in non-commercial subsistence farming systems. However, the commercial benefits of these approaches are often limited and so are funding inputs in most countries. The workers participating in the research into different novel approaches are identified as well as the different methods being studied in the different areas of LA according to their published results. In addition, the difficulties experienced during extension efforts to reach farmers and help them to adopt novel approaches for the control of parasitic nematodes in LA are discussed. The role of regulatory authorities in these countries is discussed as some methods of control might need an official confirmation of their efficacy as well as authorization prior to application as they may affect animal products (i.e. residues) and/or impose a hazard for animal welfare. The role of the pharmaceutical companies is also discussed.

Biochemical characterization of two purified proteins of the IB-16 Bacillus thuringiensis strains and their toxicity against the sheep nematode Haemonchus contortus in vitro.

The aim of this study was to characterize the biochemical composition of two main IB-16 Bacillus thuringiensis proteins and to determine their toxicity on the blood-feeder nematode, Haemonchus contortus. The soluble toxin of IB-16 strain of B. thuringiensis produces five main bands of proteins, the chemical composition of which might play an important role on the lethal activity. Two bands of proteins around 25 and 70 kDa were chosen and purified by HPLC using a hydrogel column and sephadex-beads G-50. Biochemical analysis was carried out to determine enzyme and carbohydrate moieties on purified fractions of the 25 and 70 kDa proteins. In addition, in vitro assays were carried out using H. contortus histiotropic larvae (L(4)) and the purified fractions. Biochemical results showed only enzyme activity in the purified fraction of the 25 kDa protein using gelatine as the substrate. In contrast, carbohydrate moieties were only observed in the purified fraction of the 70 kDa protein. Moreover, IB-16 B. thuringiensis purified fractions of 70 and 25 kDa showed lethal activity of 67.1% and 17.3% of toxicity on H. contortus L(4), respectively.

A technique for the quantification of Duddingtonia flagrans chlamydospores in sheep faeces.

Previous observations showed that Duddingtonia flagrans chlamydospores were visualized in McMaster chambers containing faeces of treated sheep. This trial explored the McMaster technique as a tool to quantify chlamydospores in sheep faeces. A range of individual chlamydospore doses (from 19.5 x 10(6) to 177.5 x 10(6)) were offered orally to nine lambs for 7 consecutive days. A faecal sample (5 g) was daily obtained from the rectum of each animal (from days 1 to 13) to perform the McMaster technique using a sugar flotation fluid with 1.27 g/mL density. Each chlamydospore counted in the McMaster chamber was considered as 50 chlamydospores per g of faeces (CPG). The results confirmed that the estimated CPG was associated with the daily dose offered to the animals (r(2)=0.90; P<0.001). Furthermore, the total chlamydospore dose received by each animal was strongly associated to the total quantity of CPG obtained from the bulk faeces (TCtot) (r(2)=0.96; P<0.0001). Quantification of CPG can be used as a helpful tool to determine the number of chlamydospores reaching the faeces in orally dosed animals. This could be used to evaluate the efficacy of D. flagrans for the control of gastrointestinal nematode larvae in sheep faeces.

In vitro nematicidal effects of medicinal plants from the Sierra de Huautla, Biosphere Reserve, Morelos, Mexico against Haemonchus contortus infective larvae.

Twenty extracts from plants from Sierra de Huautla Biosphere Reserve, Morelos, Mexico were evaluated against Haemonchus contortus infective larvae in an in vitro assay. The plant species evaluated were Bursera copallifera, B. grandifolia, Lippia graveolens, Passiflora mexicana, Prosopis laevigata, Randia echinocarpa and Urtica dioica. The plants were separated into their parts and macerated with different solvents (n-hexane, acetone, ethanol and methanol). An in vitro assay was used to evaluate the anthelmintic activity against unsheathed third stage H. contortus infective larvae. The experiment was carried out in 24-well cell culture plates at room temperature with three replicates per treatment and using a concentration of 20 mg ml- 1. Ten 5 microl aliquots were taken from the corresponding wells and deposited on a slide for microscopical observation at 24, 48, 72 and 96 h post-exposure. The evaluation criteria were based on the average numbers of live and/or dead larvae in the different treatments. Alive and dead larval numbers were statistically analysed through the ANOVA test (P>0.01). The Tukey test was used as a complementary tool to determine which treatment was different from the other treatments (P>0.05). The highest mortality was observed with P. laevigata hexanic extract from stem and leaves combined, which produced 51%, 81% and 86% larval mortality at 24, 48 and 72 h post-exposure, respectively. On the other hand, B. copallifera stem acetonic extract exhibited 18%, 59% and 66% nematicidal activity after 24, 48 and 72 h of exposure, respectively.

Evaluating the effectiveness of a Mexican strain of Duddingtonia flagrans as a biological control agent against gastrointestinal nematodes in goat faeces.

The use of Duddingtonia flagrans in the control of goat nematodes was investigated. Initially, the time of passage of chlamydospores through the digestive tract of goats was evaluated. Two groups of seven parasite-free kids were formed. Group A received a single dose of 3.5x10(6) D. flagrans chlamydospores (FTHO-8 strain) per kg of live weight. Group B did not receive any chlamydospores. Faeces were obtained from each kid daily from day 4 prior to inoculation until day 5 post-inoculation (PI) and were placed in Petri dishes containing water agar. Gastrointestinal nematode infective larvae were added to each Petri dish and incubated at 25 degrees C for 7 days. Petri dishes were examined to detect the fungus and trapped nematodes. A second trial evaluated the effect of D. flagrans on the number of gastrointestinal nematode larvae harvested from goat faecal cultures in naturally infected goats. Two groups of seven goats were formed. The treated group received a single dose of 3.5x10(6) D. flagrans chlamydospores per kg of liveweight. The control group did not receive any chlamydospores. Faeces were obtained twice daily from each kid. Two faecal cultures were made for each kid. One was incubated for 7 days and the other for 14 days. Gastrointestinal nematode larvae were recovered from each culture and counted. Percentage of larval development reduction was determined using a ratio of larvae/eggs deposited in the control and treated groups. Duddingtonia flagrans survived the digestive process of goats, and maintained its predatory activity, being observed from 21 to 81 h PI (3 to 4 days). A reduction in the infective larvae population in the treated group compared to the non-treated group was observed in both incubation periods (7 days: 5.3-36.0%; 14 days: 0-52.8%, P>0.05). Although a single inoculation of D. flagrans can induce a reduction of infective larvae collected from faeces, a different scheme of dosing may be needed to enhance the efficacy of D. flagrans in goats.

[In vitro predatory activity of 8 fungal isolates against the nematode Panagrellus redivivus]. / Acción depredadora in vitro de ocho aislados de hongos contra el nematodo Panagrellus redivivus.

The aim of this study was to evaluate the predacious capacity in vitro of eight isolates of nematophagous fungi: four of Arthrobotrys sp., one of Arthrobotrys oligospora, one of Duddingtonia flagrans, one of Dactylaria sp. and one Monacrosporium eudermatum. Nine groups of Petri dishes with 13 repetitions each were set up. The fungi were seeded in fluor-corn-agar media, following this each Petri dish was added with 150 larvae of the free living nematode Panagrellus redivivus. Five days after larval addition these were collected by Baermannization and were quantified. A significant difference (p < 0.05) between all treated group was observed respect with the control. Isolates FTHO-8 D. flagrans, R6 M. eudermatum, DAC Dactylaria sp. as well as FTHO-4 and FTHO-6 Arthrobotrys sp., showed an excellent predatory activity (> 90%) and they could be considered as potential bio-control agents in future field trials.

[In vitro nematophagous capacity of Duddingtonia flagrans maintained under 2 conditions of preservation]. / Capacidad nematófaga in vitro de Duddingtonia flagrans Mantenido en dos Condiciones de Preservación.

One trial was carried out to evaluate the nematophagous capacity of two Duddingtonia flagrans cultures, one maintained during one year at laboratory temperature and the other one was a recent culture, twelve Petri dishes with flour-corn-agar media were seeded with the 1YC another 12 Petri dishes were inoculated with the RC. Both were added with 150 larvae/dish of the free living nematode Panagrellus redivivus and 12 fluor-corn-agar dishes only with the free living nematode were used as a control. The results showed that the nematophagous capacity of both cultures were similar but it was statistically different (p < 0.05) with respect to the control group. It was concluded that the nematophagous capacity of D. flagrans was not affected in spite of being kept one year at laboratory temperature.

Cryopreservation of infective larvae of Haemonchus contortus.

Three freeze protectants were evaluated to preserve H. contortus infective larvae. Freezing solutions used: A) saline solution phosphate buffer pH 7.2 (PBS); B) 10% DMSO (dimethyl sulphoxide); C) 10% glycerol. Fifty thousand infective larvae were put into each of 10 vials per freeze protectant and then stored into liquid nitrogen. Results were based on the motility of the larvae under a light microscope at 30, 90, 180, and 360 days of freezing. Ten vials of each freeze protectant were removed from the liquid nitrogen at these times and immediately were put on water at 37 C during a minute. Motility percentages obtained were as follows: PBS: 36%, 20%, 7% and 39%; DMSO,: 87%, 69%, 46% and 85%; glycerol: 67%, 62%, 29% and 55%; at 30, 90, 180 and 360 days respectively. Inoculation of infectiva larvae from DMSO and glycerol to calves was successful after 28 days. DMSO was a better freeze preserver for H. contortus.