Natural resistance to worms exacerbates bovine tuberculosis severity independently of worm coinfection.

Pathogen interactions arising during coinfection can exacerbate disease severity, for example when the immune response mounted against one pathogen negatively affects defense of another. It is also possible that host immune responses to a pathogen, shaped by historical evolutionary interactions between host and pathogen, may modify host immune defenses in ways that have repercussions for other pathogens. In this case, negative interactions between two pathogens could emerge even in the absence of concurrent infection. Parasitic worms and tuberculosis (TB) are involved in one of the most geographically extensive of pathogen interactions, and during coinfection worms can exacerbate TB disease outcomes. Here, we show that in a wild mammal natural resistance to worms affects bovine tuberculosis (BTB) severity independently of active worm infection. We found that worm-resistant individuals were more likely to die of BTB than were nonresistant individuals, and their disease progressed more quickly. Anthelmintic treatment moderated, but did not eliminate, the resistance effect, and the effects of resistance and treatment were opposite and additive, with untreated, resistant individuals experiencing the highest mortality. Furthermore, resistance and anthelmintic treatment had nonoverlapping effects on BTB pathology. The effects of resistance manifested in the lungs (the primary site of BTB infection), while the effects of treatment manifested almost entirely in the lymph nodes (the site of disseminated disease), suggesting that resistance and active worm infection affect BTB progression via distinct mechanisms. Our findings reveal that interactions between pathogens can occur as a consequence of processes arising on very different timescales.

Digging for gold nuggets: uncovering novel candidate genes for variation in gastrointestinal nematode burden in a wild bird species.

The extent to which genotypic variation at a priori identified candidate genes can explain variation in complex phenotypes is a major debate in evolutionary biology. Whereas some high-profile genes such as the MHC or MC1R clearly do account for variation in ecologically relevant characters, many complex phenotypes such as response to parasite infection may well be underpinned by a large number of genes, each of small and effectively undetectable effect. Here, we characterize a suite of novel candidate genes for variation in gastrointestinal nematode (Trichostrongylus tenuis) burden among red grouse (Lagopus lagopus scotica) individuals across a network of moors in north-east Scotland. We test for associations between parasite load and genotypic variation in twelve genes previously identified to be differentially expressed in experimentally infected red grouse or genetically differentiated among red grouse populations with overall different parasite loads. These genes are associated with a broad physiological response including immune system processes. Based on individual-level generalized linear models, genotypic variants in nine genes were significantly associated with parasite load, with effect sizes accounting for differences of 514-666 worms per bird. All but one of these variants were synonymous or untranslated, suggesting that these may be linked to protein-coding variants or affect regulatory processes. In contrast, population-level analyses revealed few and inconsistent associations with parasite load, and little evidence of signatures of natural selection. We discuss the broader significance of these contrasting results in the context of the utility of population genomics and landscape genomics approaches in detecting adaptive genomic signatures.

Network model of immune responses reveals key effectors to single and co-infection dynamics by a respiratory bacterium and a gastrointestinal helminth.

Co-infections alter the host immune response but how the systemic and local processes at the site of infection interact is still unclear. The majority of studies on co-infections concentrate on one of the infecting species, an immune function or group of cells and often focus on the initial phase of the infection. Here, we used a combination of experiments and mathematical modelling to investigate the network of immune responses against single and co-infections with the respiratory bacterium Bordetella bronchiseptica and the gastrointestinal helminth Trichostrongylus retortaeformis. Our goal was to identify representative mediators and functions that could capture the essence of the host immune response as a whole, and to assess how their relative contribution dynamically changed over time and between single and co-infected individuals. Network-based discrete dynamic models of single infections were built using current knowledge of bacterial and helminth immunology; the two single infection models were combined into a co-infection model that was then verified by our empirical findings. Simulations showed that a T helper cell mediated antibody and neutrophil response led to phagocytosis and clearance of B. bronchiseptica from the lungs. This was consistent in single and co-infection with no significant delay induced by the helminth. In contrast, T. retortaeformis intensity decreased faster when co-infected with the bacterium. Simulations suggested that the robust recruitment of neutrophils in the co-infection, added to the activation of IgG and eosinophil driven reduction of larvae, which also played an important role in single infection, contributed to this fast clearance. Perturbation analysis of the models, through the knockout of individual nodes (immune cells), identified the cells critical to parasite persistence and clearance both in single and co-infections. Our integrated approach captured the within-host immuno-dynamics of bacteria-helminth infection and identified key components that can be crucial for explaining individual variability between single and co-infections in natural populations.

Responses of the humid zone ecotype of the Nigerian West African Dwarf sheep to mixed infections with Haemonchus contortus and Trichostrongylus colubriformis.

The responses of the Nigerian West African Dwarf sheep to experimental infections with two of its most important gastrointestinal (GI) nematodes, namely, Haemonchus contortus and Trichostrongylus colubriformis were studied by means of two measures of parasitological response, namely, faecal egg count (FEC) and worm burden (Wb), and three measures of host pathology, namely, packed cell volume (PCV), body weight (Bwt) and body condition score (BCS). Following exposure to weekly escalating infections (60% H. contortus and 40% T. colubriformis) starting with single doses of 500 infective larvae (L(3)) at week 1, 1,000 L(3) at week 2, 2,000 L(3) at week 3 and 4,000 L(3) at week 4, two distinctive worm burden response phenotypes were readily recognisable, namely, low Wb (LWb) and high Wb (HWb) phenotypes. The percentage of inoculums (adults and immature stages) recovered at necropsy were 3.75% and 33.08% respectively for H. contortus and T. colubriformis. The results showed that the FECs of the lambs belonging to the LWb phenotype were significantly lower than the FECs of their HWb counterpart. Among the measures of host pathology tested, the LWb phenotype sheep had similar Bwt, PCV and BCS to the uninfected control sheep, whereas the HWb phenotype sheep had significantly lower values than their LWb counterparts and the control sheep towards the end of the experiment. There was a highly significant positive correlation between worm burden transformed as log(10) (Wb + 10) and the average of D56 and D59 FECs. A highly significant negative correlation also was obtained between log(10) (Wb + 10) and the PCV, Bwt and BCS. The low percentage of H. contortus inoculums recovered at necropsy suggested that the sheep may be resistant to their most important GI nematode, H. contortus but less so to T. colubriformis.

Gastrointestinal nematode infections in German sheep.

The objective of the present study was to determine the prevalence and variation of natural gastrointestinal nematode (GIN) infections in lambs according to birth type, gender and breed based on individual faecal egg counts (FEC) from various regions in Germany. A total of 3,924 lambs (3 to 15 months old) with different genetic backgrounds (Merinoland, German Blackhead Mutton, Rhoen, Texel and Merino long-wool) were individually sampled during the grazing period between 2006 and 2008. Furthermore, pooled faecal samples from each of the farms were cultured in order to differentiate the third-stage larvae of the nematode spp. Sixty-three percent of the lambs were infected with GIN. The infections were mostly low to moderate and involved several nematode species. The Trichostrongylus spp. was the predominant species based on the percentage of larvae in faecal cultures. Only 11.4% of the lambs were free of Eimeria oocysts. Tapeworm eggs were encountered in 13.2% of all samples. The prevalence of GIN infections varied significantly (P < 0.001) among farms. A significantly higher FEC (P < 0.05) was observed in multiple-born lambs when compared with singletons. Moreover, male lambs were more susceptible to infection than females (P < 0.001). No significant differences (P > 0.05) were observed between breeds regarding FEC. Inter-individual variations were higher than inter-breed differences, which may indicate the possibility of selection within these breeds for parasites resistance as described in earlier studies.

Animal grazing selectivity and plant chemistry issues impact on the potential of Rhagodia preissii as an anthelmintic shrub.

Rhagodia preissii had shown significant in vitro anthelmintic activity in a previous study, we examined the effect of including this shrub in the diet of sheep infected with Trichostrongylus colubriformis. Worm-infected merino wethers were grazed for 7 weeks on either R. preissii or annual pasture, and faecal egg counts (FECs) were conducted weekly. Plant material was collected weekly from eaten and uneaten plants, and analysed for levels of plant secondary metabolites (tannins, oxalates, saponins) and in vitro anthelmintic activity. While mean FECs were consistently lower in sheep grazing R. preissii compared to pasture (reductions of 20-74%), the differences were not significant. There was no relationship between grazing preference (eaten or uneaten) and in vitro anthelmintic activity of plant extracts. The levels of saponins and oxalates did not correlate with grazing preference or in vitro anthelmintic activity, while tannins were not responsible for the anthelmintic activity. While the identity of the grazing deterrent and in vitro anthelmintic compounds remain unknown, the presence of plants which were both highly preferred by the sheep and showed in vitro anthelmintic activity indicates a potential to develop the species as an anthelmintic shrub through selection of shrub populations dominated by such plants.

Population genetic structure and history of a generalist parasite infecting multiple sympatric host species.

Host specificity is predicted to shape patterns of parasite gene flow between host species; specialist parasites should have low gene flow between host species, while generalists are predicted to have high gene flow between species. However, even for generalist parasites external forces, including ecological differences between host species may sometimes intervene to limit gene flow and create genetic structure. To investigate the potential for cryptic parasite genetic structure to arise under such circumstances, we examined the population genetic structure and history of the generalist nematode, Trichostrongylus axei, infecting six sympatric wild ungulate species in North America. Using genotypes for 186 T. axei larvae at two mitochondrial genes, cox1 and nad4, we found that T. axei was completely panmictic across host species, with 0% of genetic variation structured between host species and 97% within individual hosts. In addition, T. axei showed no evidence of recent genetic bottlenecks, had high nucleotide diversities (above 2%), and an effective population size estimated to be in the tens of millions. Our result that T. axei maintains high rates of gene flow between multiple sympatric host species adds to a growing body of information on trichostrongylid population genetic structure in different ecological contexts. Furthermore, the high rates of gene flow, coupled with high levels of genetic diversity and large effective population size which we observed in T. axei, point to a potentially broad capacity for rapid evolutionary change in this parasite.

Systems biology of ovine intestinal parasite resistance: disease gene modules and biomarkers.

This study reports on the molecular systems biology of gastrointestinal nematode (GIN) infection and potential biomarkers for GIN resistance in sheep. Microarray gene expression data were obtained for 3 different tissues at 4 time points from sheep artificially challenged with two types of nematodes, Haemonchus contortus (HC) and Trichostrongylus colubriformis (TC). We employed an integrated systems biology approach, integrating 3 main methods: standard differential gene expression analyses, weighted gene co-expression network analyses (WGCNA) and quantitative genetic analyses of gene expression traits of key biomarkers. Using standard differential gene expression analyses we identified differentially expressed genes (DE) which responded differently in sheep challenged with HC compared to those challenged with TC. These interaction genes (e.g. MRPL51, SMEK2, CAT, MAPK1IP1 and SLC25A20A) were enriched in Wnt receptor signalling pathway (p = 0.0132) and positive regulation of NFκß transcription factor activity (p = 0.00208). We report FCER1A, a gene encoding a high-affinity receptor for the Fc region of immunoglobulin E, which is linked to innate immunity to GIN in sheep. Using weighted gene co-expression network analysis (WGCNA) methods, we identified gene modules that were correlated with the length of infection (disease modules). Hub genes (with high intramodular connectivity) were filtered further to identify biomarkers that are related to the length of infection (e.g. CAT, FBX033, COL15A1, IGFBP7, FBLN1 and IgCgamma). The biomarkers we found in HC networks were significantly associated with functions such as T-cell and B-cell regulations, TNF-alpha, interleukin and cytokine production. In TC networks, biomarkers were significantly associated with functions such as protein catabolic process, heat shock protein binding, protein targeting and localization, cytokine receptor binding, TNF receptor binding, apoptosis and IGF binding. These results provide specific gene targets for therapeutic interventions and provide insights into GIN infections in sheep which may be used to infer the same in related host species. This is also the first study to apply the concept of estimating breeding values of animals to expression traits and reveals 11 heritable candidate biomarkers (0.05 to 0.92) that could be used in selection of animals for GIN resistance.

Detection of quantitative trait loci for internal parasite resistance in sheep. I. Linkage analysis in a Romney x Merino sheep backcross population.

This study aimed to identify putative quantitative trait loci (QTL) that significantly affect internal parasite resistance in a backcross sheep population. A Romney x Merino backcross (to Merino) flock was challenged in 3 separate infections with Trichostrongylus colubriformis (primary and secondary) and Haemonchus contortus (tertiary). Haematological parameters were measured and faecal worm egg counts (FWEC) were established to estimate parasite burden. QTL mapping was conducted for FWEC and for the changes in haematocrit following H. contortus challenge and in eosinophil numbers following T. colubriformis challenge. Animals were genotyped for 55 microsatellite markers on selected chromosomes 2, 3, 6, 11, 13, 15, 21, and 22. Four putative quantitative trait loci were found; these being for eosinophil change in the primary infection (OAR 21), for FWEC in the first infection and eosinophil change in the secondary infection (OAR 3) and for FWEC in the secondary infection (OAR 22). No significant quantitative trait loci were detected for FWEC or haematocrit change during the Haemonchus contortus infection. The position of the putative quantitative trait loci for eosinophil change on OAR 3 is consistent with other reports of parasite resistance quantitative trait loci, implying some commonality between studies.

Lysine-scanning mutagenesis reveals an amendable face of the cyclotide kalata B1 for the optimization of nematocidal activity.

Cyclotides are a family of macrocyclic peptides that combine the unique features of a head-to-tail cyclic backbone and a cystine knot motif, the combination of which imparts them with extraordinary stability. The prototypic cyclotide kalata B1 is toxic against two economically important gastrointestinal nematode parasites of sheep, Haemonchus contortus and Trichostrongylus colubriformis. A lysine scan was conducted to examine the effect of the incorporation of positive charges into the kalata B1 cyclotide framework. Each of the non-cysteine residues in this 29-amino acid peptide was successively substituted with lysine, and the nematocidal and hemolytic activities of the suite of mutants were determined. Substitution of 11 residues within kalata B1 decreased the nematocidal activity dramatically. On the other hand, six other residues that are clustered on the surface of kalata B1 were tolerant to Lys substitution, and indeed the introduction of positively charged residues into this region increased nematocidal activity. This activity was increased further in double and triple lysine mutants, with a maximal increase (relative to the native kalata B1) of 13-fold obtained with a triple lysine mutant (mutated at positions Thr-20, Asn-29, and Gly-1). Hemolytic activity correlated with the nematocidal activity of all lysine mutants. Our data clearly highlight the residues crucial for nematocidal and hemolytic activity in cyclotides, and demonstrate that the nematocidal activity of cyclotides can be increased by incorporation of basic amino acids.

Intraspecific epitopic variation in a carbohydrate antigen exposed on the surface of Trichostrongylus colubriformis infective L3 larvae.

The carbohydrate larval antigen, CarLA, is present on the exposed surface of all strongylid nematode infective L3 larvae tested, and antibodies against CarLA can promote rapid immune rejection of incoming Trichostrongylus colubriformis larvae in sheep. A library of ovine recombinant single chain Fv (scFv) antibody fragments, displayed on phage, was prepared from B cell mRNA of field-immune sheep. Phage displaying scFvs that bind to the surface of living exsheathed T. colubriformis L3 larvae were identified, and the majority of worm-binding scFvs recognized CarLA. Characterization of greater than 500 worm surface binding phage resulted in the identification of nine different anti-CarLA scFvs that recognized three distinct T. colubriformis CarLA epitopes based on blocking and additive ELISA. All anti-CarLA scFvs were specific to the T. colubriformis species of nematode. Each of the three scFv epitope classes displayed identical Western blot recognition patterns and recognized the exposed surface of living T. colubriformis exsheathed L3 larvae. Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms. Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations. These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.

Haemonchus contortus and Trichostrongylus colubriformis did not adapt to long-term exposure to sheep that were genetically resistant or susceptible to nematode infections.

We tested the hypothesis that Haemonchus contortus and Trichostrongylus colubriformis would adapt to long-term exposure to sheep that were either genetically resistant or susceptible to H. contortus. Sheep genotypes were from lines with 10 years prior selection for low (resistant, R) or high (susceptible, S) faecal worm egg count (WEC) following H. contortus infection. Long-term exposure of H. contortus and T.colubriformis to R or S genotypes was achieved using serial passage for up to 30 nematode generations. Thus, we generated four nematode strains; one strain of each species solely exposed to R sheep and one strain of each species solely exposed to S sheep. Considerable host genotype differences in mean WEC during serial passage confirmed adequate nematode selection pressure for both H. contortus (R 4900 eggs per gram (epg), S 19,900 epg) and T. colubriformis (R 5300 epg, S 13,500 epg). Adaptation of nematode strain to host genotype was tested using seven cross-classified tests for H. contortus, and two cross-classified and one outbred genotype test for T. colubriformis. In the cross-classified design, where each strain infects groups of R, S or randomly bred control sheep, parasite adaptation would be indicated by a significant host genotype by nematode strain interaction for traits indicating parasite reproductive success; specifically WEC and, for H. contortus strains, packed cell volume. We found no significant evidence of parasite adaptation to host genotype (P>0.05) for either the H. contortus or T. colubriformis strains. Therefore, we argue that nematodes will not adapt quickly to sheep bred for nematode resistance, where selection is based on low WEC, although selecting sheep using a subset of immune functions may increase adaptation risk. Our results support the hypothesis that nematode resistance is determined by many genes each with relatively small effect. In conclusion, selection of sheep for nematode resistance using WEC should be sustainable in the medium to long-term.

Nutritional sensitivity of resistance to Trichostrongylus colubriformis in lactating ewes.

It has been suggested that the periparturient breakdown of immunity to parasites has a nutritional basis. Our overall hypothesis is that it results from a prioritized scarce metabolizable protein (MP) allocation to reproductive functions (e.g. milk production) rather than to immune functions. We have earlier shown that the relationship between MP supply, milk production and resistance to the abomasal nematode Teladorsagia circumcincta provides support for this hypothesis. Because nutritional sensitivity of resistance to parasites may differ between parasite species, we used the same nutritional protocol to test the overall hypothesis with a different parasite. Thus, five levels of dietary MP, ranging from 0.60 to 1.2 times assumed requirements, were offered for 4 weeks post-parturition to twin-rearing Greyface ewes, experimentally infected with the small intestinal nematode Trichostrongylus colubriformis. We hypothesized that the initial increments of MP supply would increase milk production without affecting the degree of breakdown of immunity whilst later increments would reduce the degree of breakdown of immunity. Indeed, the data suggest that the first two increments of MP supply increased milk production, whilst final worm burdens were reduced from the second increment onwards. MP supply did not affect mucosal mast cell concentration but increased globule leukocyte concentration. These results support the overall hypothesis that scarce MP allocation is prioritized to milk production over immune functions. In addition, the contrast between effects of MP supply on resistance to T. colubriformis and to T. circumcincta supports the view that periparturient immunity to small intestinal nematodes may be less sensitive to nutrient scarcity than periparturient immunity to abomasal nematodes.

Genomic characterization of Tv-ant-1, a Caenorhabditis elegans tag-61 homologue from the parasitic nematode Trichostrongylus vitrinus.

A full-length cDNA (Tv-ant-1) encoding an adenine nucleotide translocator (ANT or ADP/ATP translocase) (Tv-ANT-1) was isolated from Trichostrongylus vitrinus (order Strongylida), an economically important parasitic nematode of small ruminants. The uninterrupted open reading frame (ORF) of 894 nucleotides encoded a predicted protein of 297 amino acids, containing characteristic motifs [RRRMMM] and PX(D,E)XX(K,R). Comparison with selected sequences from the free-living nematode Caenorhabditis elegans, cattle and human showed that Tv-ANT-1 is relatively conserved. Sequence identity was the greatest in and near the consensus sequence RRRMMM, and in the six hydrophobic regions predicted to be associated with alpha-helices and to traverse the cell membrane. Phylogenetic analyses of selected amino acid sequence data, using the neighbor-joining and maximum parsimony methods, revealed Tv-ANT-1 to be most closely related to the molecule (Ce-ANT-3) inferred from the tag-61 gene of C. elegans. Comparison of the genomic organization of the full-length Tv-ant-1 gene was similar to that of tag-61. Analysis of the region (5'-UTR) upstream of Tv-ant-1 identified some promoter components, including GATA transcription factor, CAAT and E-box elements. Transcriptional analysis by reverse transcription polymerase chain reaction (RT-PCR) showed that Tv-ant-1 was transcribed in all developmental stages of T. vitrinus, including the first- to fourth- stage larvae (L(1)-L(4)) as well as female and male adults. RNA interference, conducted by feeding C. elegans with double-stranded RNA (dsRNA) from Tv-ant-1 cDNA (using the homologous gene from C. elegans as a positive control), revealed no gene silencing. In spite of nucleotide identities of 100% in 23-30 bp stretches of sequence between the genes Tv-ant-1 and tag-61, these identities seem to be insufficient to achieve effective silencing in C. elegans using the parasite homologue/orthologue Tv-ant-1. This first insight into an ANT of T. vitrinus provides a foundation for exploring its role in developmental and/or survival processes of trichostrongylid nematodes.

Gastrointestinal nematode species burdens and host mortality in a feral sheep population.

Every few years a large proportion of the feral sheep on Hirta, St Kilda die due to food shortage. The effects of malnutrition are exacerbated by gastrointestinal nematodes. As found in sheep flocks in mainland Britain, Teladorsagia circumcincta has long been considered the predominant and most pathogenic nematode species in all age classes of Soay sheep. Previous research indicated that intensity of this species showed a negative association with host age and comprised 75% of the entire gastrointestinal burden. Here we present new data that show Trichostrongylus axei and Trichostrongylus vitrinus to be the predominant worm pathogens in young Soay sheep. In the present study, Trichostrongylus spp. burdens declined with host age whereas T. circumcincta actually increased in burden over the first few age classes. Also, male hosts had significantly higher burdens of Trichostrongylus spp. than females, with this genus making up a higher proportion of the strongyle egg producing community in male hosts than female hosts. These new findings raise questions concerning our previous interpretation of the main nematode species contributing to strongyle egg count in the population, and the contrasting infection patterns of these nematode species in unmanaged St Kilda Soay sheep compared with domestic sheep in mainland Britain.

Haemonchus contortus and Trichostrongylus colubriformis in pen-trials with Javanese thin tail sheep and Kacang cross Etawah goats.

Weight gain costs due to infection were higher in sheep than goats, 28 and 17.5%, respectively, for Trichostrongylus colubriformis and 48.7 and 32.2%, respectively, for Haemonchus contortus. The extent of bodyweight cost attributed to anorexia in sheep infected with H. contortus was higher (13.5 g/day) than in sheep infected with T. colubriformis (2.3 g/day). On the other hand, bodyweight cost due to the other pathogenic effects in sheep infected with T. colubriformis were higher (35.6 g/day) compared to sheep infected with H. contortus (10.9 g/day). A strong relationship between faecal egg count and worm count (r=0.79, P=0.006) was shown only in sheep infected with T. colubriformis. About half of the infected sheep and goats had low or zero faecal egg counts throughout the study. In about 40% the egg count rose initially but became low by weeks 10-16, whereas in about 10% counts increased progressively throughout the period of observation and these animals also had the highest numbers of worms at slaughter. Packed cell volume was reduced in sheep and goats infected with H. contortus but serum protein and haemoglobin levels were unaffected. Sheep infected with T. colubriformis had a higher level of eosinophilia after 8 weeks (18.4%) than sheep infected with H. contortus (11.4%), whereas this pattern was reversed in goats and levels were also lower (4.1 and 8.9%, respectively). There was no apparent relationship between eosinophilia and resistance to infection with H. contortus or T. colubriformis.

The responses of a tropical breed of domestic rabbit, Oryctolagus cuniculus, to experimental infection with Trichostrongylus colubriformis.

Clinical, parasitological and pathological responses of a tropical out-bred domestic rabbit to experimental Trichostrongylus colubriformis infection were used to evaluate its suitability as a laboratory host and model for studying the host-parasite relationships of T. colubriformis. In the first experiment, three groups each of 16, predominantly juvenile male, 8- to 10-week-old rabbits were given a single pulse infection with 500, 5000 or 25000 infective larvae (L3) of T. colubriformis, to represent low, medium and high levels of infection, respectively. A fourth group of 16 rabbits of similar age formed the uninfected controls. In the second experiment, two groups of 10 juvenile (8- to 10-week-old) and 10 adult (8- to 10-month-old) rabbits were similarly infected with 20000 L3, with appropriate naive controls. Prepatency was 14 and 16 days and peak faecal egg counts occurred on days 24 and 20 after infection in young and adult rabbits respectively. Peak worm counts occurred on day 14 in both age groups and at all levels of infection. Subsequently, parasite burdens declined in a highly significantly dose- and age-dependent manner. At low and moderate levels of infection, approximately 83-98% of worms were recovered from the first 60 cm of the small intestine. Worm fecundity was also significantly influenced by host age and larval dose. Host age also had a significant effect on worm length. Infections with T. colubriformis were associated with a highly significant loss of body weight, accompanied by anorexia, diarrhoea and 25% mortality at high dose levels during the patent period of infection. There were no significant changes in packed cell volume and eosinophil counts at all ages and levels of infection but significant lymphocytosis occurred at the high dose level between days 7 and 21. Parasite-specific serum IgG responses were not related to worm burden. Overall, data showed that this miniature, docile and relatively inexpensive breed of rabbit is a potentially valuable laboratory host for studying T. colubriformis infections. The larval dose, duration of infection and host age were major determinants of host responsiveness to primary infections in this rabbit genotype.

Trichostrongylus colubriformis extract upregulates TNF-alpha receptor expression and enhances TNF-alpha sensitivity of L929 cells.

Many pathogens have developed strategies to avoid the host's immune system and hence improve their long-term survival. These strategies include antigenic variation, mimicry of host regulatory proteins and production of immunoregulatory molecules. The ruminant gastrointestinal nematode Trichostrongylus colubriformis produces several factors with homology to human immunoregulatory proteins. However, direct immunomodulation by T. colubriformis proteins has not yet been unequivocally demonstrated. Results in the present paper demonstrate that soluble T. colubriformis factors promote proliferation of the TNF-susceptible mouse fibrosarcoma cell line L929, while inhibiting proliferation of all other cell types tested. In addition, T. colubriformis homogenate enhanced the susceptibility of L929 cells to the cytotoxic action of ovine TNF-alpha. Within 1 h of exposure, T. colubriformis factors bind L929 cells in a stable fashion, yet it takes up to 24 h for the cells to become sensitised to TNF-alpha. Interestingly, the increase of both TNF-alpha sensitivity and proliferation of treated L929 cells correlated with an upregulation in expression of TNF-alpha p55 and p75 receptors.

Susceptibility of high and low producer dairy goats to single experimental infection with Trichostrongylus colubriformis.

Culled dry dairy goats, which differed in their level of production in previous lactations, received a single infection with Trichostrongylus colubriformis. The objectives of the study were twofold. First, the study aimed at examining the ability of dairy goats to develop an immune response to trichostrongyle infection and the associated cellular changes developing within the intestinal mucosa. Second, a comparison between animals differing in their level of production was assessed, in order to determine whether these differed in their susceptibility to infection. No difference occurred in egg excretion, worm burden and local inflammatory cell responses between high (HP) and low (LP) producer dairy goats, in contrast to observations in previous studies. Because their nutrition was controlled and milk production absent in the goats employed in the present study, these results suggest that any genetic component associated with the selection of HP and LP goats had little influence on the development of acquired resistance to T. colubriformis. The analysis of the relationship between different cell types in the mucosa and some characteristics of the worm population show that eosinophils are negatively related to worm burden. Also, a role is suggested for mast cells and globule leukocytes in the modulation of egg excretion.

Parenterally administered gastrointestinal nematode infective larvae viable after more than 15 years in liquid nitrogen.

After cryopreservation for 13.3-15.8 years, the viability of the infective larvae (L3) of Trichostrongylus axei, T. colubriformis, Oesophagostomum columbianum, Haemonchus contortus, Ostertagia circumcincta, T. falculatus, Nematodirus spathiger, Chabertia ovina and Dictyocaulus filaria was assessed in sheep, by being deposited at their predilection sites. D. filaria was, however, an exception, in that the L3 were injected into the jugular vein. The mean development of all the species was 22.8%, but if three species (O. columbianum, C. ovina and D. filaria), that developed poorly are disregarded, then the mean development was 33.4%, similar to previous tests after shorter periods of cryopreservation. The L3 of some of the species appeared sluggish when examined 10-15 min after being thawed, and in the case of H. contortus practically all the larvae of the original batch tested in the previous trials of the series appeared dead when thawed for use in the present trial, and were replaced by another batch of L3 of the same species. When re-examined after about 8 h, however, a high percentage of the L3 of the original batch appeared to have become revitalised, and their viability was tested in a trial reported elsewhere. The intestinal cells of the majority of the L3 of N. spathiger, O. circumcincta and C. ovina were vesiculated when they were thawed. Nevertheless, the degree of development of the former two species was of the highest in the trial, and it can be concluded that this phenomenon does not necessarily impede the viability of larvae.