In silico analysis of two Haemonchus spp. serine protease peptides (S28) and their immunomodulatory activity in vitro.

The aim of this study was to evaluate the in vitro immune modulation of two de novo peptides with hypothetical identity to the serine protease family (S28) from Haemonchus spp. Expression of mRNAs encoding these peptides was confirmed by RTqPCR in L3 and adult stage parasites. Antibodies from serum samples collected from an H. contortus-infected lamb at 60 days post infection detected both peptides, as assessed by indirect ELISA. Lamb peripheral blood mononuclear cells (PBMCs) were exposed to each peptide, as well as to the peptide mixture, and cell proliferation assays were performed at 24, 48 and 72 h. The relative expression of the IL4, IL5, IL6, IL13, CXCL8 and FCεR1A genes was quantified by RTqPCR from lamb PBMCs exposed to the peptide mixture at 24 and 48 h. With respect to immune gene expression, 15- and 3-fold upregulation at 24 h was observed with IL5 and CXCL8, respectively, and 2-fold upregulation of CXCL8 at 48 h. In contrast, downregulation of IL5 was stimulated at 48 h. These data suggest that these peptides (pep-hsp and pep-pcx), which show high identity with intestinal and excretion/secretion serine proteases, can trigger immunogenic activity, and suggest that they may be useful as potential parasite vaccines.

Acyl-CoA oxidase ACOX-1 interacts with a peroxin PEX-5 to play roles in larval development of Haemonchus contortus.

Hypobiosis (facultative developmental arrest) is the most important life-cycle adaptation ensuring survival of parasitic nematodes under adverse conditions. Little is known about such survival mechanisms, although ascarosides (ascarylose with fatty acid-derived side chains) have been reported to mediate the formation of dauer larvae in the free-living nematode Caenorhabditis elegans. Here, we investigated the role of a key gene acox-1, in the larval development of Haemonchus contortus, one of the most important parasitic nematodes that employ hypobiosis as a routine survival mechanism. In this parasite, acox-1 encodes three proteins (ACOXs) that all show a fatty acid oxidation activity in vitro and in vivo, and interact with a peroxin PEX-5 in peroxisomes. In particular, a peroxisomal targeting signal type1 (PTS1) sequence is required for ACOX-1 to be recognised by PEX-5. Analyses on developmental transcription and tissue expression show that acox-1 is predominantly expressed in the intestine and hypodermis of H. contortus, particularly in the early larval stages in the environment and the arrested fourth larval stage within host animals. Knockdown of acox-1 and pex-5 in parasitic H. contortus shows that these genes play essential roles in the post-embryonic larval development and likely in the facultative arrest of this species. A comprehensive understanding of these genes and the associated ß-oxidation cycle of fatty acids should provide novel insights into the developmental regulation of parasitic nematodes, and into the discovery of novel interventions for species of socioeconomic importance.

Suppression of hyaluronidase reduces invasion and establishment of Haemonchus contortus larvae in sheep.

Haemonchus contortus is a hematophagous endoparasite of small ruminants, which is responsible for huge economic losses in livestock sector. Hyaluronidase produced by infective larvae of H. contortus can degrade hyaluronic acid present in the host's abomasal tissue. Thus, it facilitates larval tissue invasion and early establishment. We herein explored this ability of hyaluronidase in H. contortus, and tested whether hyaluronidase is utilized as a virulence factor by H. contortus while establishing the infection. We first successfully blocked the hyaluronidase gene in L3 larvae by RNA interference (RNAi), which was subsequently confirmed by qPCR, enzymatic activity, and immunohistochemistry assays. Using these larvae we then conducted in vivo and in vitro assays on sheep to assess the effects of hyaluronidase suppression on larval invasion and establishment of infection. The in vivo assay showed a significant drop in worm burden in siRNA treated group in comparison to control group. During in vitro assay we applied an ovine ex vivo model where siRNA treated group of larvae showed significantly reduced invasion of the abomasal tissue explants as compared to control group. These findings indicate that hyaluronidase plays a key role in host's tissue invasion and larval establishment, and it is used as a virulence factor by H. contortus while establishing the infection. As an invasive virulence molecule, its functional research is thus conducive to the prevention of haemonchosis.

Ruminal fermentation, microbial population and lipid metabolism in gastrointestinal nematode-infected lambs fed a diet supplemented with herbal mixtures.

The aim of this study was to evaluate the effects of medicinal herbal mixtures rich in phenolic, flavonoid and alkaloid compounds on ruminal fermentation and microbial populations, and fatty acid (FA) concentrations and lipid oxidation in tissues of lambs infected with the gastrointestinal nematode (GIN) parasite (Haemonchus contortus). Parallel in vitro and in vivo studies were performed using two different herbal mixtures (Mix1 and Mix2). The in vitro study was conducted in a 2 (infection status; non-infected versus infected) × 3 (diets; control, Mix1 and Mix2) factorial design. In the in vivo study, 24 lambs were equally divided into four treatments: non-infected lambs fed a control diet, infected lambs fed the control diet, infected lambs fed a diet with Mix1 and infected lambs fed a diet with Mix2. Herbal mixtures (100 g dry matter (DM)/d) were added to the basal diets of meadow hay (ad libitum) and a commercial concentrate (500 g DM/d). The experimental period lasted for 70 days. Ruminal fermentation characteristics and methane production were not affected by infection in vivo or in vitro. Both herbal mixture supplementation increased total volatile fatty acid (VFA) concentrations (P < 0.01) and DM digestibility (P < 0.01) in vitro. Archaea population was slightly diminished by both herbal mixtures (P < 0.05), but they did not lower methane production in vitro or in vivo (P > 0.05). Infection of H. contortus or herbal mixtures modulated FA proportion mainly in the liver, especially the long chain FA proportion. Concentrations of thiobarbituric acid reactive substances (TBARS) in serum were significantly higher after 70 days post-infection in the infected lambs. Herbal Mix1 supplementation reduced TBARS concentrations in meat after seven days of storage. In conclusion, supplementing of herbal mixtures to the diets of GIN parasite infected lambs did not affect the basic ruminal fermentation parameters. Herbal mixtures may improve few FA proportions mainly in liver as well as decrease lipid oxidation in meat.

Copper oxide and closantel prevent alterations in hepatic energetic metabolism and reduce inflammation in Haemonchus contortus infection.

The aims of this study were to evaluate if the use of copper oxide wire particles, isolated or in association with closantel, in lambs infected with Haemonchus contortus enhances the anthelmintic efficacy of closantel, as well as to evaluate the effects of treatment in hepatic energy metabolism, inflammatory markers and hematological and biochemical tests. The lambs were randomly divided into five groups (6 animals each), as follows: uninfected animals (Control); animals infected with H. contortus (HC); infected and treated with closantel (HC + CL); infected and treated with copper oxide wire particles (HC + Cu); and infected and treated with closantel plus copper oxide wire particles (HC + CL + Cu). The animals of infected groups were infected orally with H. contortus (5,000 L3 -larvae) and on day 14 post infection (p.i) the treatments were initiated. The egg per gram of feces (EPG), butyrylcholinesterase (BuChE), myeloperoxidase (MPO), adenylate kinase (AK) and pyruvate kinase (PK) activities and hematological and biochemical tests were evaluated. Treatments with copper oxide (isolated and associated) were able to reduce the EPG count on days 28, 35, 42 and 49 p.i when compared to HC group, while closantel was able to reduce EPG only from day 35 p.i. Moreover, treatment with closantel (isolated or associated) was able to prevent the inhibition of hepatic AK and PK activities caused by H. contortus infection, which may contribute to efficient intracellular energetic communication in order to maintain the balance between cellular ATP consumption and production. Butyrylcholinesterase and MPO activities were higher in infected lambs compared to uninfected, while treated groups showed lower enzymatic activity compared to the group HC. The use of all therapeutic protocols was able to reduce the EPG count. Based on these evidences, the use of copper oxide plus closantel may be considered an alternative to treat lambs infected by H. contortus.

Evaluation of antioxidant and oxidant status of goats (Capra aegagrus hircus) naturally infected with Haemonchus contortus.

The present study aimed to assess the antioxidant and oxidant status of goats naturally infected with Haemonchus contortus. Based upon the parasite burden, infection in goats was categorized as heavy (> 500 worms), mild (100-500 worms) or low (< 100 worms). Abomasal tissues from non-infected and infected goats were used for the determination of catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), aspartate (AST) and alanine (ALT) aminotransferases, acid (ACP) and alkaline (ALP) phosphatases, superoxide content (O2-), protein carbonyl (PC), malondialdehyde (MDA) and reduced glutathione (GSH). A significantly higher level of CAT, GST and GR activity and a lower level of GPx activity were recorded in infected compared to non-infected tissue. A significant increase in the level of AST, ALT, ALP and ACP was found in the abomasal tissue of the infected animals, which was related to the worm burden. The oxidative stress markers were also altered, with a significant decline in GSH levels, whereas MDA, PC and O2- concentrations showed a marked increase. In conclusion, it has been demonstrated that haemonchosis in goats resulted in considerable oxidative stress, which was directly related to the worm burden.

Supplementation with copper edetate in control of Haemonchus contortus of sheep, and its effect on cholinesterase's and superoxide dismutase activities.

The aim of this study was to evaluate the effect of copper edetate on biochemical parameters, oxidative profile, cholinesterase's activities, as well as its capacity to control gastrointestinal parasites in infected sheep. Thus, Lacaune sheep (n = 18) infected by Haemonchus contortus were used and divided into three groups of six animal each: the group A was composed of untreated animals (the control group), the group B was formed by animals treated with 0.3 mg/kg of copper edetate, and the group C was composed of animals treated with 0.5 mg/kg of copper edetate. Blood collection was performed on days 0, 10, 20 and 30 after mineral supplementation and different variables were measured. Cholinergic system was evaluated to determine the acetylcholinesterase (AChE) activity in total blood and butyrylcholinesterase (BChE) activity in serum. Eggs per gram of feces (EPG) were evaluated. There were no significant differences (P > 0.05) between groups regarding total protein, albumin, globulin and urea levels, GGT activity, as well as the hematocrit, and EPG. ALT activity decreased (P < 0.05) on groups B and C on day 30 compared to the control group (the group A). AChE activity decreased (P < 0.05) in the group C on days 10 and 30 compared to the control group, such decrease (P < 0.05) was also observed for BChE activity in the group C on day 10. ROS levels increased in the group C compared to groups A and B on day 10, while the SOD activity increased in the group C on days 20 and 30 compared to the control group (P < 0.05). CAT activity did not differ between groups (P > 0.05). In summary, the copper edetate was not efficient to control gastrointestinal parasites, but efficiently activated SOD, an important antioxidant enzyme. In addition, copper edetate was able to partially inhibit cholinesterase's activities when supplementated at its highest dose.

Histochemical study of the effects on abomasal mucins of Haemonchus contortus or Teladorsagia circumcincta infection in lambs.

Previously, chemical analysis of gastric fundic mucin showed that infection of sheep with Haemonchus contortus or Teladorsagia circumcincta changed the proportions of monosaccharides and decreased terminal mucin fucosylation and sialylation. To identify the effects of these parasites on the two mucin-secreting cell lineages, fundic and antral tissues were collected for histochemistry from 69 lambs aged from 3-4 to 9-10 months-of-age which had received a single infection of either H. contortus or T. circumcincta and euthanased at Day 21 or 28 post- infection respectively. All fundic tissues were stained separately with: (1) with Periodic Acid Schiff (PAS) for all mucins; (2) Alcian Blue (AB) pH 2.5 for acidic mucins (sialylated and sulphated); (3) AB pH 1 for sulphated mucins and (4) High Iron Diamine (HID) for sulphated mucins. Antral and fundic tissues from 24 lambs were also stained for acidic and neutral mucins or with specific lectins for α-1-linked fucose and for α-2,3- and α-2,6-linked sialic acids. Only mucin sulphation appeared to differ visually in uninfected lambs over this age range: there was weak staining with HID in tissues from lambs 3-6 months-of-age, but was generally more intense in those over 7 months-of-age. Sulphomucins were not apparent in surface mucous cells (SMC) or generally in the upper pits. Sialylomucins were located predominantly in the pits and glands, with small amounts of sialylated mucins in SMC and on the luminal surface, mainly in younger animals up to 6 months-of-age and less in the older animals. Parasitism markedly reduced the predominantly neutral surface mucin5AC of the SMC and pit cells, despite pit elongation in both antrum and fundus, whereas the acidic Muc6 secreted by mucus neck cells (MNC) increased along with MNC hyperplasia. Sulphated mucins were present mainly from the mid-pits downward and heavy staining was more common in older animals. In these sheep, the markedly reduced neutral mucin in the SMC and pit cells in both antrum and fundus contrasts with reported hypersecretion of mucus in the intestine, which is believed to aid in parasite expulsion. It has been proposed that intestinal goblet cell hypersecretion occurs only in resistant animals, therefore reduced mucins in the abomasum may be indicative of susceptibility to abomasal parasites.

Galectins and collectinis expression are increased in Haemonchus contortus-infected corriedale sheep.

Galectins and collectins are proteins classified in the lectin family that have the ability to recognize molecular patterns associated with pathogens. Studies on cattle have demonstrated high expression of these proteins during infection with gastrointestinal nematodes. The aim of this study was to investigate whether the level of Haemonchus contortus infection would alter the expression of galectins (Gal11 and Gal14) and collectins (SPA and CGN) in sheep. Twelve Corriedale sheep exposed to natural infection with nematodes were divided into two groups: group 1 (G1, n = 7) and group 2 (G2, n = 5), with low and high parasite burdens, respectively, based on fecal egg counts and abomasal parasite counts. The fecal egg counts and abomasal parasite counts were significantly different (p < 0.05) between the groups. Galectin and collectin gene expression was observed in all sheep abomasal samples. However, animals with lower infection levels showed lower expression of the genes Gal14, SPA and CGN (p < 0.05). Expression of lectins was associated with the abomasal H. contortus burden, thus suggesting that these proteins may have a role in controlling of this infection.

Galectins and collectinis expression are increased in Haemonchus contortus-infected corriedale sheep / Aumento da expressão gênica de colectinas e galectinas em ovinos corriedale infectados por Haemonchus contortus

Galectins and collectins are proteins classified in the lectin family that have the ability to recognize molecular patterns associated with pathogens. Studies on cattle have demonstrated high expression of these proteins during infection with gastrointestinal nematodes. The aim of this study was to investigate whether the level of Haemonchus contortus infection would alter the expression of galectins (Gal11 and Gal14) and collectins (SPA and CGN) in sheep. Twelve Corriedale sheep exposed to natural infection with nematodes were divided into two groups: group 1 (G1, n = 7) and group 2 (G2, n = 5), with low and high parasite burdens, respectively, based on fecal egg counts and abomasal parasite counts. The fecal egg counts and abomasal parasite counts were significantly different (p < 0.05) between the groups. Galectin and collectin gene expression was observed in all sheep abomasal samples. However, animals with lower infection levels showed lower expression of the genes Gal14, SPA and CGN (p < 0.05). Expression of lectins was associated with the abomasal H. contortus burden, thus suggesting that these proteins may have a role in controlling of this infection.

Colectinas e galectinas são proteínas da família das lectinas que possuem a capacidade de reconhecer padrões moleculares associados aos patógenos. Estudos em bovinos têm demonstrado a alta expressão dessas proteínas durante a infecção por nematoides gastrintestinais. O objetivo deste estudo foi investigar se o nível de infecção de Haemonchus contortus altera a expressão de colectinas (SPA e CGN) e galectinas (Gal11 e Gal14) de ovinos. Doze ovinos da raça Corriedale expostos a infecção natural com nematoides foram separados em dois grupos: grupo 1 (G1, n=7) com menor grau de parasitismo; e grupo 2 (G2, n=5) com maior grau, a partir da contagem do número de parasitos recuperados do abomaso e OPG. A contagem de OPG e de parasitos recuperados do abomaso dos grupos G1 e G2 apresentaram diferença estatística (p<0,05). A expressão dos genes de colectinas e galectina foi observada em todas as amostras de abomaso dos ovinos, porém animais com menor grau de infecção apresentaram menor expressão dos genes de Gal14, SPA e CGN (p<0,05). A expressão de lectinas foi associada ao número de H. contortus encontrados no abomaso de ovinos, indicando um possível papel dessas proteínas no controle da infecção.

Effects of dietary supplementation with green tea polyphenols on digestion and meat quality in lambs infected with Haemonchus contortus.

Ujumqin sheep are susceptible to infection by the gastrointestinal nematode Haemonchus contortus, which reduces productivity and total meat yield in sheep. Thus, the effects of green tea polyphenol (GTP) supplements (0, 2, 4, or 6g of GTP/kg feed) on dietary nutrient digestibility and meat quality in lambs infected with H. contortus were examined; control lambs were not infected. H. contortus infections did not affect digestion but the apparent digestibilities of nutrients were decreased by dietary 2g of GTP/kg feed supplementation. There was an interaction between treatment and sampling time on plasma total protein, urea nitrogen, and amino acid concentrations. The antioxidant activity and meat color of INFGTP0 lambs decreased. In conclusion, H. contortus infections in lambs decreased meat quality, but appropriate levels of dietary GTP supplementation diminished these negative effects though lower dose of GTP supplement showed negative effects on digestion.

Cloning and tissue distribution of novel splice variants of the ovine ghrelin gene.

BACKGROUND: The ghrelin axis is involved in the regulation of metabolism, energy balance, and the immune, cardiovascular and reproductive systems. The manipulation of this axis has potential for improving economically valuable traits in production animals, and polymorphisms in the ghrelin (GHRL) and ghrelin receptor (GHSR) genes have been associated with growth and carcass traits. Here we investigate the structure and expression of the ghrelin gene (GHRL) in sheep, Ovis aries. RESULTS: We identify two ghrelin mRNA isoforms, which we have designated Δex2 preproghrelin and Δex2,3 preproghrelin. Expression of Δex2,3 preproghrelin is likely to be restricted to ruminants, and would encode truncated ghrelin and a novel C-terminal peptide. Both Δex2 preproghrelin and canonical preproghrelin mRNA isoforms were expressed in a range of tissues. Expression of the Δex2,3 preproghrelin isoform, however, was restricted to white blood cells (WBC; where the wild-type preproghrelin isoform is not co-expressed), and gastrointestinal tissues. Expression of Δex2 preproghrelin and Δex2,3 preproghrelin mRNA was elevated in white blood cells in response to parasitic worm (helminth) infection in genetically susceptible sheep, but not in resistant sheep. CONCLUSIONS: The restricted expression of the novel preproghrelin variants and their distinct WBC expression pattern during parasite infection may indicate a novel link between the ghrelin axis and metabolic and immune function in ruminants.

Protein profile of lambs experimentally infected with Haemonchus contortus and supplemented with selenium and copper.

BACKGROUND: Gastrointestinal nematodes cause significant economic losses in the sheep industry, with frequent reports of anthelmintic resistance. Therefore, alternative methods to control these parasites are necessary. Thus, the aim of the present study was to assess the effect of treatment with selenium and copper on the protein profile of sheep that were experimentally infected with Haemonchus contortus. METHODS: Twenty-eight lambs were experimentally infected with H. contortus and divided into four experimental groups as follow: G1--untreated animals; G2--treated with sodium selenite; G3--treated with copper; G4--treated with sodium selenite and copper. The serum protein, body weight and egg count per gram of feces (EPG) were assessed at the baseline and after 20, 40, 60 and 80 days. The parasite burden was assessed 80 days after the beginning of the experiment. RESULTS: Higher levels of total protein and gamma globulin were observed in the lambs treated with sodium selenite and copper on D80. Copper acted as a growth promoter. The copper-supplemented groups exhibited higher daily and total weight gain. The association of selenium and copper altered the protein profile of sheep. Copper and selenium supplementation reduced EPG and worm burden at the end of the experiment. To the best of our knowledge, this is the first study to demonstrate the positive effect of the combined parenteral supplementation of Se and Cu on H. contortus infection. CONCLUSIONS: This injectable supplementation could be used as an auxiliary method to control H. contortus in sheep.

Influence of experimental infection by Haemonchus contortus on acetylcholinesterase activity in lymphocytes of lambs.

The aim of this study was to evaluate the acetylcholinesterase (AChE) activity in lymphocytes of lambs experimentally infected by Haemonchus contortus. A total of 14 healthy lambs were used, divided into two groups of seven animals each. Group A (negative control) represented the uninfected animals, and Group B (positive control) was formed by animals infected with 15,000 larvae of H. contortus. Blood was drawn on the days 15, 45 and 75 post-infection (PI) in order to perform the white blood cells (WBC) count, as well as the evaluation of AChE activity in lymphocytes. Parasitological stool exam (eggs per gram of feces - EPG) was performed on the same days to follow up the evolution of the infection. On day 15 PI it was verified negative EPG; however, on days 45 and 75 PI it was observed positive EPG only in the animals of group B. In the three evaluated periods was observed a lower number of leukocytes, associated with decreased lymphocytes and neutrophils in lambs infected by this gastrointestinal nematodes. Lambs infected with H. contortus showed significant (P<0.01) lower AChE activity in lymphocytes compared uninfected. Statistically, there was a positive correlation (P<0.05) between AChE activity in lymphocytes and number of lymphocytes (r=0.69). The lymphocytes are cells with direct participation in the cholinergic system; therefore, based on these results, it can be concluded that the experimental infection with H. contortus influences the number of lymphocytes, and consequently the AChE activity in these cells.

Haemonchus contortus P-glycoproteins interact with host eosinophil granules: a novel insight into the role of ABC transporters in host-parasite interaction.

Eosinophils are one of the major mammalian effector cells encountered by helminths during infection. In the present study, we investigated the effects of eosinophil granule exposure on the sheep parasitic nematode Haemonchus contortus as a model. H. contortus eggs exposed to eosinophil granule products showed increased rhodamine 123 efflux and this effect was not due to loss of egg integrity. Rh123 is known to be a specific P-glycoprotein (Pgp) substrate and led to the hypothesis that in addition to their critical role in xenobiotic resistance, helminth ABC transporters such as Pgp may also be involved in the detoxification of host cytotoxic products. We showed by quantitative RT-PCR that, among nine different H. contortus Pgp genes, Hco-pgp-3, Hco-pgp-9.2, Hco-pgp-11 and, Hco-pgp-16 were specifically up-regulated in parasitic life stages suggesting a potential involvement of these Pgps in the detoxification of eosinophil granule products. Using exsheathed L3 larvae that mimic the first life stage in contact with the host, we demonstrated that eosinophil granules induced a dose dependent overexpression of Hco-pgp-3 and the closely related Hco-pgp-16. Taken together, our results provide the first evidence that a subset of helminth Pgps interact with, and could be involved in the detoxification of, host products. This opens the way for further studies aiming to explore the role of helminth Pgps in the host-parasite interaction, including evasion of the host immune response.

Measurement of ivermectin concentrations in target worms and host gastrointestinal tissues: influence of the route of administration on the activity against resistant Haemonchus contortus in lambs.

The influence of the administration route on the relationship between efficacy and ivermectin concentration profiles achieved in the bloodstream, the gastrointestinal mucosal tissues/fluid contents and within a target abomasal parasite (Haemonchus contortus) was evaluated in lambs. Twenty-six (26) parasitized lambs were assigned into three experimental groups: untreated (control) and ivermectin treated by the subcutaneous and intraruminal route at 0.2mg/kg. Blood samples were collected between 0 and 15 days post-treatment (plasma disposition study). Four animals from each group were sacrificed at day 3 post-treatment. Mucosa and content samples from abomasum and small intestine and adult specimens of H. contortus were collected. Drug concentrations were measured by HPLC. Individual fecal egg counts were evaluated at -1, 3 and 15 days post treatment. Post-mortem examination was done at day 15 post-treatment. Adult nematodes recovered from the digestive tract were counted and identified by species. Ivermectin plasma availability was higher (P<0.05) after the subcutaneous administration (129 ng.d/ml) compared to the intraruminal treatment (58.4 ng.d/ml). However, ivermectin concentrations measured in the gastrointestinal contents were higher in lambs treated by the intraruminal route. The mean ivermectin concentrations achieved (3 days post-treatment) in the abomasal content were 143 ng/g (intraruminal) and 2.53 ng/g (subcutaneous). Ivermectin concentrations were 15-fold higher in H. contortus recovered from intraruminally treated lambs. Whereas the subcutaneous administration reduced the number of adult nematodes from 4376 to 1300, the number of adult nematodes after the treatment with ivermectin given by the intraruminal route was 206 (P<0.05). The higher ivermectin concentrations achieved in the digestive tract shortly after the intraruminal treatment may account for the observed enhanced efficacy compared to the parenteral administration against parasites of reduced susceptibility.

Proteomic analysis of the abomasal mucosal response following infection by the nematode, Haemonchus contortus, in genetically resistant and susceptible sheep.

Sheep have a variable ability to resist gastrointestinal nematode infection, but the key factors mediating this response are poorly defined. Here we report the first large-scale application of quantitative proteomic technologies to define proteins that are differentially abundant between sheep selectively bred to have an enhanced (resistant) or reduced (susceptible) ability to eliminate nematodes. Samples were collected from the abomasal mucosa three days after experimental challenge with the nematode, Haemonchus contortus. This timing reflects the initial interaction of host and parasite, and the tissue represents the immediate interface. We identified and quantified more than 4400 unique proteins, of which 158 proteins showed >1.5 fold difference between the resistant and susceptible sheep. Trefoil factor 2, a member of RAS oncogene family (RAP1A) and ring finger protein 126 were amongst the proteins found to be highly abundant in the abomasal surface of resistant sheep, whereas adenosine deaminase and the gastrokine-3 like precursor were found at higher levels in susceptible sheep. Construction of gut proteome interaction networks identified mitochondrial function and energetic partitioning as important components of an effective nematode eliminating response. The differentially abundant proteins may be useful targets for phenotypic tests that aim to identify sheep with an enhanced ability to resist nematode infection.

On being the right size: the impact of population size and stochastic effects on the evolution of drug resistance in hospitals and the community.

The evolution of drug resistant bacteria is a severe public health problem, both in hospitals and in the community. Currently, some countries aim at concentrating highly specialized services in large hospitals in order to improve patient outcomes. Emergent resistant strains often originate in health care facilities, but it is unknown to what extent hospital size affects resistance evolution and the resulting spillover of hospital-associated pathogens to the community. We used two published datasets from the US and Ireland to investigate the effects of hospital size and controlled for several confounders such as antimicrobial usage, sampling frequency, mortality, disinfection and length of stay. The proportion of patients acquiring both sensitive and resistant infections in a hospital strongly correlated with hospital size. Moreover, we observe the same pattern for both the percentage of resistant infections and the increase of hospital-acquired infections over time. One interpretation of this pattern is that chance effects in small hospitals impede the spread of drug-resistance. To investigate to what extent the size distribution of hospitals can directly affect the prevalence of antibiotic resistance, we use a stochastic epidemiological model describing the spread of drug resistance in a hospital setting as well as the interaction between one or several hospitals and the community. We show that the level of drug resistance typically increases with population size: In small hospitals chance effects cause large fluctuations in pathogen population size or even extinctions, both of which impede the acquisition and spread of drug resistance. Finally, we show that indirect transmission via environmental reservoirs can reduce the effect of hospital size because the slow turnover in the environment can prevent extinction of resistant strains. This implies that reducing environmental transmission is especially important in small hospitals, because such a reduction not only reduces overall transmission but might also facilitate the extinction of resistant strains. Overall, our study shows that the distribution of hospital sizes is a crucial factor for the spread of drug resistance.

Rumen function and digestion of Merino sheep divergently selected for genetic difference in resistance to Haemonchus contortus.

The aim of this experiment was to investigate the effect of Haemonchus contortus infection on rumen function and digestion of Merino sheep from lines divergently selected for genetic resistance to H. contortus. Rumen function and whole-tract digestibility were determined in 29 Merino weaner wethers from the CSIRO Haemonchus selection flock, increased resistance to Haemonchus (IRH), decreased resistance to Haemonchus (DRH) and random bred control (C) selection lines. Wethers were fed a restricted low quality roughage diet (5.9 MJ ME/kg DM, 29 g MP/kg DM) to maintain weight and were either uninfected (NIL) or infected with H. contortus (INF). Measurements were taken at week 7 of infection, while animals were housed in metabolic crates. Animals were then euthanased to determine abomasal worm counts. IRH had significantly lower worm egg count than DRH and C lines however, adult worm and larval counts, though lower in IRH animals, did not differ significantly. DM and OM digestibility and in sacco degradability of IRH and DRH selection lines were greater than C animals. Rumen function of animals from the IRH line was altered in response to H. contortus infection with an increase in fluid outflow and turnover rate and a decrease in propionic acid concentration. These changes may be a component of a greater host resistance and begin to explain the failure of IRH animals to translate lower worm egg count into greater bodyweight gain.

Dual oxidase 2 and glutathione peroxidase gene expression are elevated in hyperimmunized sheep challenged with Haemonchus contortus.

A sequential biopsy sampling method was used to investigate oxidant and antioxidant gene responses in resistant sheep challenged with Haemonchus contortus larvae or a sham saline challenge. The expression of key sheep oxidant and antioxidant producing genes were measured in sequential samples removed from the abomasums at days 0, 1, 2, 3, 5, 7 and 28 post challenge. Gene expression levels at each time point were compared to expression at day 0, and levels of the various genes were also correlated to other markers of infection including immune cell counts and cytokine gene expression. The early response to larval challenge infection in resistant animals was marked by a divergence of two groups of host oxidant producing genes: the dual oxidase group (DUOX2/DUOXA2) showing increases in expression to day 7, while members of the phagocytic NADPH oxidase (PHOX) group showed significant decreases in expression. The change in DUOX2 expression between days zero and seven, when host resistance to infection is mediated, was negatively correlated to final worm burden suggesting NADPH oxidase expression may play a role in parasite expulsion. Expression of the DUOX group oxidants was positively correlated to expression of the Th2 cytokine IL4. Changes in host antioxidant pathways between different members of the glutathione peroxidase family (intestinal and plasma GPX) and genes involved in glutathione metabolism were also observed. This first study of the putative roles of oxidant production by the dual oxidase group, antioxidant glutathione pathways, immune cell populations, and cytokine profiles, in the development of resistance to infection by hyperimmune sheep are discussed.