C.A. Sandoval-Castro

Direct and indirect effects of bioactive tannin-rich tropical and temperate legumes against nematode infections

Parasitic infections with gastrointestinal nematodes (GINs) represent a major pathological threat associated with the outdoor production of various livestock species. Up to now, the control of these parasitic diseases essentially relied on the use of commercial anthelmintic drugs. However, resistance to anthelmintics is nowadays widespread in worm populations. Recent results indicate that bioactive tanniniferous plants represent a valuable option as an alternative to commercial drugs for the control of GINs. The pertinent use of tannin-containing fodders as nutraceuticals supposes a clear understanding of the mode of action against the worms. The objectives of this paper are: (1) to discuss the nature and quantity of the active molecules involved in the anthelmintic activity; and (2) to review and analyze the changes provoked to the various parasitic stages. The possible involvement of some main polyphenols to explain the bioactivity of some tannin-rich plants will be discussed as well as the possible effects on the various nematode stages, relying on data obtained either with the temperate forage, sainfoin (Onobrychis viciifoliae) or with the tropical legume tree (Lysiloma latisiliquum). The information on the mode of action will be related to the potential consequences for better field applications under entirely different environmental and ecological conditions of productions.

In vitro larval migration and kinetics of exsheathment of Haemonchus contortus larvae exposed to four tropical tanniniferous plant extracts

As for some temperate forage, some tropical tanniniferous plants (TTP) from browsing might represent an alternative to chemical anthelmintic. The anthelmintic effect of four TTP (Acacia pennatula, Lysiloma latisiliquum, Piscidia piscipula, Leucaena leucocephala) on Haemonchus contortus was measured using two in vitro assays. First, the effects of increasing concentrations of lyophilized extracts (150, 300, 600, 1200 microg/ml PBS) were tested on H. contortus larvae (L(3)) using the larval migration inhibition (LMI) test. An inhibitor of tannin, polyvinyl polypyrrolidone (PVPP), was used to verify whether tannins were responsible for the AH effect. Secondly, the effects of extracts on larval exsheathment were examined. Larvae (L(3)) were in contact with extracts (1200 microg/ml) for 3h, and then were exposed to an artificial exsheathment procedure with observations of the process at 10 min intervals. A general lineal model (GLM) test was used to determine the dose effect in the LMI test and the difference of the percentage of exsheathed larvae between the control and the treatment groups. A Kruskal Wallis test was used to determine the effect of PVPP on LMI results. The LMI test showed a dose-dependent anthelmintic effect for A. pennatula, L. latisiliquum and L. leucocephala (P<0.01), which disappeared after PVPP addition, confirming the role of tannins. No effect was found for P. piscipula on H. contortus in the LMI test. However, all four plant extracts interfered with the process of L(3) exsheathment which might be involved as a mechanism of action of tannins on H. contortus larvae. A. pennatula, L. latisiliquum and L. leucocephala could be used as an anthelmintic for the control of H. contortus after confirmation based on in vivo studies.

Effect of a tropical tannin-rich plant Lysiloma latisiliquum on adult populations of Haemonchus contortus in sheep.

Bioactive plants with anthelmintic (AH) properties represent a promising alternative solution to chemical treatments. The AH effect of several Mexican tannin-rich (TR) plants has been screened in vitro. The in vivo AH effect of one TR legume, Lysiloma latisiliquum (Tzalam) on nematode larval establishment was confirmed. The present trial aimed at evaluating the direct and indirect effects of L. latisiliquum fodder consumption on adult Haemonchus contortus. Twenty-two parasite-naïve hair sheep lambs were allocated to an infected group (I) (400H. contortus L(3)/kg BW on D0) and a non-infected group (NI). From D0 to D28 post infection (PI), all the lambs were fed a complete diet. On D28, the two groups were sub-divided into four groups. Two control (C) groups maintained on the original basal diet (CI: 6 infected lambs and CNI: 5 non-infected lambs). The two treatment groups (T) received L. latisiliquum fodder ad libitum up to D36 when lambs were humanely slaughtered (TI: 6 infected lambs and TNI: 5 non-infected lambs). From D28 to D36 PI, individual fodder consumption and nematode egg excretion were measured daily. At necropsy, abomasal contents were recovered to obtain worm burdens and measure the female worm length and fecundity. Histological samples were taken from the respective abomasums and small intestines to count mucosal inflammatory cells. An increased consumption of TR fodder was observed in the TI vs. the TNI group (P<0.01). Before L. latisiliquum distribution, faecal egg excretion was similar in TI and CI groups. From D29 PI the TI group showed lower faecal egg counts compared to CI group (P<0.02). Although no differences in worm burdens were observed, worms of the TI group were smaller and, according to their size, contained fewer eggs in utero than worms from the CI group (P<0.05). Only minor differences in mucosal inflammatory cells were observed between groups, indicating that the indirect effect was not evident. Thus, a short-term consumption of L. latisiliquum can modulate directly the biology of adult H. contortus affecting the worm size and female fecundity while the worm burdens were not affected. Infected animals ate more L. latisiliquum fodder than non-infected animals.

Effects of four tropical tanniniferous plant extracts on the inhibition of larval migration and the exsheathment process of Trichostrongylus colubriformis infective stage.

The anthelmintic (AH) effect of Acacia pennatula, Leucaena leucocephala, Lisyloma latisiliquum and Piscidia piscipula was evaluated in the infective larvae (L(3)) of Trichostrongylus colubriformis. Different concentrations of lyophilized extracts were tested using the larval migration inhibition (LMI) test. An inhibitor of tannins (the polyvinyl polypyrrolidone [PVPP]) was used to verify whether these compounds were responsible for the AH effects. Then, the effect of extracts on larval exsheathment was examined by observing the exsheathment process at 10-min intervals for 70 min. The LMI test showed a dose-dependant AH effect for A. pennatula, L. leucocephala and L. latisiliquum (P<0.01), but not for P. piscipula. The restoration of L(3) migration to values similar to those of controls after the addition of PVPP, indicates that tannins are involved in AH effects. Trichostrongylus colubriformis exsheathment was partially or totally blocked by the four plants extracts. Tropical tanniniferous plants evaluated in the current study may have potential as AH for the control of T. colubriformis if in vivo investigations indicate useful effects.

Effect of the consumption of Lysiloma latisiliquum on the larval establishment of gastrointestinal nematodes in goats

The consumption of tannin-rich (TR) forages has been associated with negative effects against gastrointestinal nematodes and with an improved host resilience. It has been hypothesized that tannins affect the capacity of infective larvae to establish in the mucosae of the host. In this study, we aimed at testing this hypothesis using Lysiloma latisiliquum, a tropical TR tree. The objectives were: (i) to evaluate the effect of the consumption of L. latisiliquum on the establishment of nematode third-stage larvae (L3) in goats; (ii) to define the role of tannins in these effects in vivo by using an inhibitor (polyethylene glycol, PEG); and (iii) to examine a possible indirect effect of tannins on the inflammatory response in the digestive mucosa. Eighteen Criollo goats composed three experimental groups. The control group received fresh leaves of Brosimum alicastrum, a plant with a low level of tannins. Two groups received L. latisiliquum leaves either with (L.L.+PEG) or without (L.L.) daily addition of 25 g PEG. After a 7-day adaptation period, each goat was infected with both Haemonchus contortus and Trichostrongylus colubriformis (3000 L3 per species). The goats were slaughtered 5 days after infection and worm counts and histological analyses were performed. No difference in the voluntary feed intake of foliage was observed between the 3 groups. The consumption of L. latisiliquum significantly reduced the larval establishment of both nematode species compared to the control (P<0.01). For both worm species, the effects were totally alleviated with PEG (L.L.+PEG group), suggesting a major role of tannins in the observed effects. Only minor differences in the mucosal cellular response were observed between the 3 groups. These results confirm that the consumption of TR plants reduces the establishment of nematode larvae in the host and that a direct effect is principally involved.

Scanning electron microscopy of Haemonchus contortus exposed to tannin-rich plants under in vivo and in vitro conditions

The structural changes induced in adult Haemonchus contortus after in vitro and in vivo contact with a tannin-rich (TR) plant, either tzalam (Lysiloma latisiliquum) or sainfoin (Onobrychis viciifolia), were assessed using scanning electron microscopy (SEM). All the worms used in the study were adult females. The Haemonchus adult worms were obtained from the abomasum of infected donor goats. Adult H. contortus were kept in contact with the extracts of TR plants for 24h for the in vitro assays and were compared to worms maintained in PBS (control). For the in vivo studies, the adult H. contortus parasites were obtained from artificially infected goats. All the goats were fed a tannin-free diet until D27 post-infection when infection was patent. On D28 some goats were fed fodder of one of the TR plants for seven consecutive days. Thus, their H. contortus were in contact with TR fodder within the gastrointestinal tract. The control worms were obtained from goats fed only a tannin-free diet. In the in vitro assays and in vivo studies, the SEM observations revealed structural alterations in the worms after contact with TR plants when compared to the control worms (i.e.: longitudinal and transversal folds and thicker cuticular ridges, material aggregates around the buccal capsule and/or vulva or anus). The main changes concerned the cuticle and the buccal area. The structural changes found in the worms exposed to TR plants might affect their motility and nutrition with possible consequences on their reproduction, as suggested by previous in vivo trials.

In vitro acaricidal effect of tannin-rich plants against the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

The objectives of this study were to evaluate the in vitro acaricidal effects of lyophilized extracts of four tannin rich plants (Acacia pennatula, Piscidia piscipula, Leucaena leucocephala and Lysiloma latisiliquum) against diverse stages of Rhipicephalus (Boophilus) microplus, and to asses whether tannins were involved in the acaricidal effect using polyethylene glycol (PEG) to block tannins. Larval immersion (LIT) and adult immersion (AIT) tests were used to evaluate the acaricidal effect of each of the lyophilized extracts against larval and adult stages of R. microplus respectively. Larvae and adult ticks were exposed to increasing concentrations of each plant extract (0, 1200, 2400, 4800, 9600 and 19,200 μg ml(-1)) for 10 min. Larval mortality was recorded at 48 h post-incubation. Adult mortality was recorded daily over 14 days, at which point their reproductive efficiency was evaluated. PEG was added to the extracts to verify whether tannins were involved in the acaricidal effect. The effect on egg laying inhibition and larval mortality was analyzed using the GLM procedure in SAS. A Kruskal-Wallis test was used to assess the effect of PEG on LIT results. Calculation of the lethal concentration 50 (LC50) was performed using a probit analysis. All extracts reduced the viability of R. microplus larval stages (P<0.001), and viability was restored with the addition of PEG suggesting an important role of tannins in the acaricidal effect (P<0.001). The LC50 values of L. latisiliquum and P. piscipula plant extracts were 6.402 and 2.466 μg ml(-1). None of the tannin-rich plant extracts affected adult mortality (P>0.05). Lysiloma latisiliquum extract inhibited egg hatching of R. microplus (P<0.01). Tannin-rich plant extracts from A. pennatula, P. piscipula, L. leucocephala and L. latisiliquum showed potential acaricidal activity. Further in vivo studies are needed to confirm this finding.

Comparing the sensitivity of two in vitro assays to evaluate the anthelmintic activity of tropical tannin rich plant extracts against Haemonchus contortus.

The present trial aimed at comparing the sensitivity of two in vitro methods, i.e. the larval migration inhibition assay (LMIA) and the larval exsheathment inhibition assay (LEIA), to evaluate the anthelmintic (AH) properties of tannin-rich plant extracts against Haemonchus contortus infective larvae. The two assays were applied on the same batch of H. contortus infective larvae exposed to water/acetonic extracts obtained from four tropical plants with different tannin contents: Acacia gaumeri, Brosimum alicastrum, Havardia albicans and Leucaena leucocephala. Increasing concentrations (0, 75, 150, 300, 600, 1200 μg/ml PBS) of lyophilized extracts were used in both in vitro assays. A general lineal model test was used to determine the dose-effect in the LMIA or the difference in the percentage of exsheathed larvae between the respective control and treated groups. The LMIA showed a dose-dependent AH effect for H. albicans (P<0.001) and A. gaumeri (P<0.05), but not for L. leucocephala and B. alicastrum. In contrast, the exsheathment process was significantly affected by all doses of H. albicans and A. gaumeri extracts and a significant dose-dependent effect was found for B. alicastrum and L. leucocephala. Calculation of lethal dose (LD) was possible with LEIA using B. alicastrum and L. leucocephala but not with H. albicans and A. gaumeri as the lowest tested concentration was achieving more than 50% inhibition. Calculation of LD with the LMIA results was not feasible. These results suggest that tannin-rich plant extracts are more potent inhibitors of the exsheathment of H. contortus L(3) larvae than their motility. This information underlines the difference of sensitivity between methodological procedures to evaluate the AH properties of plant extracts on the same nematode stage.

Assessing the efficacy of Duddingtonia flagrans chlamydospores per gram of faeces to control Haemonchus contortus larvae.

The aims were (a) to quantify the number of Duddingtonia flagrans chlamydospores per gram of faeces (CPG) recovered from sheep administered with different oral doses and, (b) to describe the relationship between CPG and eggs per gram of faeces (EPG) on the efficacy to reduce Haemonchus contortus infective larvae. Three doses of chlamydospores per kg BW were orally administered during seven days: (T1) non treated control group, (T2) 1 x 10(6), (T3) 2.5 x 10(6) and (T4) 5 x 10(6). Three lambs, infected with H. contortus, were used per group. Faeces were obtained from the rectum of each lamb during the fungal administration period (days 0-6) and for six days after that period. Four coproculture replicates were made from each animal in days 2, 4, 6, 8 and 10. A higher chlamydospore dose produced higher CPG in faeces (p < 0.05), but a clear dose dependent effect was not found either in the larvae reduction or in the CPG:EPG ratio. When ratios were re-analyzed, independently of the treatment groups of origin, a better efficacy was obtained with a ratio from 5 to 10 CPG:EPG and a higher ratio (> 10 per egg) showed a lower reduction efficacy (p < 0.05). The binomial analysis showed that for each unit of increment in CPG:EPG ratio there was a reduction of larvae number until a point (between 5 and 10 CPG:EPG) where no further reduction was detected. The surface response test indicated that the number of larvae was reduced by CPG until possible saturation. The highest CPG:EPG ratios did not necessarily improve efficacy of D. flagrans.

Production responses of tropical crossbred cattle to supplementary feeding and to different milking and restricted suckling regimes.

Abstract The relationships between cow nutrition, restricted suckling and milking patterns and their effect on milk yield, milk composition and calf growth were examined under tropical conditions in south east Mexico. Eleven Bos indicus×Bos taurus cows, 14 days postpartum, with calves weighing on average 37 kg were used in a change-over experimental design. All cows were fed Cynodon nlemfuensis hay ad libitum, and either 4.0 kg Leucaena leucocephala, 2.0 kg sorghum grain and 0.25 kg sugar-cane molasses on a fresh weight basis (L1), or twice this amount of the same supplement (L2). The calves were offered the same hay ad libitum in addition to suckling milk. The milking/restricted suckling treatments were: twice-daily milking with suckling after morning milking (2×AM), and once-daily milking in the morning with afternoon suckling (1×PM). The two regimes were examined at L1 and L2 feeding levels. A suckling time of 30 min was allowed at each suckling occasion. For 2×AML1, 2×AML2, 1×PML1 and 1×PML2, respectively saleable milk yields (SMYs) (kg/day) were 5.43, 5.99, 3.47, 4.09 (S.E.M. 0.174); calf suckled milk (CSM) (kg/day) 1.36, 1.46, 3.14, 3.43 (S.E.M. 0.160); and total milk yield (kg/day) 6.79, 7.46, 6.62, 7.53 (S.E.M. 0.226). For SMYs, fat contents (g/kg) were 30.0, 29.4, 29.0, 28.7 (S.E.M. 1.11), respectively, and for CSM 59.6, 63.4, 38.8, 42.4 (S.E.M. 2.46). Protein contents (g/kg) for SMYs were 27.5, 29.1, 29.3, 30.4 (S.E.M. 0.67) and for CSM 27.9, 28.4, 29.1, 30.3 (S.E.M. 0.81). Total food intakes of cows were 10.84, 12.12, 10.99, 12.16 (S.E.M. 0.331) kg DM/day. Calf growth rates were, respectively, 0.17, 0.20, 0.41, 0.52 (S.E.M. 0.029) kg/day, and hay DM intake 0.58, 0.49, 0.41, 0.34 (S.E.M. 0.040) kg/day. It was concluded that level of supplementation and restricted suckling and milking regimes could both be used to manipulate TMY, SMY and calf performance.