Abomasal Tissue Research Articles

Microarray analysis reveals difference in gene expression profiles of hair and wool sheep infected with Haemonchus contortus

Sheep infected with the abomasal parasite, Haemonchus contortus, have reduced growth rates, decreased wool production, and anemia, and heavy infections may result in death. Anthelmintic treatment can remove worms, but the cost of treatment and prevalence of drug-resistant worms has led to greater focus on genetic resistance of the host to parasitism. Variation in parasite resistance exists within and among sheep breeds, and Caribbean hair sheep have greater resistance than most conventional wool breeds. Our objective was to investigate differences in gene expression between 24 parasite-resistant hair and 24 susceptible wool lambs to determine genetic mechanisms involved in resistance to H. contortus. Half of the animals of each breed were infected and sacrificed at 3 or 27 days post-infection; the remaining animals were uninfected controls. Breed differences in abomasum and abomasal lymph node tissue gene expression were assessed using bovine cDNA microarrays. Over 60 transcripts differed between breeds for each tissue and infection status. Genes differentially expressed between hair and wool sheep 3 days PI were assessed for gene function and mechanisms for greater immune cell infiltration, abomasal tissue repair, Th17 response, and anticoagulation were present in parasite-resistant hair sheep. By 27 days PI, hair sheep had greater expression of genes involved in gut motility, inflammatory cytokines, and cell proliferation and differentiation compared to wool sheep. Changes in these processes indicate Caribbean hair sheep have a stronger inflammatory response when infected with H. contortus which may facilitate the increased parasite resistance observed in these sheep.

Pulsatile Cerebrospinal Fluid and Plasma Ghrelin in Relation to Growth Hormone Secretion and Food Intake in the Sheep

As in other species, exogenous administration of ghrelin, an endogenous ligand for the growth hormone (GH) secretagogue receptors can stimulates feeding behaviour and GH secretion in the sheep. However, the importance of endogenous ghrelin for these two functions as well as its central or peripheral origin remained to be established. In the present study, cerebrospinal fluid (CSF) ghrelin concentrations were measured in five anoestrous ewes and found to be more than 1000-fold lower than circulating plasma levels, in keeping with the even lower concentration in hypothalamic compared to abomasum tissue extracts. Cluster analysis indicated that CSF ghrelin levels were markedly pulsatile, with a greater number of peaks than plasma ghrelin. Pulsatility parameters were closer for GH and CSF ghrelin than between GH and plasma ghrelin. Plasma ghrelin and GH levels were significantly correlated in three out of five ewes but CSF ghrelin and GH in one ewe only. Half of the CSF ghrelin episodes were preceded by a ghrelin peak in plasma with a 22-min delay. Cross-correlations between plasma GH and plasma or CSF ghrelin did not reach significance but a trend towards cross-correlation was observed from 20 to 0 min between plasma and CSF ghrelin. At 09.00 h, when food was returned to ewes, voluntary food intake did not elicit a consistent change in plasma or CSF ghrelin levels. By contrast, a peripheral ghrelin injection (1 mg, i.v.) immediately stimulated feeding behaviour and GH secretion. These effects were concomitant with a more than ten-fold increase in plasma ghrelin levels, whereas CSF ghrelin values only doubled 40-50 min after the injection. This suggests that peripherally-injected ghrelin crosses the blood-brain barrier, but only in low amount and with relatively slow kinetics compared to its effects on GH release and food intake. Taken together, the results obtained in the present study support the notion that, in the ovariectomised-oestradiol implanted sheep model, peripheral ghrelin injection rapidly induces GH secretion, and feeding behaviour, probably by acting on growth hormone secretagogue receptor subtype 1 located in brain regions in which the blood-brain barrier is not complete (e.g. the arcuate nucleus).

Ghrelin, adrenaline and neostigmine stimulate the in vitro expression of ghrelin in lambs’ abomasal tissue slices?

Ghrelin is a 28 amino acid acylated peptide secreted by several digestive organs; X/A cells of stomach's fundic region seem to be its main producers‐ Ghrelin's positive effect on food intake has a negative connotation in humans; in contrast, the same effect could be a positive factor in farm animals’ production; therefore, our aim was to evaluate the effect of administration of human synthetic ghrelin, adrenaline, and neostigmine, on ghrelin expression in lambs′ abomasal tissue in vitro. Samples from 12 lambs were used. At slaughter, tissue slices were washed with saline solution, stored in tubes with media culture (DMEM‐F12, FBS 20%, penicillin/streptomycin and amphotericin) and transported to the lab. Slices were placed in polypropylene inserts, in 24‐well multidishes with 500 μl of medium, and distributed to five treatments: control; adrenaline 10 μM; neostigmine 0.1 μM; ghrelin (500 and 1000 pg), in four exposure times (0, 1, 2, 3 h). Ghrelin expression was identified by purifying RNA from tissue slices using the RT‐PCR technique. Total RNA was extracted from the cells by QIAGEN™ protocol (RNeasy Mini Kit). RT was made using oligo (dT)12–18 primer and PCR were made using the primers: Forward 5′‐CATCTACAGCCTGCTGCTG and Reverse 5′‐CACATCCGGGTCAAACTG. The expression of ghrelin was detected in agarose gel with a band of 361 bp. The results showed no differences among treatments; pending confirmation with Real Time PCR.

Increased levels of cysteinyl leukotrienes and prostaglandin E2 in gastrointestinal tract mucus are associated with decreased faecal dry matter in Merino rams during nematode infection

Immune-mediated scouring in sheep is a mucosal hypersensitivity response incited by infective larvae of gastrointestinal nematodes in the southern half of Australia and in New Zealand, regions characterised by a Mediterranean, winter-rainfall climate. To gain insights into the underlying mechanism(s), this study investigated the relationship between nematode infection of sheep in pens, faecal dry matter and release of inflammatory mediators in the intestinal tract. Six worm-free, immune rams received a dose of 3000 Trichostrongylus third stage larvae (L3) and 3000 Ostertagia L3 per week for 6 weeks, after which they were euthanased. Three rams acted as uninfected controls. Faecal dry matter was significantly lower in the infected group from the first week of infection until the end of the trial. Concentrations of cysteinyl leukotrienes and prostaglandin E2 were higher in mucus from both the abomasum and the small intestine of infected rams compared with controls. Within the infected group, concentrations of both mediators were higher in mucus from the small intestine than from the abomasum. There were greater numbers of eosinophilic granulocytes and mucosal mast cells in abomasal and small-intestinal tissue sections from infected rams compared with the controls. The results are consistent with the hypothesis that nematode infection causes an inflammatory response in the gastrointestinal mucosa, characterised by increased fluid secretion.

Purification and characterization of milk clotting enzyme from goat ( Capra hircus)

Chymosin, the major component of rennet (milk clotting enzyme), is an acid protease produced in the fourth stomach of milk-fed ruminants including goat and sheep in the form of an inactive precursor prochymosin. It is responsible for hydrolysis of κ-casein chain in casein micelles of milk and therefore, used as milk coagulant in cheese preparation. The present investigation was undertaken to purify and characterize goat ( Capra hircus) chymosin for its suitability as milk coagulant. The enzyme was extracted from abomasal tissue of kid and purified nearly 30-fold using anion exchanger and gel filtration chromatography. Goat chymosin resolved into three major active peaks, indicating possible heterogeneity when passed through DEAE-cellulose ion exchange column. The purified enzyme had a molecular mass of 36 kDa on SDS-PAGE, which was further confirmed by Western blot analysis. The purified enzyme preparation was stable up to 55 °C with maximum activity at 30 °C. The milk clotting activity was decreased steadily as pH is increased and indicated maximum activity at pH 5.5. Proteolytic activity of goat chymosin increased with incubation time at 37 °C. Goat chymosin was found to be more thermostable than cattle chymosin and equally stable to buffalo chymosin.

Sexual Variations in Food Quality and Gastrointestinal Features of Sika Deer (Cervus nippon) in Japan During Winter: Implications for Feeding Strategy

We assessed sexual variation in food quality and gut macrostructure in adult male and pregnant female sika deer, Cervus nippon (Temminck, 1838), in Japan during winter. These variations might have important implications relative to sexual differences in habitat use, forage acquisition, and digestive strategy. According to the sexual dimorphism-body size hypothesis the larger males would feed on poorer forage and have heavier stomach contents and heavier intestine contents and longer intestines than smaller females. However, the food quality in rumen contents of males was higher than, or at least similar with, that of pregnant females. In correspondence to food quality, the relative weights of stomach contents and intestines with contents, the relative lengths of intestines to the lengths of body and total intestines in pregnant females were similar to adult males. The relative weights of omasum and abomasum tissues in pregnant females were greater than in males. Our findings suggest sexual differences in feeding strategy in sika deer in Japan during winter. To meet greater nutritional demands of high metabolic rate and gestation, pregnant females seemed to maintain a greater volume of digesta in guts and had more stomach tissues than expected by the sexual dimorphism-body size hypothesis to compensate for poorer forage quality.

Purification and characterization of chymosin and pepsin from kid

The objective of this work was to study the characteristics of the gastric aspartic proteinases chymosin and pepsin which are constituents of the kid rennet. The two enzymes were extracted from abomasal tissue of one kid from a local indigenous breed, separated from each other by DEAE-cellulose chromatography and then were purified by gel filtration and anion-exchange chromatography. The molecular weights of the purified kid chymosin and pepsin as determined by gel filtration were 36 kDa and 40 kDa respectively. The isoelectric point of kid chymosin was as multiple forms of 3-6 zones at pH 4.6-5.1, while that of kid pepsin was at pH < or =3.0. Kid pepsin contained 0.37 molecules phosphorous per molecule and was totally inhibited by 5 muM pepstatin A, being more sensitive than kid chymosin. Both enzymes were almost equally as proteolytic as calf chymosin on total casein at pH 5.6. Kid pepsin activity was more pH and temperature dependent than kid chymosin activity. In comparison with the calf chymosin temperature sensitivity, the order of increased sensitivity was: calf chymosin <kid chymosin <kid pepsin.

Relationship of abomasal histology and parasite-specific immunoglobulin A with the resistance to Haemonchus contortus infection in three breeds of sheep

This study was carried out to evaluate the relationship of abomasal inflammatory cells and parasite-specific immunoglobulin A (IgA) in mucus, with the resistance to Haemonchus contortus infection in three breeds of sheep naturally infected with gastrointestinal nematodes. The breeds were the native Santa Ines sheep, and the European Suffolk and Ile de France breeds. Mast cells, eosinophils and globule leucocytes were enumerated in abomasal mucosa. Eosinophils within the sub-mucosa also were counted separately. Histamine concentration was estimated in abomasal tissue samples. Enzyme-linked immunosorbent assay was carried out in mucus samples to determine the level of IgA anti- H. contortus third and fifth instar. There were no significant differences among group means of these variables ( P > 0.05). The correlation coefficients between fecal egg counts (FEC) × mast cells ( r = −0.490; P < 0.05) and FEC × eosinophils in sub-mucosa ( r = −0.714; P < 0.01) was significant in the Santa Ines sheep. In the Ile de France group, the correlation coefficients between globule leucocytes × FEC ( r = −0.879; P < 0.001) and histamine × worm burden ( r = −0.833; P < 0.01) were also significant. In the Santa Ines and Ile de France sheep, correlation coefficients between IgA anti-L3 × worm burden and IgA anti-L3 × FEC were negative. In general, inflammatory cells and IgA-parasite-specific in abomasum were inversely associated with H. contortus worm burden and FEC indicating that they may impair parasite development or fecundity in the three breeds of sheep. However, similar mean values of inflammatory cells and IgA were found in the resistant (Santa Ines) and in the susceptible (Suffolk and Ile de France) breeds of sheep. The enumeration of cells by histological assessment does not provide information on their functional activity, which may be different among breeds. Thus, the effect of breed on the functional activity of these and other inflammatory cells is an important area for further study.

Studies using Teladorsagia circumcincta in an in vitro direct challenge method using abomasal tissue explants

An in vitro direct challenge (IVDC) method in which abomasal tissue explants maintained at 37 °C in Hanks/Hepes solution (pH 7.6) in a high oxygen concentration (incubator gassed with pure oxygen and sealed prior to use) were-challenged with exsheathed third stage larvae of Teladorsagia circumcincta has been used to investigate the tissue association phase of larval establishment. Studies using naïve and recently challenged immune sheep and goat abomasal tissues have shown significant reductions ( P < 0.05) in the numbers of tissue associated larvae in material from immune animals. The mechanisms that affect the tissue association process appear to be labile since more larvae were recovered from the tissue digests of previously infected sheep that had not been recently exposed to larval challenge in comparison to those that had been recently challenged ( P < 0.05). The method has also been used to demonstrate the influence of protein nutrition on the establishment of larvae in abomasal tissues and region specific differences in the efficacy of exclusion mechanisms. The technique appears to have potential as a means of investigating the crucial first phase of the process of establishment.

Local immune responses in sensitized sheep following challenge infection with Teladorsagia circumcincta.

Sheep were sensitized by weekly infections with Teladorsagia circumcincta over a 9-week period. After a 12-week rest, sheep were divided into four groups and killed without challenge or 3, 5 and 10 days post challenge (DPC) with 50000 L3. Recovery of challenge larvae from abomasal scrapings was highest at 3 DPC while no parasites were recovered by 10 DPC. Abomasal lymph nodes (ALN) of challenged sheep were significantly larger at 5 DPC, coinciding with an increase in the proportion of CD4 T cells and a decrease in CD21+ cells, probably reflecting the loss of CD21 from terminally differentiated antibody secreting cells. A significant increase was observed in gammadelta-TCR+ cells at 3 DPC in the ALN, while their number slightly decreased in the abomasal tissues throughout the challenge period. The number of tissue eosinophils was dramatically increased after challenge compared with the unchallenged controls, with a peak at 3 DPC, coinciding with the peak in larval recovery. CD4+ cells significantly increased in the abomasal tissues at 5 DPC, while no changes in globule leucocytes were observed until 10 DPC. Antibody-secreting cell probes (ASC-probes) generated from the ALN showed highest reactivity against larval antigens at 5 DPC. This reactivity was predominantly directed against regions between 90 and 100 kDa and 30-35 kDa in the L3 preparation and lower molecular weight antigens in the L4. No reactivity was observed against the adult extract. The 30-35 kDa antigen seemed to exist as a high molecular weight complex in L3 homogenate and was not susceptible to protease K treatment, suggesting it may be non-protein in nature.

Mechanisms of immunity to Haemonchus contortus infection in sheep.

In two separate experiments, sheep were immunized by nine to 12 weekly immunizing infections with 4000 Haemonchus contortus third stage larva (L3), drenched with anthelminthics and maintained free of H. contortus infection for a further 12 weeks. The anamnestic cellular immune responses in both the abomasal lymph node (ALN) and mucosa of the immunized sheep were examined 3 and 5 days post challenge with 50 000 H. contortus L3. Sheep in the two experiments clearly segregated out in two distinct groups, one in which challenge larvae were obviously present in the tissues of all 12 sheep at 3 and 5 days post challenge while no challenge larvae were detected in tissues of seven of the eight sheep in the other group. In sheep in which no tissue larvae were detected, very few changes were noted in either the ALN or mucosa. In contrast, dramatic changes were observed in the cellular profiles of the ALN and mucosa after challenge infection in sheep in which larvae were observed in the abomasal tissues. In the ALN, these changes were characterized by an increase in the relative percentage of gammadelta-TCR+ T cells and B cells and an increase in the proportion of CD4+ T cells coexpressing the activation markers MHC class II and CD25. In the abomasal mucosa, an increase in the number of infiltrating CD4+ and gammadelta-TCR+ T cells and B cells was observed by 3 days postinfection and these levels were further increased at 5 days postinfection. This infiltration of the abomasal mucosa by lymphocytes was accompanied by a dramatic increase in the number of infiltrating eosinophils, which were often in intimate association with the surface of H. contortus larvae. None of these changes occurred in the mucosa of the sheep that showed no sign of challenge larvae in the tissues; however, a transient increase in gammadelta T cells in the ALN and a drop in intraepithelial globule leucocytes were uniquely observed in these sheep at 5 days post challenge. These results suggest that two different types of immune responses can be generated after challenge infection of immunized sheep, one where tissue larvae are excluded from their tissue niche as observed previously and which is associated with changes in globular leucocyte population but no mobilization of the local immune system. In contrast, when challenge larvae reach their tissue niche, dramatic changes in the local immune system occur, including a pronounced infiltration of eosinophils. These two immune mechanisms may be associated with the rapid and delayed rejection of parasite infections in immune sheep.

Isolation, characterisation and expression of mRNAs encoding the ovine CC chemokines, monocyte chemoattractant protein (MCP)-1α and -2

CC chemokines are important mediators of immune responses, orchestrating the differential recruitment of various leukocyte populations. Despite the large number of known CC chemokines in other species, no cDNA encoding ovine CC chemokines have been isolated. A homology cloning strategy was utilised to isolate the cDNA of ovine CC chemokines. Full-length monocyte chemoattractant protein (MCP)-1α and -2 cDNA have been isolated. The predicted ovine MCP-1α amino acid sequence shares 87 and 75% identity with bovine MCP-1α and porcine MCP-1, respectively. The predicted ovine MCP-2 amino acid sequence shares 92 and 85% identity with bovine and porcine MCP-2, respectively. Northern blot analysis of MCP-1α revealed that it is strongly expressed in cells isolated from mammary lavage fluid (MAL) of ewes given intramammary infusions of Haemonchus contortus. Weak signals were detected in mammary and abomasal tissue. Southern blot analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) products indicates that MCP-1α mRNA levels increase in abomasum after challenge with H. contortus. MCP-1α mRNA levels were also increased in bronchoalveolar lavage (BAL) cells and lung tissue after house dust mite extract (HDME) challenge. Similarly, MCP-2 mRNA was detected by Northern blot analysis at high levels in MAL cells after H. contortus intramammary infusion, and increased in BAL cells and lung tissue in HDME-challenged sheep. MCP-2 mRNA was not detected by Northern blots in whole mammary or abomasal tissue, but Southern blot analysis of RT-PCR products also indicates that MCP-2 mRNA increases in abomasal tissue after challenge with H. contortus. Hence, two ovine CC chemokine mRNA have been isolated that are up-regulated in response to parasite infection and allergen challenge. Ultimately the isolation of these and other ovine CC chemokines will help elucidate a wide variety of immune responses in sheep.

Age-related protective immunity after vaccination with Haemonchus contortus excretory/secretory proteins.

Protection against an experimental challenge infection by immunization with excretory/secretory products (ES) from Haemonchus contortus, containing predominantly proteins of 15 and 24 kDa, depends on the age of the sheep. Vaccinated sheep 9 and 6 months of age had reduced final worm burdens of 82 and 77, respectively. No reduction in worm burden was found in 3-month-old lambs. Nine-month-old sheep had significantly higher ES-specific serum immunoglobulin (Ig)G1 and IgA during immunizations and after challenge infection than 3-month-old lambs. There was no correlation within the 9-month-old sheep between ES-specific IgA or IgG1 levels and protection, measured as worm burden. However, when the different age groups were combined, negative correlations between percentage protection and ES-specific IgA and IgG1 levels after challenge were found. At the end of the experiment, peripheral blood eosinophils and mast cell counts in abomasal tissue were also significantly higher in the vaccinated and challenged 9-month-old sheep than in the vaccinated and challenged 3-month-old or than in the 9-month-old sheep with challenge, but without vaccination. The responses measured in young lambs were similar to the responses in sheep, but the height of these responses was in general of a lower magnitude.

Isolation and Characterization of a Novel Inducible Mammalian Galectin

A novel mammalian galectin cDNA (ovgal11) was isolated by representational difference analysis from sheep stomach (abomasal) tissue infected with the nematode parasite, Haemonchus contortus. The mRNA is greatly up-regulated in helminth larval infected gastrointestinal tissue subject to inflammation and eosinophil infiltration. Immunohistological analysis indicates that the protein is localized in the cytoplasm and nucleus of upper epithelial cells of the gastrointestinal tract. The protein is also detected in mucus samples collected from infected abomasum but not from uninfected tissue. The restricted and inducible expression of ovgal11 mRNA and limited secretion of the protein support the hypothesis that OVGAL11 may be involved in gastrointestinal immune/inflammatory responses and possibly protection against infection.

Cellular profiles in the abomasal mucosa and lymph node during primary infection with Haemonchus contortus in sheep

Cellular changes in the abomasal tissue and draining abomasal lymph nodes were examined after primary infection of lambs with Haemonchus contortus for 3, 5 or 27–36 days. Infection with H. contortus larvae resulted in a rapid and selective increase in the percentage of CD4 + T-cells in the abomasal lymph node at 3 days post-infection (PI). By 5 days PI, the lymph node weight had increased two-fold; however, the percentage of lymphocyte populations in the abomasal lymph node resembled that seen in uninfected sheep. Lymph node weights remained at increased levels in the adult nematode infected sheep and down-regulation of B-cell surface markers (sIg and MHC Class II) was apparent in this group. Significant increases in the percentage of CD4 + T-cells co-expressing MHC Class II, but not CD25, were observed in the larval infected groups except in adult nematode infected sheep. Increased numbers of eosinophils, CD4 +, γδ + T-cells and B-cells were found in the abomasal tissue by 5 days PI, but no further increases in these cell populations were observed in the adult nematode infected group. In contrast, the level of both lamina propria and intraepithelial mast cells observed in the abomasal mucosa was highest in the sheep carrying an adult nematode burden. These findings indicate that sheep are able to generate an early immune response to infection with H. contortus larvae, characterised by the activation of CD4 T-cells and B-cells in the draining lymph nodes and recruitment of eosinophils, CD4 + and γδ-TCR,WC1 + T-cells and B-cells in larval infected tissues. However, these changes do not seem to be maintained during infection with the adult parasite where increases in mast cell numbers dominate the local response, indicating that different parasite stages may induce distinct and possibly counteractive immune responses.

Histamine in lambs with abomasal bloat, haemorrhage and ulcers.

The median concentration of histamine in abomasal fluid of lambs with abomasal haemorrhage and/or ulcers (group 2) was significantly (P < 0.05) higher than the concentrations in lambs presenting abomasal bloat (group 1) and in the healthy and the diseased controls. In group 2, there was also a strong correlation (R2 = 0.81) between the histamine concentrations in abomasal tissue and abomasal fluid, although the median value of histamine in the abomasal tissue was not statistically higher in this group than in the others. The urine of lambs in group 2 also had numerically higher median concentration of histamine than the other groups. Five out of eight tested strains of Lactobacillus spp. and one out of two strains of Clostridium sordellii, isolated from abomasal contents of lambs with abomasal disease, were strong producers of histamine. Bacterial production is one possible source for the increased histamine concentrations in lambs suffering from abomasal haemorrhage and/or ulcers.

Induction of T helper 1- and T helper 2-type immune responses during Haemonchus contortus infection in sheep.

The production of cytokines by lymphoid cells, isolated from non-infected and Haemonchus contortus-infected lambs, was investigated. Particular attention was paid to differences in T helper 1- (Th1) and Th2-type immune profiles between genetically resistant and random-bred animal groups. Non-infected resistant and random-bred lambs produced equivalent levels of interferon-gamma (IFN-gamma) and interleukin-5 (IL-5), from isolated abomasal lymph node cells (ALN), mesenteric lymph node cells (MLN) and spleen cells (SC), in response to in vitro stimulation with T-cell mitogen (concanavalin A) or larval parasite antigen. ALN and MLN cells derived from infected resistant and random-bred lambs produced relatively lower levels of IFN-gamma, following in vitro stimulation with parasite antigen, when compared with their uninfected counterparts. In contrast, infected lambs of both groups showed enhanced mitogen- and antigen-stimulated production of IL-5, in comparison with uninfected controls, at days 5 and 28 postinfection (p.i.). Mitogen- and antigen-stimulated IL-5 responses were higher among resistant lambs compared with random-bred lambs, with the highest overall production of IL-5 by parasite antigen-stimulated ALN and MLN cells. Among day 28 p.i. lambs, levels of cell culture-derived parasite-specific immunoglobulin G1 (IgG1) and IgE antibodies were higher in resistant lambs than in random-bred lambs, following in vitro stimulation of SC or ALN cells with parasite antigen. Finally, after 28 days p.i., histological examination of abomasal tissue revealed higher densities of mast cells and eosinophils in the mucosa of resistant lambs than in random-bred lambs. Taken together, these data support the notion of a strong Th2-type immune response to Haemonchus infection in genetically resistant sheep, and support the claim for a Th1/Th2 dichotomy in ruminants.

Protection in lambs vaccinated with Haemonchus contortus antigens is age related, and correlates with IgE rather than IgG1 antibody.

Protection by vaccination with excretory-secretory products (ES) from Haemonchus contortus, containing predominantly proteins of 15 and 24 kDa, against an experimental challenge infection depends on the age of the sheep. Vaccinated sheep 9, 6 or 3 months of age were protected for 83%, 77% and -34%, respectively. There was a significant difference in ES-specific serum IgE response but not in IgG1 response, after the last vaccination between the different age groups. In the protected 9-month-old animals, there was an increase up to 18 times the prevaccination levels, while the increase in the unprotected 3-month-old animals was at most 1.4 times. The 6-month-old animals showed an intermediate increase of approximately six times the prevaccination level. There was no correlation within the 9-month-old sheep between ES-specific IgE levels and protection, measured as worm burden. However, when the different age groups were combined, there was a positive correlation (r = 0.38) between protection and ES-specific IgE levels 1 week after the vaccination. At the end of the experiment, peripheral blood eosinophils and mast cell counts in abomasal tissue were also significantly higher in the vaccinated and challenged 9-month-old sheep than in the vaccinated and challenged 3-month-old or than in the 9-month-old sheep with challenge, but without vaccination. Increased serum IgE levels, eosinophilia and mucosal mast cell hyperplasia are the hallmarks of a Th2 response and were all demonstrated in protected, older sheep, but not in unprotected, younger sheep.

Moxidectin in cattle: correlation between plasma and target tissues disposition.

The time of parasite exposure to active drug concentrations determines the persistence of the antiparasitic activity of endectocide compounds. This study evaluates the disposition kinetics of moxidectin (MXD) in plasma and in different target tissues following its subcutaneous (s.c.) administration to cattle. Eighteen male, 10-month old Holstein calves weighing 120-140 kg were subcutaneously injected in the shoulder area with a commercially available formulation of MXD (Cydectin 1%, American Cyanamid, Wayne, NJ, USA) at 200 micrograms/kg. Two treated calves were killed at each of the following times post-treatment: 1, 4, 8, 18, 28, 38, 48, 58 and 68 days. Abomasal and small intestine mucosal tissue and fluids, bile, faeces, lung, skin and plasma samples were collected, extracted, derivatized and analysed to determine MXD concentrations by high performance liquid chromatography (HPLC) with fluorescence detection. MXD was extensively distributed to all tissues and fluids analysed, being detected (concentrations > 0.1 ng/g; ng/mL) between 1 and 58 days post-treatment. MXD peak concentrations were attained during the first sampling day. MXD maximum concentration (Cmax) values ranged from 52.9 (intestinal mucosa) up to 149 ng/g (faeces). The mean residence time (MRT) in the different tissues and fluids ranged from 6.8 (abomasal mucosa) up to 11.3 (bile) days. MXD concentrations in abomasal and intestinal mucosal tissue were higher than those detected in plasma; however, there was a high correlation between MXD concentrations observed in plasma and those detected in both gastrointestinal mucosal tissues. MXD concentrations were markedly greater in the mucosa than in its respective digestive fluid (P < 0.01). MXD concentrations in skin were higher than those found in plasma (P < 0.01). Drug concentrations recovered in the dermis were greater than those detected in the hypodermal tissue (P < 0.05). Large concentrations of MXD were excreted in bile and faeces. These findings may contribute to an understanding of the relationship between the kinetic behaviour and the persistence of the antiparasite activity of MXD against different ecto-endoparasites in cattle.

The distribution of pepsinogen within the abomasa of cattle and sheep infected with Ostertagia spp. and sheep infected with Haemonchus contortus

The effect of nematode infections on the production of pepsinogen by ruminants was investigated immunohistochemically and biochemically. Abomasal tissues were collected from parasite-naive cattle and sheep, from sheep infected with predominantly Ostertagia circumcincta, sheep infected experimentally with Haemonchus contortus and cattle infected with Ostertagia ostertagi. Pepsinogen was also assayed biochemically in homogenates of fundic mucosae from sheep infected with predominantly O. circumcincta. Infection with Ostertagia spp. parasites was associated mainly with nodular hyperplasia, resulting in increased numbers of cells that produce both pepsinogen and mucus. Measured biochemically, nodules contained more pepsinogen than adjacent more normal mucosa ( p < 0.05), and this effect was largely attributable to the greater mass of nodules. Infection of sheep with H. contortus was associated with generalised hyperplasia, characterised by increased numbers of mucopeptic cells and in at least one animal with reductions in parietal cell numbers. At the same time, the zymogen granule content of chief cells was reduced. Similar changes were occasionally seen in sheep infected predominantly with O. circumcincta. Generalised hyperplasia is likely to be indicative of the presence of ambulatory parasitic stages as opposed to those confined to nodules. The potential for the enhanced production of pepsinogen by increased numbers of cells with a joint mucous cell and zymogenic cell phenotype may offset decreases in the numbers of chief cells or reductions in chief cell activity.