Development of an In Vivo Exsheathment Assay of Infective L3 Haemonchus contortus Larvae in Fistualated Sheep

Purpose: Gastrointestinal nematodes (GIN) hinder the sustainable production of small ruminants on pasture and parasite resistance to chemical dewormers is becoming a growing concern. Condensed tannin containing legume forages are being tested to evaluate their anti-parasitic properties and potential contribution to an overall parasite control program for small ruminants such as sheep and goats. One of the most pathogenic GIN of small ruminants is Haemonchus contortus. The final step to full infectivity of H. contortus third stage larvae (L3) is exsheathment in the rumen. The objective of this study was to establish an in-vivo exsheathment assay in fistulated sheep as a tool to evaluate the ability of several varieties of birdsfoot trefoil, a condensed tannin containing forage, to inhibit the exsheathment of H. contortus L3. Methods: The larvae used in the experiment was harvested from the feces of infected donor lambs using a modified Baermann’s technique. In addition, two in-vitro exsheathment assays using either dilute bleach solution or overnight incubation after exposure to CO2 were used for comparison against in-vivo exsheathment. Three different methods of containment of the infective L3 in a porous PVC tube within the rumen were evaluated. 1) Histology cassettes fitted with nylon mesh with a pore size of 8 microns. 3000 larvae were pipetted into the cassettes and the cassettes sealed. To prevent the escape of L3 various methods of sealing the cassettes were tried including super glue, clamps, rubber bands, and clay. 2) Dialysis tubes. 2000 larvae were pipetted into dialysis tubing with varying molecular weight cut offs (50 to 1,000 kD). 3) Small (5 ml) polypropylene sample jars with 8 micron mesh cover. 2000 L3 pipetted into polypropylene sample jars covered with an 8 micron mesh. Each method was tested by suspending the larvae in the various containment systems within a porous PVC tube and then removing a container after 60 and 120 minutes of incubation within the rumen. The percentage of viable and exsheathed infective L3 larvae were determined by microscope. Criteria used to evaluate the efficacy of containment were the percent of recovered L3 from the containment system and the percent of total L3 exsheathed. Results: Results from the histology cassette technique show that, although some L3 were exsheathed, the histology cassettes were not able to be sealed to the degree necessary to prevent L3 from escaping, thus potentially infecting the sheep. Results are still pending on the other two methods as we continue to collect data. Conclusion: Development of an in-vivo exsheathment assay is critical in determining the anthelmintic efficacy of condensed tannin containing legume forages. In order to see if the forages affect exsheathment it is necessary to have an in-vivo assay that is able to provide reliable control-exsheathment from which you can compare exsheathment statistics after being fed the forages

A New Paraprobiotic-based Treatment for Control of Haemonchus Contortus in Sheep

Haemonchus contortus is a critical parasite of goats and sheep. Infection by this blood-feeding gastrointestinal nematode (GIN) parasite has significant health consequences, especially in lambs and kids. The parasite has developed resistance to virtually all known classes of small molecule anthelmintics used to treat it, giving rise in some areas to multidrug resistant parasites that are very difficult to control. Thus, new anthelmintics are urgently needed. Bacillus thuringiensis (Bt) crystal protein 5B (Cry5B), a naturally occurring protein made by a bacterium widely and safely used around the world as a bioinsecticide, represents a new non-small molecule modality for treating GINs. Cry5B has demonstrated anthelmintic activities against parasites of monogastric animals, including some related to those that infect humans, but has not yet been studied in a ruminant. Here we show that H. contortus adults are susceptible to Cry5B protein in vitro. Cry5B produced in its natural form as a spore-crystal lysate against H. contortus infections in goats had no significant efficacy. However, a new Active Pharmaceutical Ingredient (API) paraprobiotic form of Cry5B called IBaCC (Inactivated Bacterium with Cytosolic Crystals), in which Cry5B crystals are encapsulated in dead Bt cell wall ghosts, showed excellent efficacy in vitro against larval stages of H. contortus and relative protein stability in bovine rumen fluid. When given to sheep experimentally infected with H. contortus as three 60 mg/kg doses, Cry5B IBaCC resulted in significant reductions in fecal egg counts (90%) and parasite burdens (72%), with a very high impact on female parasites (96% reduction). These data indicate that Cry5B IBaCC is a potent new treatment tool for small ruminants in the battle against H. contortus

Effect of vitamin E on the immune system of ewes during late pregnancy and lactation

The present experiment was designed to determine the effects of a regimen of repeated, intramuscular (i.m.) injections of vitamin E (VE) on innate and humoral components of the immune response of pregnant and lactating ewes. Pregnant ewes were randomly assigned to two treatments consisting of i.m. injections of either VE (30. IU d-α-tocopherol/kg BW, n=10) or equivalent volume of a placebo (emulsified base, n=8). Injections were administered at 2-week (wk) intervals to all ewes, as a group, for a total of 24. wk. Lambing occurred over a 5-wk period, starting wk 7 of the study. All ewes were administered a booster vaccination for Clostridium tetani and Clostridium perfringens (C + D) at wk 3. Blood and colostrum were analyzed for α-tocopherol and selenium content, and extracellular glutathione peroxidase (GPX3) activity as measures of antioxidant status; lysozyme activity as a measure of innate immunity and immunoglobulin G (IgG), and anti-tetanus toxoid IgG (IgG-T) concentration as a measure of humoral immune status. Three ewes in the placebo group did not lamb and were therefore excluded from all analyses. Results showed a significant treatment × week effect of VE supplementation on IgG concentration. VE supplementation increased IgG concentrations in ewes after their yearly booster vaccination against C. tetani and C. perfringens; placebo-treated ewes showed no such response. The extent, magnitude and persistence of the decreased IgG concentration during the transition period were less in VE-supplemented ewes than controls. There was no effect of VE supplementation on the concentration of IgG-T after vaccination. VE-supplementation of pregnant ewes had no effect on serum lysozyme activity. GPX3 increased in both treatment groups, peaking during the transition period. The α-tocopherol content of the colostrum of VE-supplemented ewes was greater than that of controls. In conclusion, VE supplementation of pregnant and lactating ewes had an equivocal effect on variables used to assess immune function. The benefit of VE supplementation to pregnant and lactating ewes requires further research however it may have utility in blunting the immune suppression that occurs during the transition period. © 2012 Elsevier B.V

Determining the Effects of Pelleted Cranberry Vine Grains on the Ewe and Offspring during Pregnancy and Lactation

When creating any new anti-parasitic interventions, it is important to evaluate their effects across all life stages. This study had three objectives, which were to evaluate the effect of feeding cranberry vine pellet (CVP) on (1) ewes’ body weights and BCS during late gestation and lactation; (2) ewes’ milk quality during lactation; and (3) lambs’ body weight and growth parameters from birth to 65 days of age. Across two years, 41 Dorset ewes were fed either a 50% CVP or a matching control pellet (CON) from 104 ± 1.60 days of gestation for 62.8 ± 0.68 days of lactation. Measurements were collected from ewes (BW, BCS, and milk) and lambs (BW and body size). Milk from CVP ewes exhibited reduced milk fat and solids (p p = 0.02) when evaluated for the treatment–time. There was no significant difference in the BCS, protein, lamb BW, or growth measurements for treatment–time (p ≥ 0.05). Additional research that targets blood biochemistry and metabolic assessments is needed to fully determine the impact of this pellet on ewes and lambs

Observational methods to measure behaviors of captive cotton-top tamarins (Saguinus oedipus)

The purpose of this study was to identify efficient sampling methods for establishing accurate activity budgets for zoo animals. Seven cotton-top tamarins (Saguinus oedipus) from two zoos were videotaped for multiple 90min sessions, 3 to 4 days per week for 12 weeks. An activity budget was constructed for each animal using a continuous sampling method to analyze 30 hr of video recording of each animal. These master datasets, reflecting actual behavior, were re-sampled using interval sampling lengths of 0.25, 0.5, 1, 2, 4, 5, 7, 10, 15 and 20min, and cluster sampling protocols (periodic sessions of continuous sampling) of 10 min×3, 15 min×2, 20 min×1, 15 min×1 and 10min×1 (min×repetitions/90min sample period) to construct additional activity budgets for each animal. The Canberra similarity index was used to determine the statistical relationship between these activity budgets and those based on the master datasets. As interval length increased, there was a consequent decrease in the accuracy of the associated activity budgets as compared with the master dataset. No cluster sampling protocols yielded activity budgets as accurate as the four shortest interval lengths, but all cluster sampling protocols were more accurate than the three longest interval lengths. All the tested protocols varied in ability to accurately portray animal behavior. Overall, interval sampling provided superior behavioral representations at lower observer input. Results from this study will potentially facilitate the standardization of behavior monitoring protocols at zoos. © 2009 Wiley-Liss, Inc

Effects of lactate on ground lamb colour stability and mitochondria- mediated metmyoglobin reduction

Previous research suggests that lactate\u27s colour stabilizing effect in beef is through NADH production and antioxidant activity. However, no research has assessed lactate\u27s role in lamb colour. Hence, our objectives were to evaluate the effects of lactate on lamb surface discolouration, oxygen consumption, and metmyoglobin reduction. In experiment 1, lactate (final meat concentration = 2.5% w/w) was added to ground lamb (n = 20 carcasses) and patties were stored for 3 days at 1 °C in PVC packaging. Surface colour (CIE L and a) and metmyoglobin reducing activity of ground lamb patties were measured. Addition of lactate improved colour stability and metmyoglobin reducing activity (p \u3c 0.05). In experiment 2, mitochondria were isolated from lamb longissimus muscle (n = 3). Addition of lactate-LDH-NAD to mitochondria resulted in significant oxygen consumption and metmyoglobin reduction compared with mitochondrial controls without lactate (p \u3c 0.05). Lactate can improve the colour stability of lamb, possibly by increasing metmyoglobin reducing activity. © 2010 Elsevier Ltd. All rights reserved

Na+,K+-ATPase Activity and Subunit Protein Expression: Ontogeny and Effects of Exogenous and Endogenous Steroids on the Cerebral Cortex and Renal Cortex of Sheep

We examined the effects of development, exogenous, and endogenous glucocorticoids on Na+,K+-ATPase activity and subunit protein expression in ovine cerebral cortices and renal cortices. Ewes at 60%, 80%, and 90% gestation, newborns, and adults received 4 dexamethasone or placebo injections. Cerebral cortex Na+,K+-ATPase activity was higher (P < .05) in placebo-treated newborns than fetuses of placebo-treated ewes and adults, α1-expression was higher at 90% gestation than the other ages; α2-expression was higher in newborns than fetuses; α3-expression was higher in newborns than 60% gestation; β1-expression was higher in newborns than the other ages, and β2-expression higher at 60% than 80% and 90% gestation, and in adults. Renal cortex Na+,K+-ATPase activity was higher in placebo-treated adults and newborns than fetuses. Cerebral cortex Na+,K+-ATPase activity was higher in dexamethasone- than placebo-treated adults, and α1-expression higher in fetuses of dexamethasone- than placebo-treated ewes at 60% and 80% gestation. Renal cortex Na+,K+-ATPase activity and α1-expression were higher in fetuses of dexamethasone- than placebo-treated ewes at each gestational age, and β1-expression was higher in fetuses of dexamethasone- than placebo-treated ewes at 90% gestation and in dexamethasone- than placebo-treated adults. Cerebral cortex Na+,K+-ATPase activity, α1-expression, β1-expression, and renal cortex α1-expression correlated directly with increases in fetal cortisol. In conclusion, Na+,K+-ATPase activity and subunit expression exhibit specific developmental patterns in brain and kidney; exogenous glucocorticoids regulate activity and subunit expression in brain and kidney at some ages; endogenous increases in fetal cortisol regulate cerebral Na+,K+-ATPase, but exogenous glucocorticoids have a greater effect on renal than cerebral Na+,K+-ATPase