J. Moreno-Gonzalo

Is the anthelmintic effect of heather supplementation to grazing goats always accompanied by anti-nutritional effects?

To test the hypothesis that the beneficial anthelmintic effect of consuming moderate amounts of tannins may not always be accompanied by anti-nutritional effects in goats, two experiments were conducted. In the first, 48 Cashmere goats were randomly assigned to two treatments: supplementation with tannin-containing heather (6.4% total tannins) and non-supplementation. All goats grazed continuously from May to September under farm conditions in a mountainous area of northern Spain. The mean percentage of heather incorporated into the diet of the supplemented animals was 29.1%. Supplementation reduced the mean number of nematode eggs in faeces (P < 0.001) and the goat mortality rate (P < 0.05). The rumen ammonia concentration was markedly reduced in the goats receiving the heather supplement (160 v. 209 mg/l; P < 0.01), while volatile fatty acid (VFA) concentrations were significantly greater (63.0 v. 53.6 mmol total VFA/l; P < 0.05). The heather-supplemented goats also showed a lower loss of live weight (P < 0.01) and body condition score (P < 0.001). In the second experiment, batch cultures of rumen microorganisms with rumen fluid from nine goats whose diet included 29% heather - or not, were used to incubate three substrates (pasture, pasture + heather and pasture + heather + polyethylene glycol) to investigate in vitro ruminal fermentation. Differences (P < 0.01) among substrates were observed in terms of dry matter disappearance (DMD), in vitro true substrate digestibility (ivTSD), gas production and ammonia concentration, the greatest values always associated with the pasture substrate. Cultures involving rumen inoculum derived from goats receiving the heather-containing diet showed slightly lower DMD (46.9 v. 48.5 g/100 g; P < 0.05), ivTSD (64.6 v. 65.9 g/100 g; P < 0.10) and gas production (105 v. 118 ml/g; P < 0.001) values, but much greater total VFA concentrations (48.5 v. 39.3 mmol/l; P < 0.05), and suggest that the efficiency of ruminal fermentation in these animals was probably improved. Together, the results support the absence of a clear nutritional cost counteracting the beneficial anthelmintic effect of supplementing the diet of grazing goats with tannin-containing heather.

Placental thrombosis in acute phase abortions during experimental Toxoplasma gondii infection in sheep

After oral administration of ewes during mid gestation with 2000 freshly prepared sporulated oocysts of T. gondii isolate M4, abortions occurred between days 7 and 11 in 91.6% of pregnant and infected ewes. Afterwards, a further infection was carried out at late gestation in another group of sheep with 500 sporulated oocysts. Abortions happened again between days 9 and 11 post infection (pi) in 58.3% of the infected ewes. Classically, abortions in natural and experimental ovine toxoplasmosis usually occur one month after infection. Few experimental studies have reported the so-called acute phase abortions as early as 7 to 14 days after oral inoculation of oocysts, and pyrexia was proposed to be responsible for abortion, although the underline mechanism was not elucidated. In the present study, all placentas analysed from ewes suffering acute phase abortions showed infarcts and thrombosis in the caruncullar villi of the placentomes and ischemic lesions (periventricular leukomalacia) in the brain of some foetuses. The parasite was identified by PCR in samples from some placentomes of only one sheep, and no antigen was detected by immunohistochemical labelling. These findings suggest that the vascular lesions found in the placenta, and the consequent hypoxic damage to the foetus, could be associated to the occurrence of acute phase abortions. Although the pathogenesis of these lesions remains to be determined, the infectious dose or virulence of the isolate may play a role in their development.

In vitro effect of heather extracts on Trichostrongylus colubriformis eggs, larvae and adults

This study was carried out to evaluate the in vitro effects of different heather species on Trichostrongylus colubriformis eggs, larvae and adult worms, and obtain scientific evidence to attribute these effects to the action of their phenolic compounds and/or tannins. Total phenolic extracts of three heather species (Calluna vulgaris, Erica cinerea, and Erica umbellata) and an equal mixture of these three extracts were tested in vitro in the three development stages of T. colubriformis using an egg hatching assay (EHA), larval exsheathment inhibition assay (LEIA), and adult motility inhibition assay (AMIA). The egg hatching rate was measured after incubation with heather extracts for 48 h at 25°C. Infective third-stage larvae (L3) were incubated for 3h at 25°C with heather extracts. The evolution of artificial exsheathment over time was measured with repeated observations at 20-min intervals for 60 min. Adult worms were obtained from one donor goat and incubated with the extracts at 37°C for 5 days in 48-multiwell plates. Worm motility was measured at 0, 19, 24, 43, 48, 67, 72, 96 and 115 h after the beginning of the experiment. The extracts were tested at concentrations of 75, 150, 300, 600 and 1,200 μg/ml. All extracts significantly (P<0.001) inhibited egg hatching and the effect was dose dependent. All extracts inhibited or delayed the exsheathment of T. colubriformis L3, and the effect was dose dependent for C. vulgaris. Incubation with heather extracts induced a reduction in adult worm motility compared to control, although significant (P<0.05) differences were only found at the highest concentrations. Additional studies showed that purified tannins of the same heather species disturbed T. colubriformis larval exsheathment. All these results confirm the anthelmintic properties of heather against T. colubriformis, and suggest that not only tannins but also some other phenolic compounds might be involved.

In vitro effect of heather (Ericaceae) extracts on different development stages of Teladorsagia circumcincta and Haemonchus contortus.

The aim of the present study was to evaluate the in vitro effects of heather (Ericaceae) phenolic extracts on the abomasal nematodes Teladorsagia circumcincta and Haemonchus contortus. Extracts of three heather species (Calluna vulgaris, Erica cinerea, Erica umbellata and a balanced mixture of all three) were tested in vitro on different development stages of T. circumcincta (eggs, infective larvae and adult worms) and H. contortus (eggs and infective larvae) using an egg hatching assay (EHA), a larval exsheathment inhibition assay (LEIA) and an adult motility inhibition assay (AMIA). The egg hatching rate was measured after incubation with heather extracts for 48 h at 25 °C. Ensheathed infective larvae were incubated for 3h at 20 °C with heather extracts. Artificial exsheathment was induced in vitro by adding hypochloride solution to the larval suspension. The progress of exsheathment over time was measured by repeated observations at 10-min (T. circumcincta) and 20-min (H. contortus) intervals for 60 min. Adult T. circumcincta worms were obtained from two donor goats and incubated with the extracts at 37 °C for 3 days in 48-well multiwell plates. Worm motility was measured at 0, 19, 24, 43, 48, 67 and 72 h. The extracts were tested at concentrations of 75, 150, 300, 600 and 1200 μg/mL. Incubation with E. cinerea, E. umbellata and mixed heather extracts had a significant (P<0.01) dose-dependent effect on T. circumcincta egg hatching. H. contortus egg hatching was significantly (P<0.01) inhibited only by the E. cinerea extract. All extracts had a significant (P<0.01) dose-dependent effect on the exsheathment of T. circumcincta and H. contortus infective larvae. The incubation with all heather extracts induced a reduction in adult T. circumcincta motility compared to the control, although significant (P<0.05) differences were only found at the highest concentration (1200 μg/mL). The effect of the mixed extract was significant at all concentrations and significant effects were also observed for C. vulgaris and E. umbellata at 600 μg/mL. These results show anthelmintic properties of heather phenolic extracts against T. circumcincta and H. contort, thus confirming observations from previous in vivo studies.

Development of a murine vertical transmission model for Toxoplasma gondii oocyst infection and studies on the efficacy of bumped kinase inhibitor (BKI)-1294 and the naphthoquinone buparvaquone against congenital toxoplasmosis

Objectives Establishment of a mouse model for congenital toxoplasmosis based on oral infection with oocysts from Toxoplasma gondii ME49 and its application for investigating chemotherapeutic options against congenital toxoplasmosis. Methods CD1 mice were mated, orally infected with 5, 25, 100, 500 or 2000 oocysts and monitored for clinical signs and survival of dams and pups until 4 weeks post partum . The parasite burden in infected mice was quantified by real-time PCR in lungs, brains and, in the case of surviving pups, also in eyes. Seroconversion was assessed by ELISA. T. gondii cysts in brain were identified by immunofluorescence. In a second experiment, pregnant CD1 mice challenged with 20 oocysts/mouse were treated with buparvaquone or the calcium-dependent protein kinase 1 inhibitor bumped kinase inhibitor (BKI)-1294 and the outcome of infection was analysed. Results T. gondii DNA was detected in the brain of all infected animals, irrespective of the infection dose. Seroconversion occurred at 3 weeks post-infection. Most pups born to infected dams died within 1 week post partum , but a small fraction survived until the end of the experiment. T. gondii DNA was detected in the brain of all survivors and half of them exhibited ocular infection. Chemotherapy with both compounds led to dramatically increased numbers of surviving pups and reduced cerebral infection. Most efficient were treatments with BKI-1294, with 100% survivors and only 7% brain-positive pups. Conclusions BKI-1294 and buparvaquone exert excellent activities against transplacental transmission in pregnant mice.

Effect of the consumption of heather on incoming larvae and established population of Teladorsagia circumcincta in experimentally infected Cashmere goats

This study was performed in Cashmere goats that were experimentally infected with Teladorsagia circumcincta to investigate the effects of heather consumption on the establishment of incoming infective larvae (experiment 1) and on an adult nematode population (experiment 2). In experiment 1, 24 non-lactating goats were divided into 2 groups: heather-supplemented vs. non-supplemented. After 2 weeks of adaptation to the diet, all of the goats were experimentally infected with 6000 infective larvae of T. circumcincta. Twelve animals (6 controls and 6 supplemented with heather) were slaughtered at 6 days and at 3 weeks post-infection (pi). After slaughter, the worms were counted and the female worm fecundity and development were determined. Heather consumption was associated with a significant reduction in larval establishment at 6 days (P=0.033) and at 3 weeks (P=0.041) pi. No differences in worm counts between the slaughter times were found. In the goats slaughtered at 3 weeks pi, the number of eggs in utero and length of the female worms were significantly (P<0.001) lower than those of control group. In experiment 2, 24 non-lactating goats were experimentally infected with 10,000 T. circumcincta infective larvae daily for 5 consecutive days (total infection of 50,000 larvae). After 3 weeks, 2 groups were established: control and heather-supplemented. The faecal egg output of each animal was measured at 2-days interval during the experimental period. The goats were slaughtered after 3 weeks of heather administration for parasitological studies. Heather administration was associated with a significant decrease in egg excretion between 25 and 29 days pi. The worm counts were similar in both groups, but the female length and fecundity were significantly (P<0.001) lower in supplemented goats. These results show that heather consumption reduces the establishment of T. circumcincta larvae in goats and the development and fecundity of female adult parasites.

Experimental ovine toxoplasmosis: influence of the gestational stage on the clinical course, lesion development and parasite distribution

The relation between gestational age and foetal death risk in ovine toxoplasmosis is already known, but the mechanisms involved are not yet clear. In order to study how the stage of gestation influences these mechanisms, pregnant sheep of the same age and genetic background were orally dosed with 50 oocysts of Toxoplasma gondii (M4 isolate) at days 40 (G1), 90 (G2) and 120 (G3) of gestation. In each group, four animals were culled on the second, third and fourth week post infection (pi) in order to evaluate parasite load and distribution, and lesions in target organs. Ewes from G1 showed a longer period of hyperthermia than the other groups. Abortions occurred in all groups. While in G2 they were more frequent during the acute phase of the disease, in G3 they mainly occurred after day 20 pi. After challenge, parasite and lesions in the placentas and foetuses were detected from day 19 pi in G3 while in G2 or G1 they were only detected at day 26 pi. However, after initial detection at day 19 pi, parasite burden, measured through RT-PCR, in placenta or foetus of G3 did not increase significantly and, at in the third week pi it was lower than that measured in foetal liver or placenta from G1 to G3 respectively. These results show that the period of gestation clearly influences the parasite multiplication and development of lesions in the placenta and foetus and, as a consequence, the clinical course in ovine toxoplasmosis.