Procyanidin A2 in the Australian plant Alectryon oleifolius has anthelmintic activity against equine cyathostomins in vitro.

There is a need to investigate new methods of controlling cyathostomins in horses due to increasing anthelmintic resistance amongst these parasites. In a previous study we identified the Australian plant Alectryon oleifolius as having anthelmintic activity towards cyathostomins. This study aimed to isolate and identify the bioactive compound(s) responsible for all or part of this anthelmintic activity and quantify its activity in vitro. The condensed tannin procyanidin A2 was isolated from the plant through a process of bioassay guided fractionation and identified using 1D and 2D nuclear magnetic resonance spectroscopy and high performance liquid chromatography with mass spectrometry. Procyanidin A2 demonstrated significant anthelmintic activity in larval development assays, completely inhibiting development from egg to third larval stage at concentrations as low as 50µg/mL and having an IC50 value of 12.6µg/mL. Procyanidin A2 also significantly inhibited larval migration at concentrations of 25µg/mL. This study indicates that procyanidin A2 is the principal anthelmintic compound in extracts from A. oleifolius, and further highlights the potential for the use of this plant as a component of cyathostomin control programs in the future.

Surgical and postoperative events during permanent fistulation of sheep rumen by the Schalk and Amadon method.

OBJECTIVE: To describe and report on the course of events during and after surgical fistulation of sheep rumen by the Schalk and Amadon method and on improvements to address current trends in animal health, care and welfare. METHODS: A permanent re-entry fistula was created in 13 sheep using a method in which a fold of rumen is exteriorised and held by a metal clamp. Following surgery, sheep were monitored daily for any abnormalities for 4 weeks, then weekly for 6 months. RESULTS: Permanent fistulation was achieved in all 13 sheep; 2 sheep had minor complications: one during the surgery (rumen perforation) and one on the day after surgery (slipping of the metal clamp), but both recovered within 48 h with treatment. Over the 4 weeks post-surgery, 7 sheep had mildly elevated rectal temperatures, which resolved with non-steroidal anti-inflammatory drugs, systemic antibiotics and/or topical antiseptic cream. There was an 8-day variation among sheep in the time from surgery to creation of the fistula. CONCLUSION: Rumen fistulation of sheep by the Schalk and Amadon method, as described here, is a comparatively simple, safe and ethical procedure, with minimal effect on or complications for the animal.

Australian plants show anthelmintic activity toward equine cyathostomins in vitro.

Anthelmintic resistance in gastrointestinal parasites of horses is an increasing problem, particularly in cyathostomins, and there is a need to find alternative means for the control of these parasites. We screened crude extracts from 37 species of Australian native plants for their anthelmintic activity in vitro against cyathostomin larvae (development from egg to third larval stage), with the aim of identifying those species that may be suitable for incorporation into sustainable parasite management programs. Water extracts from seven species, namely Acacia baileyana, Acacia melanoxylon, Acacia podalyriifolia, Alectryon oleifolius, Duboisia hopwoodii, Eucalyptus gomphocephala and Santalum spicatum completely inhibited larval development (100% inhibition compared to the control), while another 10 species caused 90% inhibition at the initial screening concentration of 1400 µg of extractable solids/mL. The seven most potent extracts produced IC50 values (concentration of extract which resulted in a 50% inhibition of development) in the range 30.9-196 µg/mL. Fourteen extracts were incubated with polyvinylpolypyrrolidone (PVPP) before the assays, which removed the anthelmintic activity from 12 of these extracts, indicating that tannins were likely to be the bioactive compound responsible for the effect, while in two species, i.e. A. melanoxylon and D. hopwoodii, compounds other than tannins were likely to be responsible for their anthelmintic action. Our results suggest that a number of Australian native plants have significant anthelmintic activity against cyathostomin larval development in vitro. There is potential for these plants to be used as part of sustainable parasite control programs in horses, although more research is needed to identify the compounds responsible for the anthelmintic effects and confirm their activity in vivo.

Eremophila glabra is an Australian plant that reduces lactic acid accumulation in an in vitro glucose challenge designed to simulate lactic acidosis in ruminants.

Lactic acidosis is a major welfare issue affecting animal health and production systems such as dairy and feedlot beef. We used two bioassays to identify bioactive plants of Australia with the potential to prevent acidosis in ruminants. In the first bioassay, a potentially acidotic environment was induced by adding glucose to rumen fluid and pH and gas production were used to estimate the effect on acid production and microbial fermentation after 5-h incubation. Australian plants (n = 104) were screened for their ability to prevent a decline in the pH without inhibiting normal gas production, and five plants namely Eremophila glabra, Kennedia eximia, Acacia saligna, Acacia decurrens and Kennedia prorepens with such properties were identified. We investigated further the two top ranking plants, E. glabra and K. prorepens, in the second bioassay to determine the extent of their effect in vitro, by extending the incubation to 24 h and measuring d-lactate, and volatile fatty acids (VFA) in addition to pH and gas production. These were measured at 0, 5, 10, 16 and 24 h after inoculation. Eremophila glabra maintained pH values that were higher and d-lactate concentrations that were lower than the control (P < 0.001), and comparable to the antibiotic-protected environment (AB; 12 µg of virginiamycin/ml). Eremophila glabra and AB treatments did not restrict fermentation, as judged by gas production and VFA. Kennedia prorepens slowed the decline in pH and reduced the accumulation of lactate but inhibited gas production. We concluded that, in vitro, E. glabra was effective at controlling events that can lead to acidosis and the effect was comparable to that of virginiamycin, while K. prorepens was less effective than E. glabra and also inhibited fermentation.

Extrusion of wheat or sorghum and/or addition of exogenous enzymes to pig diets influences the large intestinal microbiota but does not prevent development of swine dysentery following experimental challenge.

A study was made of dietary influences on the large intestinal microbiota of pigs and on the incidence of swine dysentery (SD) after experimental infection with Brachyspira hyodysenteriae, the aetiological agent of SD. Animals were fed diets based either on wheat (expts 1 and 2) or sorghum (expt 2). Grains were ground and fed either raw or after high temperature and pressure extrusion and/or after addition of exogenous enzymes to the whole diet to reduce the starch and soluble non-starch polysaccharides available for fermentation in the large intestine. Limiting fermentation creates conditions that apparently reduce the incidence of SD after infection with B. hyodysenteriae. The diets were fed to weaned pigs for 4-6 weeks, then half the animals on each diet were killed and gut samples collected for microbiology. The treatments had little effect on bacterial numbers. In expt 1, dietary extrusion of wheat reduced lactobacilli in the large intestine. Addition of enzymes to extruded wheat-based diets in expt 2 reduced facultative anaerobes and increased non-sporing anaerobes. Addition of enzymes to a raw sorghum diet in expt 3 decreased numbers of facultative anaerobes, while extrusion of sorghum increased total anaerobes. Bacteroides spp. and Fusobacterium spp., which act in synergy with B. hyodysenteriae in SD, were isolated at a higher percentage in pigs fed the untreated wheat diet than in pigs fed the treated wheat diets. Following experimental infection the incidence of SD amongst pigs fed treated wheat diets was slightly lower than those fed the untreated diet, but with sorghum-based diets the opposite was found. Overall, the different dietary treatments used did not significantly reduce SD.

Confirmation of the role of rapidly fermentable carbohydrates in the expression of swine dysentery in pigs after experimental infection.

Two experiments were conducted to test the hypothesis that soluble non-starch polysaccharides (NSP) and resistant starch (RS) cause swine dysentery (SD) in pigs experimentally infected with the spirochete Serpulina hyodysenteriae. In Experiment 1, a source of soluble NSP (guar gum; GG), insoluble NSP (oat chaff; OC), resistant starch (retrograde cornstarch; RS) or a combination of GG and RS (GG + RS) was added to a diet containing cooked white rice (R), soybean meal (SBM) and animal protein (meat and bone meal, bloodmeal, fishmeal). A diet containing only cooked white rice, SBM and the sources of animal protein (AP) was also fed. In Experiment 2, three rice-based diets containing different levels of RS were fed to pigs. In Experiment 1, the pH of digesta in the cecum, proximal colon and distal colon of pigs fed diets R-GG, R-RS and R-GG + RS was lower (P < 0.001), and volatile fatty acid concentration higher (P < 0.001), than in pigs fed diets R-OC and R-AP. Pigs fed diets with RS and GG + RS had greater (P < 0.05) concentrations of ATP in the large intestine than pigs fed other diets. There were no significant differences in any fermentation indices measured in Experiment 2. In Experiment 1, pigs fed diets R-GG, R-RS and R-GG + RS were colonized with S. hyodysenteriae after experimental infection. However, only pigs consuming diets R-GG (4 of 5) and R-GG + RS (5 of 5) showed clinical signs of SD. Spirochetes were isolated from the feces of all pigs fed diets containing RS in Experiment 2. However, and in contrast to Experiment 1, 80-100% of pigs infected with S. hyodysenteriae displayed clinical signs of SD. These data confirm the role of fermentable carbohydrate in the pathogenesis of SD.

Changes in bacterial populations in the colon of pigs fed different sources of dietary fibre, and the development of swine dysentery after experimental infection.

Swine dysentery (SD) is a disease which can be controlled by feeding a diet low in dietary fibre. The influence of source and inclusion level of dietary fibre both on bacterial populations in the colon, and on subsequent development of SD in pigs experimentally infected with Serpulina hyodysenteriae was evaluated. In Experiment 1, pigs were fed a low-fibre diet based on cooked rice and a animal protein supplement, or the same diet containing added insoluble (iNSP, fed as oaten chaff) or soluble (sNSP, fed as guar gum) non-starch polysaccharides, resistant starch (RS), or a combination of the last two (sNSP/RS). In Experiment 2, different levels of RS were added to the diet. With the base rice diet and with the addition of iNSP, the total number of colonic bacteria was low, the Gram-positive population predominated, S. hyodysenteriae did not colonize and SD did not develop. Synergistic bacteria (Fusobacterium necrophorum and Fus. nucleatum), which have been reported to facilitate colonization by S. hyodysenteriae, were found only among isolates from pigs fed the sNSP/RS diet, and these animals developed SD. Addition of RS to the diet increased total bacterial counts and stimulated growth of Gram-negative bacteria in the colon. In Experiment 1, this permitted colonization by S. hyodysenteriae, but not expression of SD. In contrast, in Experiment 2, this level of inclusion and two others allowed both colonization and development of SD. In conclusion, the addition of sNSP and/or RS to an otherwise protective rice-based diet generated changes in the large intestine microbiota which might have some influence on proliferation of S. hyodysenteriae and the development of SD.

The incidence of swine dysentery in pigs can be reduced by feeding diets that limit the amount of fermentable substrate entering the large intestine.

Two experiments were conducted to test the hypothesis that feeding diets which limit the amount of fermentable substrate entering the large intestine would protect pigs against experimental infection with Serpulina hyodysenteriae, the causative agent of swine dysentery. Experiment 1 examined the effect of grain processing (hammer milling vs. steam flaking) and grain type (barley, groats, corn, sorghum and wheat) on indices of fermentation in the large intestine and the incidence of swine dysentery. Experiment 2 examined the role of five diets, steam-flaked corn, steam-flaked sorghum, hammer-milled wheat, extruded wheat and cooked white rice, on these same measures. All diets contained an animal protein supplement and no antibiotics. Pigs fed diets based on steam-flaked corn and steam-flaked sorghum had a lower incidence of disease (11-33%) than pigs fed diets based on other grains (75-100%). Pigs fed the diet based on cooked white rice were fully protected against swine dysentery. Both the soluble non-starch polysaccharide (NSP) concentration and the total NSP concentration of the diets explained a significant proportion of the variation in swine dysentery (R2 = 0.56, P = 0.016, and R2 = 0.71, P = 0.002, respectively), such that pigs eating diets containing <1.0 g/100 g soluble NSP showed reduced disease. However, pigs fed corn, sorghum and steam-flaked sorghum (Experiment 2), which contained only 0.4-0.5 g/100 g soluble NSP, still had a high incidence of disease (>50%). This was attributable to a higher level of resistant starch present in these grains. These data provide evidence that the expression of swine dysentery is associated with an increased concentration of fermentable substrate entering the large intestine.