Analysis of P-gp genes relative expression associated to ivermectin resistance in Haemonchus contortus larval stages from in vitro cultures (L3 and xL3) and from gerbils (Meriones unguiculatus) (L4) as models of study.

The aim of the study was to compare the relative gene expression of Haemonchus contortus P-glycoprotein genes (Hco-pgp) between fourth (L4), infective (L3), and transitory infective (xL3) larval stages as laboratory models to study ivermectin (IVM) resistance. The H. contortus resistant to IVM (IVMr) and susceptible to IVM (IVMs) strains were used to develop xL3in vitro culture and to infect Meriones unguiculatus (gerbils) to collect L4 stages. Morphometric differences were evaluated from 25 individuals of H. contortus from each strain. Relative gene expression from xL3 and L4 was determined between comparison of IVMr stages and from IVMr vs IVMs stages. Seven Hco-pgp genes (1, 2, 3, 4, 9, 10, and 16) were analysed by RT-qPCR using L3 stage as control group, per strain, and GAPDH and ß-tubulin as constitutive genes. Morphological changes were confirmed between xL3 and L4 developing oral shape, oesophagus, and intestinal tube. In addition, the body length and width showed statistical differences (p < 0.05). The Hco-pgp1, 2, 3, and 4 genes (p < 0.05) were upregulated from 7.1- to 463.82-fold changes between IVMr stages, and Hco-pgp9 (13.12-fold) and Hco-pgp10 (13.56-fold) genes showed differences between L4 and xL3, respectively. The comparative study between IVMr vs IVMs strains associated to xL3 and L4 displayed significant upregulation for most of the Hco-pgp genes among 4.89-188.71 fold-change. In conclusion, these results suggest the use of H. contortus xL3 and L4 as suitable laboratory models to study IVMr associated with Hco-pgp genes to contribute to the understanding of anthelmintic resistance.

Phytochemical profile and nematicidal activity of a hydroalcoholic extract from Cazahuate flowers (Ipomoea pauciflora M. Martens & Galeotti) against Haemonchus contortus infective larvae.

Haemonchus contortus (Hc) is a hematophagous parasite affecting the health and productivity of flocks. The administration of chemical anthelmintic drugs (AH) is the common method of deworming; however, generates resistance in the parasites to AH and it is a public health risk due to drug residues in milk, meat and sub-products. Natural compounds from plants are explored to diminish this parasitosis, improving their health and productivity, without the negative effects of AH. Ipomoea genus is a group of climbing plants belonging to the Convulvulaceae family possessing perennial leaves and tuberous roots. Medicinal properties has been attributed to this plant including nutritional agents, emetics, diuretics, diaphoretics, purgatives and pesticides. The objective of this study was assessing the in vitro nematocidal activity of a hydroalcoholic extract (HA-E) obtained from Ipomoea pauciflora (Cazahuate) flowers against Hc infective larvae (L3) and to identify its phytochemical profile (PhC-P). The assay was carried out using microtiter plates (MTP). Four HA-E concentrations were assessed and Ivermectin and distilled water were used as positive and negative control groups, respectively. Approximately 100 Hc L3 were deposited in each well (n=12) and incubated at 25-35°C for 7 days. Data were analyzed using ANOVA and a General Linear Model (GLM) followed by Tukey test (P<0.05). The treatments showing a concentration-dependent effect (CDE) were analyzed to identify their 50% and 90% lethal concentrations (CL50, 90) via a Probit Analysis. The highest mortality was observed at 50 mg/mL (82.64 ± 0.71%) and the lowest at 6.25 mg/mL (56.46 ± 2.49%), showing a CDE with increasing mortality from 6.25 to 50 mg/mL. The PhC-P revealed the presence of alkaloids, coumarins, flavonoids, tannins and triterpenes/ sterols. A HA-E from flowers of I. pauciflora will be considered to assess its potential use in the control of haemonchosis in small ruminants.

Effect of three feeding levels on the pathogenesis and establishment of Haemonchus contortus in parasite-naïve Pelibuey hair sheep lambs during their first infection.

This study evaluated the effect of three feeding levels on the pathogenesis and establishment of H. contortus upon the first infection of parasite-naïve Pelibuey hair sheep lambs. Forty-two 6-month-old hair sheep lambs (24 ± 4 kg) raised parasite free from birth were used. The lambs were assigned to 3 groups (n = 14), and each was fed a diet designed for different daily weight gain (DWG): 75 g/d (Diet 1), 125 g/d (Diet 2) and 200 g/d (Diet 3). After four weeks of diet adaptation, 10 lambs/group were infected with 450 L3H. contortus/kg BW (infected), and 4 lambs/group were kept parasite-free (NInf). DWG, hematocrit (Ht), hemoglobin (Hb), peripheral eosinophils (EOS), IgG concentration against H. contortus, and eggs per gram (EPG) of feces were measured in each lamb from day 14 before infection until day 29 postinfection (PI). On day 29 PI, the lambs were slaughtered to determine the total number of adult parasites (TAW), the length of the female worms, and the number of eggs in utero (EIU). Each group reached the expected DWG (P = 0.001), and there was no effect of infection or the diet × infection interaction. Ht was lower in infected lambs than in NInf lambs, and this difference was significant for animals on Diets 1 and 2 (P = 0.044). From day 14 PI onward, Hb was lower in the infected lambs than in the NInf lambs (P = 0.001). Furthermore, compared with NInf lambs, the infected lambs had higher EOS from day 7 PI and higher IgG from day 14 PI. Neither EOS nor IgG were affected by diet. Lambs on Diet 3 had lower EPG during patency than those fed Diets 1 or 2 (days 25 and 28 PI; P = 0.002). Furthermore, lambs fed Diet 3 had lower TAW (Diet 1 vs 3 P = 0.037; Diet 2 vs 3 P = 0.049) and EIU (P = 0.004) than lambs fed Diet 1 or 2. Lambs were resilient to infection regardless of diet. Although EOS and IgG were higher in all infected animals than in Ninf animals, EPG, TAW and EIU decreased only in lambs fed Diet 3. Thus, a diet targeting a DWG of 200 g/d can significantly limit the establishment of H. contortus in Pelibuey lambs infected for the first time.

Taenia hydatigena larvae vesicular concentrate reduces the egg shedding of Haemonchus contortus associated with the overexpression of abomasal cytokines in lambs.

The expression patterns of some cytokines were compared by RT-qPCR between lambs with and without Taenia hydatigena larvae vesicular concentrate (ThLVC) administration and subsequent infection with Haemonchus contortus. Lambs that received ThLVC prior to infection with H. contortus showed lower (p < 0.03) cumulative FEC (AUC = 18450 ± 3384) than infected lambs who did not receive ThLVC (AUC = 31081 ± 3277). Lambs infected with H. contortus, in general, overexpressed Th1 and Th2 cytokines in abomasal mucosa and abomasal lymph nodes, which seems to indicate a generalized and nonpolarized activation of the immune response by H. contortus. The main immunomodulatory effects of ThLVC were observed in the abomasal fundic region. The lambs that were given ThLVC prior to infection strongly overexpressed most of the studied cytokines representing the Th1 (IFNγ and IL2) and Th2 profiles (IL4, IL5, IL6 and IL10), proinflammatory cytokines (SOD1 and PRDX6) and IgE receptor; in contrast, lambs that were infected but did not receive ThLVC only moderately overexpressed IFNγ, IL4 and IL6. The absence of the significant overexpression of cytokines in lambs that only received ThLVC suggests that this derived from T. hydatigena does not have a stimulating effect per se; however, the presence of H. contortus did produce the highest expression (p < 0.01) cytokine profile among lambs that received ThLVC prior to infection compared to those who did not receive it, so its effect seems to be immunomodulatory and not only immunostimulatory.

Artificial Haemonchus contortus infection as a strategy to induce protective immune response to natural infection in Pelibuey lambs.

The objective of this study was to evaluate the reduction in nematode faecal egg count (FEC) in Pelibuey lambs segregated as resistant (RES), susceptible (SUS) and intermediate (INT) to gastrointestinal nematodes. Twenty-nine weaned Pelibuey lambs, aged five months old, free of nematode infection, were used. Nine lambs were RES, six were SUS and 14 were INT lambs. The study consisted of two phases: in Phase 1 the lambs were infected experimentally with Haemonchus contortus. In Phase 2, the lambs were naturally infected by grazing. Faecal and blood samples were taken every week. The packed cell volume and total protein were quantified. The FEC value (FECmax) per lamb was recorded together with a natural reduction in FEC in the two phases. The data were analysed with a model of measures repeated over time. During Phase 1, the RES lambs showed the lowest FEC (1061 ± 1053) compared to the other groups (INT: 2385 ± 1794 eggs per gram of faeces (EPG); and SUS: 3958 ± 3037 EPG). However, in Phase 2 no significant differences (p > 0.05) were observed between the groups of lambs (RES: 275 ± 498 EPG; SUS: 504 ± 1036 EPG; and INT: 603 ± 1061 EPG). At the end of Phase 1, the FEC of RES lambs was naturally reduced by 75.5% in respect to FECmax (p < 0.05), and at the end of Phase 2 the reduction in FEC was 90% in respect to FECmax (p > 0.05); the same behaviour was observed in RES and SUS lambs. It is concluded that the artificial infection in the lambs induced a more rapid immune response in RES than SUS lambs, and all lambs developed high acquired resistance by continuous infection.

Molecular mechanisms of cell death induced in glioblastoma by experimental and antineoplastic drugs: New and old drugs induce apoptosis in glioblastoma.

Glioblastoma multiforme (GBM) is one of the most aggressive astrocytic tumors; it is resistant to most chemotherapeutic agents currently available and is associated with a poor patient survival. Thus, the development of new anticancer compounds is urgently required. Herein, we studied the molecular mechanisms of cell death induced by the experimental drugs resveratrol and MG132 or the antineoplastic drugs cisplatin and etoposide on a human GBM cell line (D54) and on primary cultured mouse astrocytes (PCMAs). Caspases, Bcl-2, inhibitors of apoptosis proteins (IAP) family members, and p53 were identified as potential molecular targets for these drugs. All drugs had a cytotoxic effect on D54 cells and PCMAs, with a similar inhibitory concentration (IC50) after 24 h. However, MG132 and cisplatin were more effective to induce apoptosis and autophagy than resveratrol and etoposide. Cell death by apoptosis involved the activation of caspases-3/7, -8, and -9, increased lysosomal permeability, LC3 lipidation, poly-(ADP-ribose) polymerase (PARP)-1 fragmentation, and a differential expression of genes related with apoptosis and autophagy like Mcl-1, Survivin, Noxa, LC3, and Beclin. In addition, apoptosis activation was partially dependent on p53 activation. Since experimental and antineoplastic drugs yielded similar results, further work is required to justify their use in clinical protocols.

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.

Influence of the physiological stage of Blackbelly sheep on immunological behaviour against gastrointestinal nematodes.

The influence of the physiological stage of a Blackbelly flock against natural infection by gastrointestinal parasitic nematodes (GIN) was investigated. Data on serum and saliva IgA levels and peripheral cellular immune response were recorded in 51 sheep for 6 months. The flock was divided based on their physiological stage as follows: a) pregnant ewes, b) lactating ewes, c) non-pregnant ewes, d) pre-weaning lambs, e) growing lambs and f) replacement sheep. Additionally, sheep were classified based on the parasite infection as stabled or grazing management system as follows: g) non-infected stabled sheep, h) infected and stabled sheep and i) infected grazing sheep. Faeces and blood samples were collected every fifteen days. The percentage of packed cell volume (PCV), plasma protein, (PP) and leucocyte differential counts (LDC) were recorded. Likewise, serum and saliva IgA levels were measured by the indirect ELISA technique. The number of GIN eggs excreted per g of faeces (EPG) was also determined using the McMaster technique. Data were analysed using the SAS program to identify fixed effect of physiological stages, infection time and interactions. Results showed high EPG values in stabled lactating ewes (2764) and in growing lambs (2950-4550 EPG). Pregnant ewes had higher PCV values (27%) than lactating ewes (23.5%). Infected lambs showed low PCV values (28.5%) and high PP values (5.9 g/dL) compared with non-infected lambs (31% PCV and 5.6 g/dL PP). The two most abundant GIN species identified were Haemonchus contortus (91%) and Trichostrongylus colubriformis (81%). Pregnant and non-pregnant ewes showed 29% and <20% of IgA values, respectively. High EPG, low IgA and eosinophils counts were observed until 180 days of age in infected lambs. In conclusion, the group of infected lactating ewes and lambs showed susceptibility to GIN than the other groups, including the pregnant ewes.

Immune response in Blackbelly lambs to Haemonchus contortus and Trichostrongylus colubriformis mixed infection in a hot and humid climate.

The main objective was to determine the immune response of Blackbelly lambs infected with Haemonchus contortus, Trichostrongylus colubriformis, or both species. In the first stage, an experimental challenge was performed with 200 infective larvae (L3)/kg body weight (BW) of H. contortus (Hct, n = 8). Additionally, eight animals were infected with 500 L3/kg BW of T. colubriformis (Tcl), eight lambs with the mixed infection at the same dose (HctTcl), and lambs without infection were the control group. In the second infection, the same lambs were reinfected with high doses: 400 L3/kg BW of Hct and 900 L3/kg BW of Tcl. The third stage consisted of a natural reinfection in grazing. Faecal samples were taken to determine the faecal egg count (FEC) of gastrointestinal nematodes (GINs). IgA was determined in serum and saliva by an indirect Enzyme-Linked Immuno Sorbent Assay (ELISA). The haematological parameters were recorded. With the first challenge, it was not possible to promote an immune response, whereas in the second infection, FEC were higher than 1000 eggs per gram of faeces (EPG) in infected animals. During the third stage, FECs were higher in lambs infected with Tcl. Eosinophils (EOS) did not show differences in the first and second stages, but during grazing the infected lambs had higher counts than the control group (P andlt; 0.05). IgA activity values showed the same trend, and lambs infected with HctTcl had a higher response (33% of the positive standard) than groups mono-infected (16.5% and 22.6%, respectively).

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.

In vitro larvicidal and in vivo anthelmintic effects of Oxalis tetraphylla (Oxalidaceae) hydroalcoholic extract against Haemonchus contortus in lambs.

The in vitro larvicidal and in vivo anthelmintic effects of Oxalis tetraphylla hydroalcoholic extract (HE), against Haemonchus contortus in experimentally infected lambs, were assessed. We used a microtitration plate method, comprising the following two stages. Stage 1: 20 µl of water containing 200 sheathed H. contortus infective larvae (ShHcl) were deposited in every well of three series; then, the series 2 and 3 wells were treated with 80 µl 1% ivermectin and O. tetraphylla HE at 20 mg/ml, respectively. Stage 2: the same procedure was performed replacing the ShHcl with exsheathed larvae (ExShHcl). Evaluations were performed after 24 and 48 h. The total numbers of dead and live larvae were counted. A second experiment evaluated the reduction in nematode egg populations in the faeces of lambs treated orally with the O. tetraphylla HE. The 27 lambs used were divided into Groups 1, 2 and 3 (n = 9), which were administered water (positive control), levamisole 1 m (7.5 mg/kg body weight (BW), as a unique dose) and O. tetraphylla HE (20 mg/kg BW), respectively. The plant HE was administered daily for 8 days. The in vitro assay showed 80.9% and 86.5% larval mortality of ShHcl after 24 and 48 h, respectively, while the corresponding mortality values for ExShHcl were 97 and 99%, respectively. The in vivo assay showed variability in the eggs/gram of faeces (epg) values; however, at the end of the trial, the average reduction in the epg values of the O. tetraphylla HE group was 45.6% (P < 0.05). Oxalis tetraphylla HE contains compounds that belong to the flavonol group with anthelmintic activity.

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.

Cytokine and antioxidant gene profiles from peripheral blood mononuclear cells of Pelibuey lambs after Haemonchus contortus infection.

The expression profiles of cytokines and antioxidant genes were determined from an experimental infection with H. contortus in Pelibuey lambs. The infection was followed for 34 days (d) to determine the number of eggs per gram (epg) and the packed cell volume (PCV). Differential white cell counts and expression profile estimations of IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, FCεR1A, GPX and SOD1 were determined at 0 hour, 4 hours, 2 days and 14 days post-infection (PI) in infected and control groups. Comparison of the fold change between 0 and 4-hours, 4-hours and 2-days and 2- and 14-days periods was performed. Significant differences (P<.05) between epg (>2000) and PCV (>30%) were determined after 21 days and were also observed with regard to monocyte and lymphocyte cells after 2 and 7 days PI. At 0 hour and 14 days PI, the GPX and IL-2 genes showed a 0.37- and 0.49-fold decrease in expression, respectively. In contrast, upregulation was observed at 4 hours of IL-8 (2.58) and FCεR1A (2.71), at 2 days for IL-4 (2.14) and IL-8 (4.02) and at 14 days for IL-2 (0.41), IL-10 (2.35) and FCεR1A (2.28). The comparison between the intervals of infection showed high expression values against H. contortus infection in Pelibuey sheep after the 2nd period of PI involving a dichotomy T cells.

Consumption of nutritional pellets with Duddingtonia flagrans fungal chlamydospores reduces infective nematode larvae of Haemonchus contortus in faeces of Saint Croix lambs.

Two groups of six Haemonchus contortus infected Saint Croix lambs each received different diets for 11 weeks: control group, commercial food, molasses and lucerne hay; and treated group, nutritional pellets (NPs) containing Duddingtonia flagrans at 2 × 106 chlamydospores/kg body weight (BW), sorghum and lucerne hay. Mean BW gain (BWG), body condition score (BCS) and packed cell volume (PCV) and also eggs/g of faeces (EPG) and recovered L3 were compared using a repeated measures across time model. Groups had similar BWG (control 139.7 ± 0.035 g/day and treated 167.7 ± 0.041 g/day), BCS (control 3.6 ± 0.39 and treated 3.4 ± 0.46) and PCV (control 32.5 ± 1.68% and treated 30.0 ± 1.68%). The mean EPG of the control group was 1215 ± 1040 and in the treated group it was 2097.91 ± 2050. No reduction in larval population was observed during weeks 2 and 3. The greatest larval population reduction in the faeces of treated lambs was observed during the first week (70.5%) and from weeks 6 to 11, with a mean value close to 70% (P < 0.05). In general, both experimental groups showed a similar feed conversion. It was concluded that both diets resulted in similar lamb growth, PCV, BCS and H. contortus EPG. However, NP consumption significantly reduced the H. contortus L3 population in lamb faeces.

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.

Duddingtonia flagrans chlamydospores in nutritional pellets: effect of storage time and conditions on the trapping ability against Haemonchus contortus larvae.

The study evaluated the effect of storage time and conditions of nutritional pellets (NP) containing Duddingtonia flagrans chlamydospores on its in vitro trapping ability against Haemonchus contortus L3 larvae. The treated batch (200 NP) contained 4 × 106 chlamydospores of the FTH0-8 strain, whereas the control batch (200 NP) was produced without spores. Both NP batches were exposed to four experimental storage conditions: (T1) shelves (indoors); (T2) refrigeration (4°C); (T3) outdoors under a roof; and (T4) 100% outdoors. Each group comprised 48 NP with spores and 48 NP without spores (control). The ability of D. flagrans spores to trap H. contortus L3 larvae was evaluated for 8 weeks for each storage condition. For that purpose, six randomly selected NP with spores were compared to their respective control NP. Each NP was individually crushed. The crushed material (1 g) was placed on the surface of a 2% water agar plate with 200 H. contortus L3 larvae. Plates were sealed and were incubated at room temperature for 8 days. The whole content of every plate was transferred to a Baermann apparatus to recover the remaining larvae. There was a clear larval reduction in the NP with spores, compared to the respective control NP in the four storage conditions (P< 0.05). The mean reductions ( ± SEM) of the storage conditions were 67 ± 4.9 (T2), 77 ± 6.1 (T1), 81.5 ± 3.8 (T4) and 82.1 ± 2.5 (T3). Larval reductions were similar at all times and were not affected by storage conditions or storage time (R 20.05). The long-term shelf-life of the chlamydospores in the NP suggests that this spore dosage technology is a viable option.

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.

Evaluation of predation of the mite Lasioseius penicilliger (Aracnida: Mesostigmata) on Haemonchus contortus and bacteria-feeding nematodes.

Predation by the mite Lasioseius penicilliger was studied on three nematode species, i.e. infective larval stages (L3) of Haemonchus contortus and adults of Panagrellus redivivus and Rhabditis sp. Experiments were carried out in 5.5-cm diameter Petri dishes containing 2% water-agar over a period of 5 days. Batches of up to 1500 third-stage larvae (L3) of H. contortus and 1000 adult nematodes of P. redivivus and Rhabditis sp. were exposed to five mites in separate Petri dishes. Upon contact, each mite used its pedipalp and legs to identify and hold its prey and then used its chelicerae to feed upon the prey. Predation by L. penicilliger was chance dependent but mites became aggregated around any injured/damaged prey, thereby suggesting some form of chemoperception. The rate of predation on the three species of nematodes was high but L3 of H. contortus and adult Rhabditis sp. were preferred.

Ability of the fungus Duddingtonia flagrans to adapt to the cyathostomin egg-output by spreading chlamydospores.

The analysis of the capability of the nematode trapping-fungus Duddingtonia flagrans to adapt to the cyathostomin egg-output in horses was evaluated. Fecal samples from 196 pasturing autochthonous Pura Raza Galega horses were collected from the rectum and then divided according to the egg-output into three groups: ≤ 300, 310-800 and >800 eggs per gram feces. Four doses of chlamydospores (0.1, 0.2, 0.4 and 0.8 × 10(6)/100g feces) were directly spread onto fecal pats on the ground, remaining one without treatment as control. Fecal pats confirmed the presence of gastrointestinal nematode larvae belonging to strongylid cyathostomins (Cyathostomum and Gyalocephalus spp). An overall 94% (95% CI 91, 97) percentage of reduction was obtained, and an increase in the activity of the trapping-fungi simultaneously to the rising in the number of cyathostomin eggs and larvae in the coprocultures was detected. A significantly highest reduction of the cyathostomin L3 in the coprocultures with more than 800 EPG was found, which indicates that Df trapping activity is larvae nematode density-dependant. The present research showed the high biological activity of D. flagrans against nematode larvae can adjust to the cyathostomin egg-output, and underlines its efficacy as a practical method for the control of these parasites in grazing horses.

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.