Electrochemical enzyme-linked immunosorbent assay (e-ELISA) for parasitic nematode Ostertagia ostertagi (brown stomach worm) infections in dairy cattle.

A sensitive electrochemical immunoassay (e-ELISA) has been developed for the detection of the gastrointestinal parasitic nematode Ostertagia ostertagi (brown stomach worm) in infected and control serum samples. An antigen-indirect immunoassay format was employed to detect the presence of O. ostertagi antibodies, coupled with an anti-species monoclonal horseradish peroxidase (HRP) conjugate. ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and TMB (3,3',5,5'-tetramethylbenzidine/hydrogen peroxide) were investigated as both chromogenic visualising reagents for optical ELISA and electroactive substrates for electrochemical ELISA in the HRP catalysed oxidation reaction. Coulometry was applied for the detection of O. ostertagi antibodies (via TMB electrochemistry) and compared with the commercial optical ELISA (ABTS based SVANOVIR® O. ostertagi-Ab ELISA kit). Cost-effective in-house sensors were designed and fabricated using polyester and chemical adhesive materials with the aid of stencil printing and laser machining techniques. The performance of the electrochemical ELISA and sensor was evaluated by investigating redox mediators (ABTS vs. TMB), stop solutions (sodium dodecyl sulfate vs. sulfuric acid) and incubation times (150 min vs. 70 min vs. 25 min). For a total assay incubation time of 70 minutes, the TMB/H2SO4 based e-ELISA was able to differentiate between positive (P) and negative (N) control serum samples, with a P/N70 control ratio 1.6 times higher than that of optical ELISA (TMB/H2SO4 combination) and 2.9 times higher than that of the commercial ELISA kit (ABTS/SDS combination). Furthermore, the e-ELISA approach is quicker and required only 25 min (total incubation time) with even better response (P/N25 = 14.7), which is approximately 4-fold higher than the optical immunoassay (P/N25 = 3.8). The proposed e-ELISA is specific (selective Ab-Ag interactions) and highly sensitive - capable of detecting up to 16-fold dilutions of a positive control serum sample. The electrochemical ELISA approach has the potential for rapid sample screening in a portable, disposable format, contributing to the quest for effective prevention and control of parasitic Ostertagia ostertagi infections in cattle.

Exploring the mechanisms of resistance to Teladorsagia circumcincta infection in sheep through transcriptome analysis of abomasal mucosa and abomasal lymph nodes.

The present study exploited the RNA-seq technology to analyze the transcriptome of target tissues affected by the Teladorsagia circumcincta infection in two groups of adult ewes showing different statuses against gastrointestinal nematode (GIN) infection with the aim of identifying genes linked to GIN infection resistance in sheep. For this, based on the accumulated faecal egg count of 18 adult Churra ewes subjected to a first experimental infection with T. circumcincta, six ewes were classified as resistant and six others as susceptible to the infection. These 12 animals were dewormed and infected again. After humanitarian sacrifice of these 12 animals at day 7 post-infection, RNA samples were obtained from abomasal mucosa and lymph node tissues and RNA-Seq datasets were generated using an Illumina HiSeq 2000 sequencer. The distribution of the genes based on their expression level were very similar among the two different tissues and conditions. The differential expression analysis performed with two software (DESeq and EdgeR) only identified common differentially expressed genes (DEGs), a total of 106, in the lymph node samples which were considered as GIN-activated. The enrichment analysis performed for these GIN-activated genes identified some pathways related to cytokine-mediated immune response and the PPARG signaling pathway as well as disease terms related to inflammation and gastro-intestinal diseases as enriched. A systematic comparison with the results of previous studies confirmed the involvement of genes such as ITLN2, CLAC1 and galectins, in the immune mechanism activated against T. circumcincta in resistant sheep.

Herd-level seroprevalence of Fasciola hepatica and Ostertagia ostertagi infection in dairy cattle population in the central and northeastern Poland.

BACKGROUND: Fasciola hepatica and Ostertagia ostertagi infections are widespread in cattle population of Europe, however data on their prevalence in Poland are only fragmentary. Therefore, the cross-sectional study was carried out to determine the herd-level seroprevalence of F. hepatica and O. ostertagi infection in dairy cattle population in the central and north-eastern provinces Poland, and to identify basic local risk factors for these infections. In total, 598 herds were enrolled, 394 (65.9%) in the north-eastern province and 204 (34.1%) in the central province. In each herd the questionnaire survey was conducted and bulk-tank milk (BTM) sample was collected and screened using two indirect immunoenzymatic tests. Optical density ratio (ODR) was regarded as the quantitative proxy of exposure to either of the two parasites. RESULTS: Both Fasciola and Ostertagia ELISA ODR in the north-eastern province was significantly higher than ODR in the central province. At the cut-off value of ODR = 0.27 the true herd-level seroprevalence of F. hepatica was 79.6% (95% CI: 74.0%, 84.3%) in the north-eastern province and 13.0% (95% CI: 5.3%, 21.7%) in the central province. At the cut-off of ODR = 0.50151 of 188 herds (80.3%, 95% CI: 74.1%, 85.4%) were seropositive for O. ostertagi in the north-eastern province and only 70 of 136 herds (51.5%, 95% CI: 43.1%, 59.7%) were seropositive in the central province. Location of a herd in the north-eastern province, longer grazing period practiced in a herd and > 50%-share of grazing grass in roughage were all positively related to the increase in exposure to both parasites. Moreover, the use of hay or haylage as main roughage proved to be positively related to the increase in exposure to F. hepatica. CONCLUSIONS: F. hepatica and O. ostertagi are widespread in cattle population in Poland, however their occurrence at a herd-level varies between different regions of Poland. This diversity can only partly be explained by different herd management, and appears linked to environmental and climate conditions typical for these regions.

A longitudinal study of gastrointestinal parasites in English dairy farms. Practices and factors associated with first lactation heifer exposure to Ostertagia ostertagi on pasture.

The gastrointestinal nematode Ostertagia ostertagi is an important cause of lost production, health, and welfare in cattle. Detailed records were obtained over a 5-yr period (2010-2015) by questionnaires and qualitative interviews to investigate the practices adopted by dairy farmers to control cattle helminth infections and the factors associated with heifer exposure to O. ostertagi on pasture. In total, 1,454 heifers' individual milk samples were collected over a 1-yr period (2014-2015) in 43 dairy farms in England and tested for O. ostertagi antibody by ELISA. Multilevel linear regression models were used to investigate the association between individual milk optical density ratio (ODR) against O. ostertagi and heifer management from birth to time of sampling. Farm and heifer median ODR against O. ostertagi were 0.98 (interquartile range = 0.76-1.02) and 0.64 (interquartile range = 0.42-0.84), respectively. The majority of heifers (88%) received an anthelmintic treatment before sampling in this study. After controlling for the effect of anthelmintic treatments, heifer individual milk ODR against O. ostertagi significantly increased with high stocking rate at first grazing and co-grazing with adult cows before calving. Conversely, heifer individual milk ODR against O. ostertagi significantly decreased when heifers had co-grazed with sheep and pasture grass had frequently been mowed. Overall, these results provide evidence to support targeting grazing management toward limiting the use of anthelmintics in dairy young stock to enable sustainable control of cattle helminth infections in England. However, to be accepted and adopted by farmers, these best practices would need to take into account farmers' perspectives and contextual challenges.

A stochastic model to investigate the effects of control strategies on calves exposed to Ostertagia ostertagi.

Predicting the effectiveness of parasite control strategies requires accounting for the responses of individual hosts and the epidemiology of parasite supra- and infra-populations. The first objective was to develop a stochastic model that predicted the parasitological interactions within a group of first season grazing calves challenged by Ostertagia ostertagi, by considering phenotypic variation amongst the calves and variation in parasite infra-population. Model behaviour was assessed using variations in parasite supra-population and calf stocking rate. The model showed the initial pasture infection level to have little impact on parasitological output traits, such as worm burdens and FEC, or overall performance of calves, whereas increasing stocking rate had a disproportionately large effect on both parasitological and performance traits. Model predictions were compared with published data taken from experiments on common control strategies, such as reducing stocking rates, the 'dose and move' strategy and strategic treatment with anthelmintic at specific times. Model predictions showed in most cases reasonable agreement with observations, supporting model robustness. The stochastic model developed is flexible, with the potential to predict the consequences of other nematode control strategies, such as targeted selective treatments on groups of grazing calves.

Analysis of the mucosal immune responses induced by single and trickle infections with the bovine abomasal nematode Ostertagia ostertagi.

The purpose of this study was to provide more information on the kinetics of the immunological changes occurring in the abomasal mucosa after single and trickle infections with the bovine parasite Ostertagia ostertagi. The time course analysis of gene expression revealed that the major changes coincided with the emergence of adult worms from the gastric glands. These changes consisted of a simultaneous upregulation of Th1- and Th2-type cytokines. In addition, a single O. ostertagi infection elicited an upregulation of the epithelial-derived cytokine IL33, while TSLP expression levels were not impacted. Apart from the massive increase in inflammatory cytokines IL6, IL17 and IL21, O. ostertagi infection also elicited an upregulation of the immunosuppressors TGFB, IL10 and ARG1, as well as NK and γδ-T cell markers. Furthermore, the cytotoxic factors granulysin, perforin and granzyme B were upregulated following an O. ostertagi infection. Analysis of cytokine transcript levels in animals receiving trickle infections for 60 days showed a similar trend as observed following a single infection except for IL33, IL6, GATA-3, TBX21 and NCR1, which were no longer upregulated after trickle infections. Finally, the long trickle infections were associated with mucosal eosinophilia and mastocytosis.

Structure of Ostertagia ostertagi ASP-1: insights into disulfide-mediated cyclization and dimerization.

The cysteine-rich secretory/antigen 5/pathogenesis-related 1 (CAP) protein superfamily is composed of a functionally diverse group of members that are found in both eukaryotes and prokaryotes. The excretome/secretome of numerous helminths (parasitic nematodes) contains abundant amounts of CAP members termed activation-associated secreted proteins (ASPs). Although ASPs are necessary for the parasitic life cycle in the host, the current lack of structural and functional information limits both understanding of their actual role in host-parasite interactions and the development of new routes in controlling parasitic infections and diseases. Alleviating this knowledge gap, a 1.85 Å resolution structure of recombinantly produced Oo-ASP-1 from Ostertagia ostertagi, which is one of the most prevalent gastrointestinal parasites in cattle worldwide, was solved. Overall, Oo-ASP-1 displays the common hallmark architecture shared by all CAP-superfamily members, including the N-terminal CAP and C-terminal cysteine-rich domains, but it also reveals a number of highly peculiar features. In agreement with studies of the natively produced protein, the crystal structure shows that Oo-ASP-1 forms a stable dimer that has been found to be primarily maintained via an intermolecular disulfide bridge, hence the small interaction surface of only 306.8 Å(2). Moreover, unlike any other ASP described to date, an additional intramolecular disulfide bridge links the N- and C-termini of each monomer, thereby yielding a quasi-cyclic molecule. Taken together, the insights presented here form an initial step towards a better understanding of the actual biological role(s) that this ASP plays in host-parasite interactions. The structure is also essential to help to define the key regions of the protein suitable for development of ASP-based vaccines, which would enable the current issues surrounding anthelmintic resistance in the treatment of parasitic infections and diseases to be circumvented.

Analysis of cell hyperplasia and parietal cell dysfunction induced by Ostertagia ostertagi infection.

Infections in cattle with the gastric nematode Ostertagia ostertagi are associated with decreased acid secretion and profound physio-morphological changes of the gastric mucosa. The purpose of the current study was to investigate the mechanisms triggering these pathophysiological changes. O. ostertagi infection resulted in a marked cellular hyperplasia, which can be explained by increased transcriptional levels of signaling molecules related to the homeostasis of gastric epithelial cells such as HES1, WNT5A, FGF10, HB-EGF, AREG, ADAM10 and ADAM17. Intriguingly, histological analysis indicated that the rapid rise in the gastric pH, observed following the emergence of adult worms, cannot be explained by a loss of parietal cells, as a decrease in the number of parietal cells was only observed following a long term infection of several weeks, but is likely to be caused by an inhibition of parietal cell activity. To investigate whether this inhibition is caused by a direct effect of the parasites, parietal cells were co-cultured with parasite Excretory/Secretory products (ESP) and subsequently analyzed for acid production. The results indicate that adult ESP inhibited acid secretion, whereas ESP from the L4 larval stages did not alter parietal cell function. In addition, our data show that the inhibition of parietal cell activity could be mediated by a marked upregulation of inflammatory factors, which are partly induced by adult ESP in abomasal epithelial cells. In conclusion, this study shows that the emergence of adult O. ostertagi worms is associated with marked cellular changes that can be partly triggered by the worm's Excretory/secretory antigens.

Exploring the abomasal lymph node transcriptome for genes associated with resistance to the sheep nematode Teladorsagia circumcincta.

This study exploited Blackface lambs that varied in their resistance to the abomasal nematode parasite, Teladorsagia circumcincta. Infection of these lambs over 3 months identified susceptible (high adult worm count, high faecal egg count and low IgA antibody) and resistant animals that had excluded all parasites. Previous work had shown that susceptibility and resistance is dependent on the differential immune response to the parasite, which occurs within the abomasal (gastric) lymph node (ALN) that drains the site of infection. The Affymetrix ovine gene array was used to interrogate the transcriptome of the ALN to identify genes and physiological pathways associated with resistance. We used a bovine RT-qPCR array of 84 genes to validate the gene array, and also report digital gene expression analysis on the same tissues, reanalysed using the Oar v3.1 sheep genome assembly. These analyses identified Humoral Immune Response, Protein Synthesis, Inflammatory Response and Hematological System Development and Function as the two top-ranked networks associated with resistance. Central genes within these networks were IL4, IL5, IL13RA2 and in particular IL13, which confirmed that differential activation of Th2 polarized responses is critical to the resistance phenotype. Furthermore, in resistant sheep there was up-regulation of genes linked to control and suppression of inflammation. The identity of differentially-expressed chemokines and receptors in the resistant and susceptible sheep also begins to explain the cellular nature of the host response to infection. This work will greatly help in the identification of candidate genes as potential selectable markers of genetic resistance.

Relationship between susceptibility of Blackface sheep to Teladorsagia circumcincta infection and an inflammatory mucosal T cell response.

Teladorsagia circumcincta is the most economically important gastrointestinal (abomasal) nematode parasite of sheep in cool temperate regions, to which sheep show genetically-varying resistance to infection. Lambs, from parents with genetic variation for resistance, were trickle infected with L3 larvae over 12 weeks. 45 lambs were identified with a range of susceptibilities as assessed by: adult worm count at post mortem, faecal egg count (FEC) and IgA antibody levels. This project investigated the correlation of T cell cytokine expression and resistance to infection at the mature stage of response, when the resistant lambs had excluded all parasites.Histopathology showed only minor changes in resistant animals with a low level lymphocyte infiltration; but in susceptible lambs, major pathological changes were associated with extensive infiltration of lymphocytes, eosinophils and neutrophils.Absolute quantitative RT-qPCR assays on the abomasal lymph node (ALN) revealed a significant positive correlation between IL6, IL21 and IL23A transcript levels with adult worm count and FEC. IL23A was also negatively correlated with IgA antibody levels. Significantly positive correlation of TGFB1 levels with adult worm count and FEC were also seen in the abomasal mucosa. These data are consistent with the hypothesis that the inability to control L3 larval colonization, adult worm infection and egg production is due to the activation of the inflammatory Th17 T cell subset.

Obligate larval inhibition of Ostertagia gruehneri in Rangifer tarandus? Causes and consequences in an Arctic system.

Larval inhibition is a common strategy of Trichostrongylidae nematodes that may increase survival of larvae during unfavourable periods and concentrate egg production when conditions are favourable for development and transmission. We investigated the propensity for larval inhibition in a population of Ostertagia gruehneri, the most common gastrointestinal Trichostrongylidae nematode of Rangifer tarandus. Initial experimental infections of 4 reindeer with O. gruehneri sourced from the Bathurst caribou herd in Arctic Canada suggested that the propensity for larval inhibition was 100%. In the summer of 2009 we infected 12 additional reindeer with the F1 and F2 generations of O. gruehneri sourced from the previously infected reindeer to further investigate the propensity of larval inhibition. The reindeer were divided into 2 groups and half were infected before the summer solstice (17 June) and half were infected after the solstice (16 July). Reindeer did not shed eggs until March 2010, i.e. 8 and 9 months post-infection. These results suggest obligate larval inhibition for at least 1 population of O. gruehneri, a phenomenon that has not been conclusively shown for any other trichostrongylid species. Obligate inhibition is likely to be an adaptation to both the Arctic environment and to a migratory host and may influence the ability of O. gruehneri to adapt to climate change.

Development of Bovine Gastric Organoids as a Novel In Vitro Model to Study Host-Parasite Interactions in Gastrointestinal Nematode Infections.

Gastro-intestinal nematode (GIN) parasites are a major cause of production losses in grazing cattle, primarily through reduced growth rates in young animals. Control of these parasites relies heavily on anthelmintic drugs; however, with growing reports of resistance to currently available anthelmintics, alternative methods of control are required. A major hurdle in this work has been the lack of physiologically relevant in vitro infection models that has made studying precise interactions between the host and the GINs difficult. Such mechanistic insights into the infection process will be valuable for the development of novel targets for drugs, vaccines, or other interventions. Here we created bovine gastric epithelial organoids from abomasal gastric tissue and studied their application as in vitro models for understanding host invasion by GIN parasites. Transcriptomic analysis of gastric organoids across multiple passages and the corresponding abomasal tissue showed conserved expression of tissue-specific genes across samples, demonstrating that the organoids are representative of bovine gastric tissue from which they were derived. We also show that self-renewing and self-organising three-dimensional organoids can also be serially passaged, cryopreserved, and resuscitated. Using Ostertagia ostertagi, the most pathogenic gastric parasite in cattle in temperate regions, we show that cattle gastric organoids are biologically relevant models for studying GIN invasion in the bovine abomasum. Within 24 h of exposure, exsheathed larvae rapidly and repeatedly infiltrated the lumen of the organoids. Prior to invasion by the parasites, the abomasal organoids rapidly expanded, developing a 'ballooning' phenotype. Ballooning of the organoids could also be induced in response to exposure to parasite excretory/secretory products. In summary, we demonstrate the power of using abomasal organoids as a physiologically relevant in vitro model system to study interactions of O. ostertagi and other GIN with bovine gastrointestinal epithelium.

Infection with the gastrointestinal nematode Ostertagia ostertagi in cattle affects mucus biosynthesis in the abomasum.

The mucus layer in the gastrointestinal (GI) tract is considered to be the first line of defense to the external environment. Alteration in mucus components has been reported to occur during intestinal nematode infection in ruminants, but the role of mucus in response to abomasal parasites remains largely unclear. The aim of the current study was to analyze the effects of an Ostertagia ostertagi infection on the abomasal mucus biosynthesis in cattle. Increased gene expression of MUC1, MUC6 and MUC20 was observed, while MUC5AC did not change during infection. Qualitative changes of mucins, related to sugar composition, were also observed. AB-PAS and HID-AB stainings highlighted a decrease in neutral and an increase in acidic mucins, throughout the infection. Several genes involved in mucin core structure synthesis, branching and oligomerization, such as GCNT3, GCNT4, A4GNT and protein disulphide isomerases were found to be upregulated. Increase in mucin fucosylation was observed using the lectin UEA-I and through the evaluation of fucosyltransferases gene expression levels. Finally, transcription levels of 2 trefoil factors, TFF1 and TFF3, which are co-expressed with mucins in the GI tract, were also found to be significantly upregulated in infected animals. Although the alterations in mucus biosynthesis started early during infection, the biggest effects were found when adult worms were present on the surface of the abomasal mucosa and are likely caused by the alterations in mucosal cell populations, characterized by hyperplasia of mucus secreting cells.

A differential interplay between the expression of Th1/Th2/Treg related cytokine genes in Teladorsagia circumcincta infected DRB1*1101 carrier lambs.

Substantial debate exists on whether the immune response between sheep resistant and susceptible to gastrointestinal nematodes can be differentiated into a Th1 and Th2 phenotype. The present study addresses the hypothesis that variation in resistance to Teladorsagia circumcincta between DRB1*1101 (associated with reduced faecal egg count and worm burden) carriers and non-carriers is due to a differential interplay in the expression of Th1/Th2 and regulatory T (Treg) related cytokine genes. Lambs from each genotype were either slaughtered at day 0 (un-infected control) or infected with 3 × 10(4) Teladorsagia circumcincta L3 and slaughtered at 3, 7, 21, and 35 days later. Lambs carrying the DRB1*1101 allele had a significantly lower worm burden (P < 0.05) compared to the non-carriers. Abomasal mucosal cytokine gene expression was evaluated by quantitative real-time PCR and comparison made for time and genotype effects. The response generated varied through the course of infection and was affected by genotype. DRB1*1101 carriers had an up-regulated expression of the Th1-related cytokine genes (IL-1ß, TNFα, and IFN-γ) at day 3, but this was replaced by an up-regulated expression of Th2-related cytokine genes (IL-10 and IL-13) and Treg-related cytokine genes (IL-2RA-CD25, TGFα, TGFß, Arg2, MIF and FOXP3) by day 7. Conversely, in the non-carriers these changes in gene expression were delayed until days 7 and 21 post infection (pi), respectively. It is concluded that resistance to Teladorsagia circumcincta in animals carrying the DRB1*1101 allele is influenced by an earlier interplay between Th1, Th2 and T regulatory immune response genes.

The dynamic influence of the DRB1*1101 allele on the resistance of sheep to experimental Teladorsagia circumcincta infection.

Suffolk sheep carrying the DRB1*1101 (previously referred to as-DRB1*0203 or G2) allele have been reported to show increased resistance to natural Teladorsagia circumcincta infection compared to non-carriers. The objective of this study was to compare the biochemical and physiological responses of DRB1*1101 carrier and non-carrier twin lambs to an experimental infection with 3 × 10(4) L3 Teladorsagia circumcincta. The variables studied included worm burden, faecal egg count, abomasal mast cells, IgA, IgE, IgG1 plus IgG2 and haematological parameters at 0, 3, 7, 21 and 35 days post infection (dpi), and duodenal smooth muscle contractility at 0 and 35 dpi. DRB1*1101 carrier lambs had significantly lower worm burden, higher mast cell and plasma platelet counts than the DRB1*1101 non-carriers (P < 0.05). Before infection, the non-carrier lambs exhibited significantly higher mucosal levels of all antibody isotypes measured compared to the carriers; these levels remained relatively stable over the course of infection in the non-carriers while there was a slow build up of these antibodies in the carriers up to day 21 post infection (pi). The DRB1*1101 non-carrier lambs had a significantly higher plasma lymphocyte count, and produced greater duodenal contractile force relative to the carrier lambs (P < 0.05). There was no significant difference between genotypes in the level of plasma eosinophils, monocytes, neutrophils or FEC. This evidence suggests that resistance conferred by DRB1*1101 is acquired rather than innate, depends on worm expulsion rather than fecundity and is dependent on mucosal mast cell proliferation, platelet activation, and IgA and IgE antibody responses.

Molecular and phenotypic characterisation of fenbendazole resistance in a field-derived isolate of Ostertagia ostertagi.

The prevalence of anthelmintic resistance in the bovine nematode Cooperia oncophora has been well documented globally but lack of efficacy against the more pathogenic nematode species Ostertagia ostertagi is less common. The sensitivity of an O. ostertagi isolate to the benzimidazole class of anthelmintic was investigated using classical parasitological techniques following apparent clinical failure of controlled release fenbendazole capsule administration in first season grazers at pasture. A controlled efficacy test (CET) was conducted in conjunction with sequencing of the ß-tubulin isotype 1 gene of larvae pre- and post-fenbendazole administration. Twelve helminth-naïve calves were infected experimentally with 20,000 third stage larvae; six received oral fenbendazole (7.5 mg/kg bodyweight) 28 days post infection. Total abomasal nematode burdens were compared between treatment and control groups to determine efficacy. Fenbendazole resistance in O. ostertagi was confirmed with a total treatment failure in reducing worm burden: efficacy of 0%. Sequence analysis of the ß-tubulin isotype-1 gene from forty-five infective larvae from both control and treated groups was performed. The three commonest single nucleotide polymorphisms (SNPs) associated with benzimidazole resistance, namely F167Y, E198A and F200Y, were examined. The predominant resistance-associated SNPs were F200Y (78 % control and 79 % treated groups) and F167Y (remaining genotypes) and emphasises the importance of these SNPs in clinical disease in this isolate. The development of diagnostic molecular tools based on a characterised field-derived isolate of benzimidazole-resistant Ostertagia will enable future prevalence surveys to be undertaken to assess the possible risk posed by resistance in this economically important species.

Associations between blood gastrin, ghrelin, leptin, pepsinogen and Ostertagia ostertagi antibody concentrations and voluntary feed intake in calves exposed to a trickle infection with O. ostertagi.

Twenty-five, castrated male Holstein-cross calves, between 4 and 5 months of age, weighing 156.5+/-12.2 kg and reared under conditions designed to minimise the risk of parasitic infection, were allocated to one of the five treatment groups on the basis of initial bodyweight. The groups were (1) ad libitum (ad lib) fed controls (ALC); (2) ad lib fed infected (INF) and treated with topical eprinomectin on Day 56; (3) controls pair-fed with the INF group (PFC); (4) ad lib fed controls treated with eprinomectin on Days 0 and 56 (E-ALC) and (5) ad lib fed, infected and treated with eprinomectin on Days 0 and 56 (E-INF). Infection comprised a trickle infection with the equivalent of 10,000 larvae of Ostertagia ostertagi per day from Day 0 to Day 56 and the study concluded on Day 77. Parameters measured throughout the study included: liveweight, feed intake, faecal egg counts; plasma pepsinogen, gastrin, ghrelin and leptin; plasma antibodies to adult O. ostertagi. No significant differences in feed intake or liveweight gain were observed between any of the different groups, a finding thought to result from the high quality of feed offered. Significant differences between the INF and control groups however were observed in faecal egg counts, plasma pepsinogen, gastrin and O. ostertagi antibodies, which were all elevated, and leptin, which was reduced. Values of these parameters for the E-INF group were intermediate between the INF and ALC groups. Plasma ghrelin showed no association with either feed intake or parasitism. Further studies are needed to fully elucidate the roles of various biochemical and neuroendocrine mediators for inappetence in ruminants with parasitic gastroenteritis.

Characterization of IL-10-producing neutrophils in cattle infected with Ostertagia ostertagi.

IL-10 is a master regulator of immune responses, but its cellular source and function in cattle during the initial phase of immune priming have not been well established. Despite a massive B cell response in the abomasal draining lymph nodes in Ostertagia ostertagi (OO)-infected cattle, protective immunity is slow to develop, and partial protection requires years of repeated exposure. In addressing this problem, our initial hypothesis was that B cells produce IL-10 that downregulates the host protective immune response. However, our results showed that neutrophils made up the majority of IL-10-producing cells in circulation and in secondary lymphoid tissues, particularly the spleen (80%). Conversely, IL-10-producing B cells were rare. In addition, approximately 10% to 20% of the neutrophils in the blood and spleen expressed MHC II and were IL-10 negative, suggesting that neutrophils could also participate in antigen presentation. In vitro investigation of bovine neutrophils revealed that exposure thereof to OO extract increased IL-10 and MHC II expression in these cells in a dose-dependent manner, consistent with IL-10+/MHC II+ neutrophils detected in cattle shortly after experimental OO infection. Co-culture of untreated neutrophils with anti-CD3 antibody (Ab)-stimulated CD4+ T cells led to enhanced T cell activation; also, IL-10 depletion with neutralizing Ab enhanced the stimulatory function of neutrophils. OO extract depressed neutrophil stimulation of CD4+ T cells in the presence of IL-10-neutralizing Ab, suggesting that OO utilizes both IL-10-dependent and independent mechanisms to manipulate the bovine immune response. Finally, contact and viability were required for T cell-stimulatory neutrophil function. This report, to the best of our knowledge, is the first to demonstrate that neutrophil-derived IL-10 is directly involved in T cell regulation in cattle. Our data suggest that neutrophils and neutrophil-derived IL-10 are co-opted by nematode parasites and other pathogens to attenuate host immune responses and facilitate pathogen survival.

Does suckling offer protection to the lamb against Teladorsagia circumcincta infection?

This study tested the hypothesis that milk has a direct effect on the establishment of infection with Teladorsagia circumcincta, and provides information on the effects of suckling on resilience to infection in young lambs. Of 46 six-week-old twin-born lambs, one from each twin was allowed to continue suckling (S-) and its twin-weaned (W-) while both were concurrently infected with an average of either 0 (-0; n=7/group), 250 (-250; n=8/group) or 1000 (-1000; n=8/group) third stage infective larvae (L3) of T. circumcincta per day, providing six treatment groups. All groups grazed minimally contaminated pasture, and after 42 days were slaughtered for necropsy. Low pasture larval contamination was confirmed in W0 and S0 lambs by faecal egg counts (FEC) of less than 30 eggs per gram (EPG) and burdens of less than 140 worms. There was no difference in FEC between weaned and suckled lambs. Within infection regime, total worm burdens were 55-60% greater in the weaned compared with their suckled counterparts (P=0.05), and represented 27 and 17%, respectively, net establishment of larvae. The greater worm burdens of both groups of weaned animals, which compared with their suckled counterparts, and of those infected with 1000 compared with 250 larvae per day, were associated with shorter female adults that had fewer eggs in utero, perhaps indicating intra-worm population regulation, but highlighting the limitation of FEC in assessing nematode burdens of such young lambs. There was no effect of infection on live weight gain of either weaned or suckled groups and the possibility was raised that, in such young lambs, immune unresponsiveness may be responsible. The major benefit of continued milk consumption appears to lie more in providing nutrients for enhanced growth rather than in improving resilience of the lambs to infection.

A survey of the exposure to Ostertagia ostertagi in dairy cow herds in Europe through the measurement of antibodies in milk samples from the bulk tank.

Measurement of antibodies to Ostertagia ostertagi in bulk tank milk (BTM) has value as a diagnostic indicator for potential production losses and anthelmintic treatment responses in dairy herds. Most of the recent data on O. ostertagi antibodies in milk have been generated in Belgium and Canada; the purpose of this study was to determine the range of O. ostertagi antibody levels in several European countries. BTM samples were collected during the autumn of 2005 and 2006 from a total of 1185 dairy herds from dairy farming regions in Denmark, Germany, Ireland, Italy, the Netherlands, Portugal, Spain and the United Kingdom. Antibody titres to O. ostertagi were determined by indirect ELISA and expressed as optical density ratios (ODR). In addition, relationships between ODR and management practices were investigated. For each country the mean ODR and the 25th-75th percentile values were determined. Mean BTM ODR values in herds with access to yards, paddocks and pastures ranged from 0.3 in Italy to 0.6 in Portugal and the UK/Ireland. The BTM ODR values obtained in this study were generally lower than those described in the literature for Belgium, but comparable with those in Canada. Variations between different European countries appeared to reflect different husbandry practices, particularly those related to access to pasture. The association analyses showed correlations between the BTM O. ostertagi ODR, outside access and grazing management, consistent with the publications from Belgium and Canada. When diagnostic values appropriate for different production situations and environments have been further validated, the test will provide an objective, quantitative assessment of the O. ostertagi status of a dairy herd and the possible impact this may have on performance and potential responses to anthelmintic treatment. This represents a significant step forward in evidence-based medicine for dairy veterinarians, advisors and farmers.