Comparing the responses of grain-fed feedlot cattle under moderate heat load and during subsequent recovery with those of feed-restricted thermoneutral counterparts: blood cells and inflammatory markers.

Given the climate projections for livestock rearing regions globally, understanding the inflammatory status of livestock under various heat loads will be informative to animal welfare and management. A survey of plasma inflammatory markers was conducted, and blood leucocyte counts followed to investigate the capacity of the ~ 500 kg grain fed Black Angus steer to respond to and recover from a moderate heat load challenge. Two sequential cohorts of 12 steers were housed in climate-controlled rooms (CCR) for 18 days. A thermally challenged (TC) group (n = 2 × 6) experienced five consecutive periods: PreChallenge, Challenge, and Recovery within the CCR, and 40 days in outdoor pens (PENS and Late PENS). PreChallenge (5 days) and Recovery (7 days) delivered thermoneutral conditions, whereas in Challenge the TC steers experienced a diurnal temperature range of 28-35 °C. A feed-restricted thermoneutral (FRTN) treatment (n = 2 × 6) was run concurrently to differentiate between responses to reduced feed intake alone and moderate heat stress. Blood neutrophil counts were particularly sensitive to moderate heat load with higher numbers during Challlenge and in PENs. The plasma concentrations of TNFα and IL-1ß were depressed in the TC group compared to the FRTN counterparts and remained so for 40 days after Challenge. Linear relationships of the concentrations of IL-1ß, IL-10, and haptoglobin with rumen temperature or dry matter intake detected in the FRTN group were altered or absent in the TC group. The findings suggest significant impacts of moderate heat load on the inflammatory status of feedlot cattle.

Comparing the responses of grain fed feedlot cattle under moderate heat load and during subsequent recovery with those of feed restricted thermoneutral counterparts: metabolic hormones.

We set out to determine the impact of moderate heat load on the plasma concentrations of a suite of hormones involved in regulating energy metabolism and feed intake. The responses of the thermally challenged (TC) feedlot steers were compared to those of feed restricted thermoneutral (FRTN) steers. Two sequential cohorts of twelve 518 ± 23 kg Black Angus steers on finisher grain ration were housed in climate-controlled rooms (CCR) for 18 days and returned to outdoor pens for 40 days. The TC group was subjected to a diurnal range of 28-35 °C for 7 days (Challenge) but held in thermoneutral conditions beforehand (PreChallenge), and in Recovery (after Challenge). The FRTN group was held in thermoneutral conditions and feed restricted throughout. Blood was collected over the three periods in CCR and two periods in outdoor pens for 40 days (PENS and Late PENS). Plasma concentrations of prolactin, thyroid stimulating hormone, insulin, leptin, adiponectin and thyroxine (T4) were determined during the five periods. Whilst the pituitary hormones were relatively stable, there were differences in plasma leptin, adiponectin and T4 between the two groups during Challenge and Recovery, and occasionally in PENS. The interaction of the plasma hormone concentrations and rumen temperature and DMI were also investigated. Whilst the positive relationship between DMI and leptin was confirmed, we found a strong negative relationship between adiponectin and rumen temperature, and a strong positive relationship between adiponectin and dry matter intake (DMI) in the TC steers only.

Associations between bone and energy metabolism in cows fed diets differing in level of dietary cation-anion difference and supplemented with cholecalciferol or calcidiol.

Bone-derived hormones play an important role in metabolism. This study examined the hypothesis that interactions between bone and energy metabolism, particularly those involving osteocalcin, are present in dairy cattle and have feedback mechanisms over time. Associations between metabolites in blood were examined in 32 Holstein cows blocked by parity and milk yield and randomly allocated to diets containing either 0.27 mg/kg dry matter (DM) calcidiol or cholecalciferol for an anticipated intake of 3 mg/d (120,000 IU/d) at 11 kg of DM, and positive (+130 mEq/kg DM) or negative (-130 mEq/kg DM) dietary cation-anion difference (DCAD) from 252 d of gestation to calving. Blood was sampled every 3 d, from 9 d prepartum to 30 d postpartum, and plasma concentrations of vitamin D3, 25-hydroxyvitamin D3, adiponectin, C-telopeptide of type 1 collagen (CTX1), glucose, insulin-like growth factor 1 (IGF1), insulin, undercarboxylated osteocalcin (uOC), and carboxylated osteocalcin (cOC) were determined. Feeding calcidiol compared with cholecalciferol increased plasma concentrations of 25-hydroxyvitamin D3 pre- (264.2 ± 8.0 vs. 61.3 ± 8.0 ng/mL) and postpartum (170.8 ± 6.2 vs. 51.3 ± 6.2 ng/mL) but decreased concentrations of vitamin D3 pre- (1.2 ± 0.6 vs. 14.5 ± 0.6 ng/mL) and postpartum (1.9 ± 0.4 vs. 3.2 ± 0.6 ng/mL). Prepartum, cows fed the negative DCAD diet had reduced concentrations of vitamin D3 and glucose compared with cows fed a positive DCAD. The combination of negative DCAD and cholecalciferol reduced IGF1 concentrations prepartum. The DCAD treatment had no effect on postpartum concentrations of metabolites. Nulliparous cows had increased concentrations of OC, CTX1, IGF1, glucose, and insulin compared with parous cows. Time series analysis identified associations between metabolites on the same day and over 3-d lags up to ±9 d that suggest feedback between 25-hydroxyvitamin D3 and vitamin D3 in the negative lags, indicating that 25-hydroxyvitamin D3 may exert feedback on vitamin D3 but not vice versa. We found evidence of a feedback mechanism between vitamin D3 and IGF1, with positive effect size (ES) on the same day and 3 d later, and negative ES 9 d later, that was more evident in cholecalciferol-fed cows. This suggests an important role of IGF1 in integrating bone metabolism with energy and protein metabolic pathways. Evidence of feedback was found between uOC and particularly cOC with IGF1, with positive ES on the same day but negative ES 6 d before and 6 d after. An association between uOC or cOC and IGF1 has not been previously identified in cattle and suggests that both uOC and cOC may have marked biological activity. Associations between OC and insulin identified in mice were not observed herein, although associations between OC and glucose were similar to those between IGF1 and glucose, supporting associations between glucose, OC, and IGF1. We provide further statistical evidence of crosstalk between vitamin D compounds, bone hormones, and energy metabolism in cattle. In particular, associations between uOC or cOC and IGF1 may provide links between prepartum diets and observations of prolonged increases in milk production and allow better control of peripartum metabolism.

Biochemical analysis of recombinant glutathione S-transferase of Fasciola hepatica.

Four cDNAs encoding GST (rGST1, rGST7, rGST47 and rGST51) of Fasciola hepatica were expressed in Escherichia coli and the rGST proteins purified for biochemical analyses. The rGST proteins are 95% pure as indicated by Coomassie staining of proteins separated by SDS-PAGE. Molecular sieving by HPLC infers that, like the native protein, the rGST proteins form homodimers under non-denaturing conditions. The rGST proteins are recognised by antisera raised to the native GST of F. hepatica. All four rGST proteins from F. hepatica actively conjugate glutathione to the universal substrate, 1-chloro-2,4-dinitrobenzene. The activity of the rGSTs was also measured for substrates which have been shown to have partial specificity for the Alpha, Mu or Pi classes of mammalian GSTs (trans-4-phenyl-3-buten-2-one, ethacrynic acid), for substrates known to be products of lipid peroxidation (trans-2-nonenal, trans,trans-2,4-decadienal) and for epoxy-3-(p-nitrophenoxy)-propane (EPNP), a known substrate for the theta class of GST. No rGST were active with EPNP. rGST47 and 51 showed activity with the other four substrates. rGST7 was active with three substrates whereas rGST1 showed relatively low activity with all substrates except trans,trans-2,4-decadienal. The sensitivity of the rGST activity to inhibition by the GST inhibitors triphenyltin chloride and bromosulphophthalein also varied among the rGSTs with rGST1 showing a 800-fold difference in sensitivity between the inhibitors. These results show that F. hepatica expresses a family of GST isoenzymes which exhibit unique substrate and inhibitor profiles.

Identification and characterisation of the excreted/secreted serine proteases of larvae of the old world screwworm fly, Chrysomya bezziana.

Serine proteases are the major proteolytic activity excreted or secreted from Chrysomya bezziana larvae as demonstrated by gelatin gel analyses and the use of specific substrates, benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Serine proteases were identified through their inhibition by 4-(2-aminoethyl)-benzene sulphonyl fluoride and classified as trypsin- and chymotrypsin-like on the basis of inhibition by tosyl-L-lysine chloromethyl ketone and tosyl-L-phenylalanine chloromethyl ketone, respectively. Like most insect serine proteases, the C. bezziana enzymes were active over broad pH range from mildly acidic to alkaline. The excreted or secreted serine proteases were purified by affinity chromatography using soybean trypsin inhibitor. A different subset of the serine proteases was isolated by salt elution from washed larval peritrophic matrices. Amino-terminal sequencing identified both trypsin and chymotrypsin-like sequences in the excreted or secreted pool with the latter being the dominant protease, whereas trypsin was the dominant species in the peritrophic matrix eluant. These results suggest that trypsin was possibly preferably adsorbed by the peritrophic matrix and may act as a final proteolytic processing stage as partially digested and ingested polypeptides pass through the peritrophic matrix. Immunoblot analysis on dissected gut tissues indicated that the anterior and posterior midguts were the main source of the serine proteases, although a novel species of 32 kDa was predominantly associated with the peritrophic matrix. Proteases are a target for a partially protective immune response and understanding the complexity of the secreted and digestive proteases is a necessary part of understanding the mechanism of the host's immunological defence against the parasite.

Peritrophins of adult dipteran ectoparasites and their evaluation as vaccine antigens.

Several peritrophins of larvae of Lucilia cuprina (sheep blowfly) have demonstrated potential as vaccine antigens, and some have been characterised and cloned. These proteins are tightly associated with the peritrophic matrix, a chitinous tube or sac lining the lumen of the gut of most insects. The peritrophins require strong denaturants for their removal from peritrophic matrix. We now report the preliminary characterisation of peritrophins of the adult stage of L. cuprina and Haematobia irritans exigua (buffalo fly). Similar SDS-PAGE profiles were obtained for proteins extracted in SDS or urea from isolated adult peritrophic matrices of both species. Radioiodination of urea-extracted peritrophins improved sensitivity, indicating numerous proteins of 15-75 kDa. Direct radioiodination of L. cuprina peritrophic matrix preferentially labelled high molecular weight complexes and proteins of 80-90 kDa. Two-dimensional gel analyses of a urea extract of adult L. cuprina peritrophic matrix revealed that most proteins were moderately acidic. Antibodies produced against SDS-extracted peritrophins, or against sonicated peritrophic matrices of these two flies were crossreactive. The sera also appeared to recognise SDS-extracted components of Triton X-100 treated and washed adult peritrophic matrix of the mosquito, Aedes vigilax (Skause). This profile altered as the peritrophic matrix matured. In concordance with extracts from the adult L. cuprina and H.i. exigua peritrophic matrices, proteins in the 50-75 kDa region were immunodominant. The vaccine potential of the peritrophins of these Diptera were examined following vaccination of cattle and rabbits with adult H.i. exigua or L. cuprina peritrophins. When the adult life stages of H.i. exigua or two mosquitoes, A. vigilax and A. aegypti (Linnaeus), were fed on the sera or blood of vaccinated hosts, there were no detrimental effects to any life cycle stages of these Diptera.

Peritrophic matrix proteins.

The peritrophic matrix (or peritrophic membrane) lines the gut of most insects at one or more stages of the life cycle. It has important roles in the facilitation of the digestive processes in the gut and the protection of the insect from invasion by microorganisms and parasites. The traditional view of the peritrophic matrix as a relatively insert sieve, composed largely of proteins and glycosaminoglycans embedded in a chitinous matrix, is under revision as more is learned about the molecular characteristics of the peritrophic matrix proteins. This review summarizes emerging knowledge of the main protein constituents of the peritrophic matrix. The availability of the first sequences of integral peritrophic matrix proteins has coincided with the explosion of information in sequence databases. It is therefore possible to examine common structural themes in this family of proteins as well as in proteins of unknown location and function from a variety of other insects, nematodes and viruses. The review concludes with speculation about the biological functions of the proteins in this matrix.

Expression of angiotensin-converting enzyme-related carboxydipeptidases in the larvae of four species of fly.

HieACE, a soluble 70 kDa protein related to the angiotensin-converting enzyme (ACE) has recently been identified, characterized and cloned from the adult buffalo fly (Haematobia irritans exigua). HieACE is enzymatically similar to the mammalian ACEs and its predicted amino acid sequence has 42% identity with the mammalian testicular ACEs. In adult H.i. exigua, HieACE expression is restricted to the compound ganglion and posterior midgut, and the maturing male reproductive system. Western blot analysis was used to investigate the expression of HieACE and its homologues in the larvae of H.i. exigua, Drosophila melanogaster, the sheep blowfly (Lucilia cuprina), the Old World screwworm fly (Chrysomya bezziana) and a secondary strike fly, Chrysomya rufifacies. Dipteran ACE homologues of 65-70 kDa were detected in all the larval instars investigated. Most of the immunoreactive proteins were concentrated in the soluble fraction. The first and second larval instars of L. cuprina and C. bezziana appeared to express two ACE homologues. These larvae were also found to secrete (or excrete) the ACE homologue in larval cultures. The presence of ACE-like enzymes in these larvae was confirmed by the measurement of carboxydipeptidase activity that was inhibited by the specific ACE inhibitor, captopril. The tissue distributions of the ACE homologues in the third instar larvae of H.i. exigua and L. cuprina were examined. As in adult H.i. exigua, HieACE was detected in the larval ganglion, but in contrast to the restricted distribution in the adult stage midgut, HieACE was found throughout the digestive system, and in the salivary glands of H.i. exigua larvae. The expression pattern in the gut of L. cuprina larvae was similar despite the differences in diet and habitat. The most striking difference from the adult stage H.i. exigua was the expression of HieACE and its L. cuprina homologues in the hindgut and Malpighian tubules of these larvae. These results suggest that the role(s) played by the dipteran ACE-like enzymes differ between the adult and larval stages.

Biochemical and molecular characterization of serine proteases from larvae of Chrysomya bezziana, the Old World Screwworm fly.

The diversity of serine proteases secreted from Chrysomya bezziana larvae was investigated biochemically and by PCR and sequence analysis. Cation-exchange chromatography of purified larval serine proteases resolved four trypsin-like activities and three chymotrypsin-like activities as discerned by kinetic studies with benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Amino-terminal sequencing of the three most abundant fractions gave two sequences, which were homologous to other Dipteran trypsins and chymotrypsins. Analysis of products generated by PCR of cDNA from whole larvae using specific primers based on the amino-terminal sequences and generic serine protease primers identified 22 different sequences, while phylogenetic analysis of the deduced amino acid sequences differentiated two trypsin-like and four chymotrypsin-like families. Phylogenetic comparisons with Dipteran and mammalian serine protease sequences showed that all the Chrysomya bezziana sequences clustered with Dipteran sequences. The Chrysomya bezziana chymotrypsin-like sequences segregated within a Dipteran cluster of chymotrypsin sequences, but were well dispersed amongst these sequences. The largest Chrysomya bezziana serine protease family, the trypB family, clustered tightly as a group, and was closely related to a Lucilia cuprina trypsin but distinct from Drosophila melanogaster alpha and beta trypsins. The trypB family contains ten highly homologous sequences and probably represents an example of concerted evolution of a trypsin gene in Chrysomya bezziana.

Secretion of the type 2 peritrophic matrix protein, peritrophin-15, from the cardia.

The midgut of most insects is lined with a peritrophic matrix, which is thought to facilitate digestion and protect the midgut digestive epithelial cells from abrasive damage and invasion by ingested micro-organisms. The type 2 peritrophic matrix is synthesised by a complex and highly specialised organ called the cardia typically located at the junction of the cuticle-lined foregut and midgut. Although the complex anatomy of this small organ has been described, virtually nothing is known of the molecular processes that lead to the assembly of the type 2 peritrophic matrix in the cardia. As a step towards understanding the synthesis of the peritrophic matrix, the synthesis and secretion of the intrinsic peritrophic matrix protein, peritrophin-15 has been followed in the cardia of Lucilia cuprina larvae using immuno-gold localisations. The protein is synthesised by cardia epithelial cells, which have abundant rough endoplasmic reticulum, Golgi, and vesicles indicative of a general secretory function. Peritrophin-15 is packaged into secretory vesicles probably produced from Golgi and transported to the cytoplasmic face of the apical plasma membrane. The vesicles fuse with the plasma membrane at the base of the microvilli and release peritrophin-15 into the inter-microvilli spaces. The protein then becomes associated with the nascent peritrophic matrix, which lies along the tips of the epithelial cell microvilli. It is proposed that peritrophin-15 binds to the ends of chitin fibrils present in the nascent peritrophic matrix, thereby protecting the fibril from the action of exochitinases.

Cloning and characterisation of angiotensin-converting enzyme from the dipteran species, Haematobia irritans exigua, and its expression in the maturing male reproductive system.

The angiotensin-converting enzymes (ACE) are involved in the regulation of the specific maturation or degradation of a number of mammalian bioactive peptides. A carboxydipeptidase similar to mammalian ACE has now been identified in the adult stage of the haematophagous fly, Haematobia irritans exigua (buffalo fly), a close relative of the horn fly of North America. The enzyme was purified by lectin-affinity chromatography and ion-exchange chromatography and migrated as a doublet of 70 kDa upon reducing SDS/PAGE. Unlike mammalian ACE, the fly carboxydipeptidase (HieACE) is not membrane bound. The amino acid sequence of an internal peptide from HieACE and a conserved amino acid region present in all mammalian ACE were used to design degenerate oligonucleotide primers suitable for PCR. A DNA fragment amplified from adult buffalo fly cDNA was used to identify a cDNA clone that encoded the enzyme. The cDNA sequence encodes a carboxydipeptidase with 41-42% amino acid identity to the mammalian testicular ACE. The active-site regions of mammalian ACE are conserved in the deduced amino acid sequence of HieACE. Enzymatically, HieACE is very similar to its mammalian counterparts, with comparable Km and V(max) values for the synthetic substrate, benzoylglycylglycylglycine, and similar patterns of inhibition by EDTA, ACE inhibitor peptide and captopril. HieACE also specifically activates angiotensin I to angiotensin II and degrades other mammalian ACE substrates such as bradykinin, substance P and cholecystokinin-8. In the adult fly, HieACE is expressed in the compound ganglion and in the posterior region of the midgut. Similar to the mammalian system, expression of this enzyme is induced in the maturing male reproductive system, which suggests conservation of ACE function in these species.

A novel family of chitin-binding proteins from insect type 2 peritrophic matrix. cDNA sequences, chitin binding activity, and cellular localization.

The peritrophic matrix is a prominent feature of the digestive tract of most insects, but its function, formation, and even its composition remain contentious. This matrix is a molecular sieve whose toughness and elasticity are generated by glycoproteins, proteoglycans, and chitin fibrils. We now describe a small, highly conserved protein, peritrophin-15, which is an abundant component of the larval peritrophic matrices of the Old World screwworm fly, Chrysomya bezziana, and sheep blowfly, Lucilia cuprina. Their deduced amino acid sequences code for a 8-kDa secreted protein characterized by a highly conserved and novel register of six cysteines. Two Drosophila homologues have also been identified from unannotated genomic sequences. Recombinant peritrophin-15 binds strongly and specifically to chitin; however, the stoichiometry of binding is low (1:10,000 N-acetyl glucosamine). We propose that peritrophin-15 caps the ends of the chitin polymer. Immunogold studies localized peritrophin-15 to the peritrophic matrix and specific vesicles in cells of the cardia, the small organ of the foregut responsible for peritrophic matrix synthesis. The vesicular contents are disgorged at the base of microvilli underlying the newly formed peritrophic matrix. This is the first time that the process of synthesis and integration of a peritrophic matrix protein into the nascent peritrophic matrix has been observed.

Vaccination against the Old World screwworm fly (Chrysomya bezziana).

Chrysomya bezziana is an endemic pest of livestock or a threat to livestock production in large areas of Africa, the Middle East, southern and south-east Asia and Australia. Its control is difficult. The feasibility of vaccinating against this pest has now been explored. In-vitro and in-vivo assays have been established. Using these assays, it has been shown that first instar larvae, third instar peritrophic membrane and cardia are all sources of material able to induce immunological reactions in sheep which lead to significant reductions in larval growth. In-vitro assays following vaccination with peritrophic membrane also show larval mortality. Taken together, these effects lead to an 82% reduction in the weight of recovered larvae in vitro and 45% reduction in vivo. Preliminary evidence suggests that the mechanism of protection may be complex.

A universal protein-protein interaction motif in the eubacterial DNA replication and repair systems.

The interaction between DNA polymerases and sliding clamp proteins confers processivity in DNA synthesis. This interaction is critical for most DNA replication machines from viruses and prokaryotes to higher eukaryotes. The clamp proteins also participate in a variety of dynamic and competing protein-protein interactions. However, clamp-protein binding sequences have not so far been identified in the eubacteria. Here we show from three lines of evidence, bioinformatics, yeast two-hybrid analysis, and inhibition of protein-protein interaction by modified peptides, that variants of a pentapeptide motif (consensus QL[SD]LF) are sufficient to enable interaction of a number of proteins with an archetypal eubacterial sliding clamp (the beta subunit of Escherichia coli DNA polymerase III holoenzyme). Representatives of this motif are present in most sequenced members of the eubacterial DnaE, PolC, PolB, DinB, and UmuC families of DNA polymerases and the MutS1 mismatch repair protein family. The component tripeptide DLF inhibits the binding of the alpha (DnaE) subunit of E. coli DNA polymerase III to beta at microM concentration, identifying key residues. Comparison of the eubacterial, eukaryotic, and archaeal sliding clamp binding motifs suggests that the basic interactions have been conserved across the evolutionary landscape.

Characterization of a 95,000 molecule on sheep leucocytes homologous to murine Pgp-1 and human CD44.

The phagocyte glycoprotein-1 (Pgp-1) antigen of mice is a 94,000 MW molecule with a wide tissue distribution, but no attributed function. We produced a monoclonal antibody (mAb) termed 25-32 which recognizes the Pgp-1 molecule of numerous mammalian species, including humans and sheep. Preclearing experiments with I42/5, a rat anti-mouse Pgp-1 mAb that cross-reacts with human Pgp-1, established the specificity of 25-32 for human and sheep Pgp-1. Moreover, an antibody recognizing human CD44, termed F10-44-2, also reacted with the same molecule as that recognized by 25-32 and I42/5, so establishing the co-identity of CD44 and Pgp-1. Within the sheep thymus, Pgp-1 was expressed most strongly by medullary thymocytes and stromal cells, and by small numbers of cells at the subcapsular cortex. Pgp-1 was expressed early in thymic ontogeny; all 35-40-day gestation fetal sheep thymocytes were intensely Pgp-1+, but by 80 days the number of reactive thymocytes had decreased to adult levels. The expression of Pgp-1 on lymphocytes was markedly increased after stimulation with mitogens, or with phorbol esters and ionomycin. The highly conserved nature of Pgp-1 through evolution, its expression on virtually all cell types within the body, and its increased expression on rapidly dividing cells indicate that this molecule mediates an important function, possibly serving as a hormone or metabolite receptor.

Genetic and biochemical characterization of antigens encoded by the Ly-24 (Pgp-1) locus.

Three allele-specific monoclonal antibodies to Pgp-1 (Ly-24) were used to biochemically characterize the cell surface structures with which they reacted and to map the gene(s) coding for these antigens. The targets of these three monoclonal antibodies (mAb) were shown to be encoded by a gene situated on chromosome 2 close to beta 2m [gene order (Pgp-1-beta 2m-a)] and no recombination between the loci detected by the three antibodies was revealed by genetic analysis. The genetic mapping of loci and tissue distribution of these antigens suggested that they might all correspond to a particular allelic form of the mouse phagocyte glycoprotein-1 (Pgp-1) antigen. Biochemical and serological analysis confirmed that this was indeed the case and revealed that all three mAbs were directed to one epitope. It is surprising that the tissue distribution defined by one mAb (Ly-24A) was different from that for the two other (Ly-24B) antibodies, despite the serological and biochemical identity of their respective targets. The possible reason for this unusual finding is discussed.

Fasciola hepatica: characterization and cloning of the major cathepsin B protease secreted by newly excysted juvenile liver fluke.

Proteolytic activity present in the excreted/secreted (ES) material of newly excysted juvenile (NEJ) Fasciola hepatica was biochemically analyzed. By gelatin substrate SDS-PAGE, only one region of activity was observed in the NEJ ES material at a molecular mass of 29 kDa. Both the secreted cathepsin L from adult fluke and the 29-kDa proteolytic activity of NEJ ES show a common pH optimum of 7.5, a cysteine protease inhibition profile, and preference for the N-benzyloxycarbonyl (Z)-Phe-Arg-NHMec fluorogenic substrate over Z-Arg-Arg-NHMec and Z-Arg-NHMec. In vitro analysis revealed that the NEJ protease activity digested sheep immunoglobulin heavy chain and bovine serum albumin but not bovine hemoglobin. Amino-terminal protein sequence analysis of the 29-kDa NEJ protease band revealed two sequences with homology to the cathepsin B family of proteases. Using degenerate oligonucleotides designed from the N-terminal sequence, reverse transcriptase polymerase chain reaction with NEJ RNA amplified a cDNA sequence encoding the first 236 amino acids of mature cathepsin B. Using this cDNA fragment an overlapping cDNA was isolated from a LambadaZAP cDNA library constructed with poly(A)+ RNA from immature 5-week-old liver fluke. Together with the N-terminal sequence, these cDNAs predict a mature cathepsin B sequence of 254 amino acids which shows 48-51% sequence identity to mammalian and Schistosoma mansoni cathepsin B. We conclude that, in contrast to the major proteases released by adult fluke, the major secreted protease of NEJ of F. hepatica is of the cathepsin B class.

The secreted cathepsin L-like proteinases of the trematode, Fasciola hepatica, contain 3-hydroxyproline residues.

The cysteine proteinases synthesized by the adult stage of the trematode Fasciola hepatica were found to be a very heterogeneous group of proteins as demonstrated by one- and two-dimensional gel analyses. N-terminal amino acid sequencing indicated the presence of at least two distinct gene products among the secreted cysteine proteinases. Enzymic studies and peptide sequence analysis of the excreted/secreted cysteine proteinases suggested a close relationship to the plant thiol cathepsins and the mammalian cathepsin L subfamily. The cloning of a representative cDNA for a putative Fasciola cathepsin confirmed similarities to the cathepsin L subfamily but revealed low identity with the cathepsin-like proteinases of the related trematode, Schistosoma, nematode cathepsins and the mammalian cathepsin B subfamily. Furthermore, peptide and protein sequencing revealed the modification of certain highly conserved prolines to unusual 3-hydroxyproline derivatives. This is the first report of modified prolines in any proteinase. This finding, as well as the high activities of these cathepsins at neutral to alkaline pH values, raises a number of questions as to the physiological function of these thiol cathepsins and their interaction with host tissues.

Fasciola hepatica: localisation of glutathione S-transferase isoenzymes in adult and juvenile liver fluke.

Four cDNA clones (GST-1, -7, -47, and -51) encoding isoenzymes of the detoxification enzyme glutathione S-transferase (GST) have previously been identified and characterised from Fasciola hepatica. In the present study, antisera were generated to synthetic peptides of regions unique to each of the four GST proteins predicted by the cDNAs. The antisera were characterised, and two were found to distinguish GST-1 from GST-7, GST-47, and GST-51 as a group. These two antisera were used to localise different GSTs in adult and newly excysted juvenile F. hepatica. The antiserum to GST-1 was specific and localised GST-1 to the parenchyma of adult fluke but not to the lamellae of the intestinal caeca. The antiserum to a GST-51 peptide, which cross-reacted with GST-7 and GST-47 but not GST-1, localised the other GSTs not only to the parenchyma but also to the intestinal lamellae of adult fluke. This appears to be the first evidence of tissue-specific expression of GST isoenzymes in trematodes. In contrast to adult fluke, immunolocalisation of the GSTs in juvenile F. hepatica revealed the binding of both the GST-1 and GST-51 antisera to the parenchymal cytoplasm, to cytoplasmic extensions of the parenchyma cells in the subtegumental area, as well as the excretory ducts. No labeling was observed in the intestinal epithelium of the juvenile fluke. These results demonstrate that adult F. hepatica, in contrast to juvenile flukes, contain a GST, which is not GST-1, associated with the lamellae of the gut and suggest that GSTs in adult fluke may play a role in the absorptive function of the adult gut.

Glutathione S-transferase. Novel vaccine against Fasciola hepatica infection in sheep.

The potential of GST as a vaccine candidate against liver fluke infection in ruminants was studied by vaccinating sheep (n = 9) with GST purified from adult worms of Fasciola hepatica and challenging with 500 F. hepatica metacercariae. The immunization induced a high antibody response to GST in contrast to the poor or undetectable response to this Ag observed in naturally infected sheep. Throughout the trial, the progress of the fluke infection was monitored by measuring RBC hemoglobin levels, the extent of liver damage and the fecal egg output in the sheep. This analysis indicated that a subpopulation (n = 4) of the GST vaccinated animals exhibited no anemia, reduced liver damage and a lower mean fecal egg count relative to the infected control group suggesting a lower fluke burden in these animals. Worm burdens in the livers of the GST vaccine group (107 +/- 22) were 57% lower than in the infected control group (250 +/- 25). The subpopulation of the GST vaccine group demonstrated a 78% reduction in mean worm burdens relative to the control group. These results show that GST of adult F. hepatica is a novel Ag that can significantly protect sheep against liver fluke infection. The results suggest that the immune response to GST is directed to the juvenile worm reducing the number of worms that can establish in the liver of the vaccinated animals.