Hepatic transcriptomic analysis reveals differential regulation of metabolic and immune pathways in three strains of chickens with distinct growth rates exposed to mixed parasite infections.

During parasite infections, the liver may prioritise immune-related pathways over its metabolic functions. Intestinal infections caused by Ascaridia galli and Heterakis gallinarum impair feed intake, nutrient absorption, and weight gain. Histomonas meleagridis, vectored by H. gallinarum, can also damage liver tissues, potentially impairing liver functions. This study examined the hepatic gene expression in three strains of chickens: Ross-308 (R), Lohmann Brown Plus (LB), and Lohmann Dual (LD), 2 weeks after an experimental infection (n = 18) with both A. galli and H. gallinarum or kept as uninfected control (n = 12). Furthermore, H. gallinarum infection led to a co-infection with H. meleagridis. The mixed infections reduced feed intake and the average daily weight gain (P < 0.001). The infections also increased the plasma concentrations of alpha (1)-acid glycoprotein and the antibody titre against H. meleagridis (P = 0.049), with no strain differences (P > 0.05). For host molecular response, 1887 genes were differentially expressed in LD, while 275 and 25 genes were differentially expressed in R and LB, respectively. The up-regulated genes in R and LD were mostly related to inflammatory and adaptive immune responses, while down-regulated genes in LD were involved in metabolic pathways, including gluconeogenesis. Despite performance differences among the strains, worm burdens were similar, but hepatic molecular responses differed significantly. Moreover, there was an indication of a shift in hepatic functions towards immune-related pathways. We, therefore, conclude that the liver shifts its functions from metabolic to immune-related activities in chickens when challenged with mixed parasite species.

Upcycling of recycled minerals from sewage sludge through black soldier fly larvae (Hermetia illucens): Impact on growth and mineral accumulation.

Phosphorous (P) resources are finite. Sewage sludge recyclates (SSR) are not only of interest as plant fertilizer but also as potential source of minerals in animal nutrition. However, besides P and calcium (Ca), SSR contain heavy metals. Under EU legislation, the use of SSR derivatives in animal feed is not permitted, but given the need to improve nutrient recycling, it could be an environmentally sound future mineral source. Black soldier fly larvae (BSFL) convert low-grade biomass into valuable proteins and lipids, and accumulate minerals in their body. It was hypothesized that BSFL modify and increase their mineral content in response to feeding on SSR containing substrates. The objective was to evaluate the upcycling of minerals from SSR into agri-food nutrient cycles through BSFL. Growth, nutrient and mineral composition were compared in BSFL reared either on a modified Gainesville fly diet (FD) or on FD supplemented with either 4% of biochar (FD + BCH) or 3.6% of single-superphosphate (FD + SSP) recyclate (n = 6 BSFL rearing units/group). Larval mass, mineral and nutrient concentrations and yields were determined, and the bioaccumulation factor (BAF) was calculated. The FD + SSP substrate decreased specific growth rate and crude fat of BSFL (P < 0.05) compared to FD. The FD + SSP larvae had higher Ca and P contents and yields but the BAF for Ca was lowest. The FD + BCH larvae increased Ca, iron, cadmium and lead contents compared to FD. Larvae produced on FD + SSP showed lower lead and higher arsenic concentration than on FD + BCH. Frass of FD + BCH had higher heavy metal concentration than FD + SSP and FD (P < 0.05). Except for cadmium and manganese, the larval heavy metal concentration was below the legally permitted upper concentrations for feed. In conclusion, the SSR used could enrich BSFL with Ca and P but at the expense of growth. Due to the accumulation of Cd and Mn, BSFL or products thereof can only be a component of farmed animal feed whereas in BSFL frass heavy metal concentrations remained below the upper limit authorized by EU.

Diurnal variation in egg excretion by Heterakis gallinarum.

Periodicity in nematode egg excretion may be of evolutionary origin as it can favour dispersal of the eggs in the environment. We investigated whether egg excretion by Heterakis gallinarum shows a repeatable pattern of periodicity. The faecal egg concentration and total number of eggs excreted within 4-h intervals were significantly affected by the sampling time within 1 day, but remained unaffected by the sampling day or interaction effects. By contrast, the total number of eggs excreted within 24 h did not differ among the 4 days of the study, collectively indicating repeatable egg excretion patterns. Both host feces and parasite egg excretion increased from night to late afternoon, followed by a decrease in the evening, resulting in higher egg excretion during daytime than the dark period. Feces excretion and worm fecundity showed overlapping diurnal rhythms with similarly timed phases, suggesting the existence of synchronicity between the host feces and nematode egg excretion patterns. We conclude that egg excretion by H. gallinarum is synchronized with host feces excretion and is higher during the daytime than during the dark period. This overlaps with the maximum activity of the day-active host and allows a maximal dispersal of the eggs in the environment.

Methane prediction based on individual or groups of milk fatty acids for dairy cows fed rations with or without linseed.

Milk fatty acids (MFA) are a proxy for the prediction of CH4 emission from cows, and prediction differs with diet. Our objectives were (1) to compare the effect of diets on the relation between MFA profile and measured CH4 production, (2) to predict CH4 production based on 6 data sets differing in the number and type of MFA, and (3) to test whether additional inclusion of energy-corrected milk (ECM) yield or dry matter intake (DMI) as explanatory variables improves predictions. Twenty dairy cows were used. Four diets were used based on corn silage (CS) or grass silage (GS) without (L0) or with linseed (LS) supplementation. Ten cows were fed CS-L0 and CS-LS and the other 10 cows were fed GS-L0 and GS-LS in random order. In feeding wk 5 of each diet, CH4 production (L/d) was measured in respiration chambers for 48 h and milk was analyzed for MFA concentrations by gas chromatography. Specific CH4 prediction equations were obtained for L0-, LS-, GS-, and CS-based diets and for all 4 diets collectively and validated by an internal cross-validation. Models were developed containing either 43 identified MFA or a reduced set of 7 groups of biochemically related MFA plus C16:0 and C18:0. The CS and LS diets reduced CH4 production compared with GS and L0 diets, respectively. Methane yield (L/kg of DMI) reduction by LS was higher with CS than GS diets. The concentrations of C18:1 trans and n-3 MFA differed among GS and CS diets. The LS diets resulted in a higher proportion of unsaturated MFA at the expense of saturated MFA. When using the data set of 43 individual MFA to predict CH4 production (L/d), the cross-validation coefficient of determination (R2CV) ranged from 0.47 to 0.92. When using groups of MFA variables, the R2CV ranged from 0.31 to 0.84. The fit parameters of the latter models were improved by inclusion of ECM or DMI, but not when added to the data set of 43 MFA for all diets pooled. Models based on GS diets always had a lower prediction potential (R2CV = 0.31 to 0.71) compared with data from CS diets (R2CV = 0.56 to 0.92). Models based on LS diets produced lower prediction with data sets with reduced MFA variables (R2CV = 0.62 to 0.68) compared with L0 diets (R2CV = 0.67 to 0.80). The MFA C18:1 cis-9 and C24:0 and the monounsaturated FA occurred most often in models. In conclusion, models with a reduced number of MFA variables and ECM or DMI are suitable for CH4 prediction, and CH4 prediction equations based on diets containing linseed resulted in lower prediction accuracy.

Stable production of cyanophycinase in Nicotiana benthamiana and its functionality to hydrolyse cyanophycin in the murine intestine.

Food supplementation with the conditionally essential amino acid arginine (Arg) has been shown to have nutritional benefits. Degradation of cyanophycin (CGP), a peptide polymer used for nitrogen storage by cyanobacteria, requires cyanophycinase (CGPase) and results in the release of ß-aspartic acid (Asp)-Arg dipeptides. The simultaneous production of CGP and CGPase in plants could be a convenient source of Arg dipeptides. Different variants of the cphB coding region from Thermosynechococcus elongatus BP-1 were transiently expressed in Nicotiana benthamiana plants. Translation and enzyme stability were optimized to produce high amounts of active CGPase. Protein stability was increased by the translational fusion of CGPase to the green fluorescent protein (GFP) or to the transit peptide of the small subunit of RuBisCO for peptide production in the chloroplasts. Studies in mice showed that plant-expressed CGP fed in combination with plant-made CGPase was hydrolysed in the intestine, and high levels of ß-Asp-Arg dipeptides were found in plasma, demonstrating dipeptide absorption. However, the lack of an increase in Asp and Arg or its metabolite ornithine in plasma suggests that Arg from CGP was not bioavailable in this mouse group. Intestinal degradation of CGP by CGPase led to low intestinal CGP content 4 h after consumption, but after ingestion of CGP alone, high CGP concentrations remained in the large intestine; this indicated that intact CGP was transported from the small to the large intestine and that CGP was resistant to colonic microbes.

Effects of rutin and buckwheat seeds on energy metabolism and methane production in dairy cows.

Flavonoids are secondary plant metabolites with several health promoting effects. As dairy cows often suffer from metabolic imbalance and health problems, interest is growing in health improvements by plant substances such as flavonoids. Our group has recently shown that the flavonoids quercetin and rutin (a glucorhamnoside of quercetin) are bioavailable in cows when given via a duodenal fistula or orally, respectively, affect glucose metabolism, and have beneficial effects on liver health. Furthermore, flavonoids may reduce rumen methane production in vitro through their antibacterial properties. To test the hypothesis that rutin has effects on energy metabolism, methane production, and production performance in dairy cows, we fed rutin trihydrate at a dose of 100mg/kg of body weight to a group of 7 lactating dairy cows for 2 wk in a crossover design. In a second experiment, 2 cows were fed the same ration but were supplemented with buckwheat seeds (Fagopyrum tartaricum), providing rutin at a dose comparable to the first experiment. Two other cows receiving barley supplements were used as controls in a change-over mode. Blood samples were taken weekly and respiration measurements were performed at the end of each treatment. Supplementation of pure rutin, but not of rutin contained in buckwheat seeds, increased the plasma quercetin content. Methane production and milk yield and composition were not affected by rutin treatment in either form. Plasma glucose, ß-hydroxybutyrate, and albumin were increased by pure rutin treatment, indicating a possible metabolic effect of rutin on energy metabolism of dairy cows. In addition, we did not show that in vivo ruminal methane production was reduced by rutin. In conclusion, we could not confirm earlier reports on in vitro methane reduction by rutin supplementation in dairy cows in established lactation.

Response to Ascaridia galli infection in growing chickens in relation to their body weight.

It was hypothesized that chickens with extremely varying body weights (BW) from an otherwise homogeneous host sample cope differently with Ascaridia galli (Schrank 1788) infection. Small and large birds, falling into either the lower or the upper 5% quantiles of BW distribution of a parent stock flock, were selected at an age of 4 weeks, housed separately and fed restrictively with the same amount of feed. At week 5, all the small and large birds (635 and 1,297 g/bird, respectively; P < 0.001) were inoculated with 1,000 A. galli eggs and euthanized 52 days post-infection. Small birds had higher daily weight gains (P = 0.004) but final BWs of larger birds were still higher (P < 0.001) at slaughter. Prevalence, intensity of infection as well as worm abundance were higher in small birds when compared with the large birds (P < 0.05), whereas plasma concentrations of A. galli-specific antibodies and worm length remained unaffected (P > 0.05). In conclusion, large birds resist A. galli infection more effectively than do small ones, possibly through different mechanisms acting on allocation of available nutrient and body reserves under the exposure of the infection.

Effects of feeding earthworm or vermicompost on early life performance of broilers under challenging dietary conditions.

We investigated if feeding earthworms (EW) or vermicompost (VC) to broilers improves performance and aids in coping with dietary challenges from a soluble non-starch polysaccharide (NSP)-enriched diet (negative control diet; CON-). Newly-hatched male Cobb-500 birds (N = 480) were fed either a positive (+) control diet (CON+, n = 240) or CON+ supplemented with either 1% EW (CON+EW; n = 120) or 1% VC in DM (CON+VC; n = 120) for 8 d (Period 1; P1). At the end of P1, blood and intestinal samples were taken from half the birds in each group. Half of remaining birds on CON+ stayed on CON+ for further 8 d (P2; d9-16) or switched to CON-. Birds on CON+EW and CON+VC in P1 were switched to CON- in P2 (CON-EW and CON-VC, respectively). The CON+VC improved (P < 0.05) BW and ADG in P1 through an elevated feed intake (FI) (P < 0.05) with no effect on FCR. CON+EW did not differ from the CON+ in terms of growth and FI in P1. In P2 CON- did not affect growth or DMI relative to CON+. In the end of P2, 10% of CON+ birds had pasty vent (PV). CON- increased incidence of PV and CON-VC aggravated this effect (P < 0.05), whereas CON-EW did not differ from CON+. CON- diet reduced proportion of 16S rDNA in colon digesta (P = 0.049), while CON-EW and CON-VC did not differ from CON+. Compared to CON-, CON-EW tended to decrease (P = 0.072) incidence of PV. Ceca were heavier (P < 0.05) in CON-EW than in CON+ fed birds. In conclusion, the challenge diet induced PV and reduced bacterial 16S rDNA in colon digesta, likely due to soluble NSP-induced anti-nutritive effects. VC supplementation enhanced early growth by increasing feed intake. Provision of EW did not impact performance but decreased incidence of PV and increased cecal size, suggesting that potential inoculation with beneficial microorganisms may counteract NSP effects.

Ascaridia galli - An old problem that requires new solutions.

Reports of Ascaridia galli in laying hens in Europe have increased since the ban on conventional battery cages in 2012. As this parasite is transmitted directly via the faecal-oral route by parasite eggs containing a larva, it is reasonable to assume that the escalating problem is related to the increased exposure now occurring in modern welfare-friendly cage-free housing systems. On many farms, A. galli reappears in subsequent flocks, even though the birds have no access to the outdoors, biosecurity is high and empty houses are cleaned and disinfected during downtime. Since the egg production cycle lasts only ≈80 weeks and recombinant antigen production for helminth vaccines has not yet been solved, the development of a vaccine seems to be an unrealistic option. Therefore, disrupting the life cycle of the parasite by other means, including the strategic use of dewormers, appears to be the key to controlling infection. Of concern is that only one class of anthelmintics is licenced for poultry in Europe and that are usually administered indiscriminately through the birds' drinking water and often too late when the parasite is already established. If current calendar-based parasite control strategies are not changed, there is a risk that resistance to anthelmintics may develop, as has already been demonstrated with nematodes in livestock. We insist that treatments can be more effective and the risk of developing drug resistance can be mitigated if we invest in a better understanding of A. galli responses to more prudent and judicious use of anthelmintics. This review identifies knowledge gaps and highlights aspects of sustainable parasite control that require further research to support commercial egg producers.

Pattern and repeatability of ascarid-specific antigen excretion through chicken faeces, and the diagnostic accuracy of coproantigen measurements as compared with McMaster egg counts and plasma and egg yolk antibody measurements in laying hens.

BACKGROUND: A coproantigen enzyme-linked immunosorbent assay (ELISA) has recently been proposed for detecting ascarid infections in chickens. The excretion pattern of ascarid antigens through chicken faeces and the consistency of measurements over the course of infections are currently unknown. This study evaluates the pattern and repeatability of worm antigen per gram of faeces (APG) and compares the diagnostic performance of the coproantigen ELISA with a plasma and egg yolk antibody ELISA and McMaster faecal egg counts (M-FEC) at different weeks post-infection (wpi). METHODS: Faecal, blood and egg yolk samples were collected from laying hens that were orally infected with a mix of Ascaridia galli and Heterakis gallinarum eggs (N = 108) or kept as uninfected controls (N = 71). Measurements including (a) APG using a coproantigen ELISA, (b) eggs per gram of faeces (EPG) using the McMaster technique and (c) ascarid-specific IgY in plasma and in egg yolks using an ascarid-specific antibody ELISA) were performed between wpi 2 and 18. RESULTS: Time-dependent significant differences in APG between infected and non-infected laying hens were quantified. At wpi 2 (t(164) = 0.66, P = 1.00) and 4 (t(164) = -3.09, P = 0.094) no significant differences were observed between the groups, whereas infected hens had significantly higher levels of APG than controls by wpi 6 (t(164) = -6.74, P < 0.001). As indicated by a high overall repeatability estimate of 0.91 (CI = 0.89-0.93), APG could be measured consistently from the same individual. Compared to McMaster and antibody ELISA, coproantigen ELISA showed the highest overall diagnostic performance (area under curve, AUC = 0.93), although the differences were time-dependent. From wpi 6 to 18 coproantigen ELISA had an AUC > 0.95, while plasma IgY ELISA showed the highest diagnostic performance in wpi 2 (AUC = 0.95). M-FEC had the highest correlation with total worm burden, while APG had highest correlations with weights and lengths of A. galli. CONCLUSION: Ascarid antigen excretion through chicken faeces can be measured with high accuracy and repeatability using a coproantigen ELISA. The antigen excretion increases over time, and is associated with worm maturation, particularly with the size of A. galli. Our results suggest the necessity of complementary use of different diagnostic tools for a more accurate diagnosis of infections.

1H-NMR based-metabolomics reveals alterations in the metabolite profiles of chickens infected with ascarids and concurrent histomonosis infection.

BACKGROUND: Gut infections of chickens caused by Ascaridia galli and Heterakis gallinarum are associated with impaired host performance, particularly in high-performing genotypes. Heterakis gallinarum is also a vector of Histomonas meleagridis that is often co-involved with ascarid infections. Here, we provide a first insight into the alteration of the chicken plasma and liver metabolome as a result of gastrointestinal nematode infections with concomitant histomonosis. 1H nuclear magnetic resonance (1H-NMR) based-metabolomics coupled with a bioinformatics analysis was applied to explore the variation in the metabolite profiles of the liver (N = 105) and plasma samples from chickens (N = 108) experimentally infected with A. galli and H. gallinarum (+H. meleagridis). This was compared with uninfected chickens at different weeks post-infection (wpi 2, 4, 6, 10, 14, 18) representing different developmental stages of the worms. RESULTS: A total of 31 and 54 metabolites were quantified in plasma and aqueous liver extracts, respectively. Statistical analysis showed no significant differences (P > 0.05) in any of the 54 identified liver metabolites between infected and uninfected hens. In contrast, 20 plasma metabolites including, amino acids, sugars, and organic acids showed significantly elevated concentrations in the infected hens (P < 0.05). Alterations of plasma metabolites occurred particularly in wpi 2, 6 and 10, covering the pre-patent period of worm infections. Plasma metabolites with the highest variation at these time points included glutamate, succinate, trimethylamine-N-oxide, myo-inositol, and acetate. Differential pathway analysis suggested that infection induced changes in (1) phenylalanine, tyrosine, and tryptophan metabolism, (2) alanine, aspartate and glutamate metabolism; and 3) arginine and proline metabolism (Pathway impact > 0.1 with FDR adjusted P-value < 0.05). CONCLUSION: In conclusion, 1H-NMR based-metabolomics revealed significant alterations in the plasma metabolome of high performing chickens infected with gut pathogens-A. galli and H. gallinarum. The alterations suggested upregulation of key metabolic pathways mainly during the patency of infections. This approach extends our understanding of host interactions with gastrointestinal nematodes at the metabolic level.

Effects of kefir on coccidial oocysts excretion and performance of dairy goat kids following weaning.

The aim of this study was to investigate effects of kefir, a traditional source of probiotic, on coccidial oocysts excretion and on the performance of dairy goat kids following weaning. Twin kids were randomly allocated to one of two groups at weaning. Kids of the first group received 20 ml of kefir daily for 6 weeks (KEF), while kids in the control group were given a placebo (CON). Individual faecal samples were regularly (n = 18 per kid) taken to quantify the number of coccidial oocysts per gram of faeces (OpG). There were no differences between the groups in terms of body weight development (P > 0.05) and feed consumption. Kids of both groups were not able to consume enough feed to meet their nutrient requirements during the first 3 weeks following weaning. KEF had a lower frequency of OpG positive samples than CON (P = 0.043). Kefir did not affect the maximum oocyst excretion and age of the kids at the highest oocyst excretion (P > 0.05). KEF shed numerically 35% lower coccidial oocysts than the controls, which corresponded to a statistical tendency (P = 0.074) in lowering Log-OpG in comparison to CON. While KEF had a lower frequency of OpG positive samples and tended to shed lower OPG by around one-third, the frequency of diarrhea, level of highest oocyst excretion, and performance of the kids remained unaffected. Therefore, it is concluded that overall effects of kefir do not have a significant impact on sub-clinical infection and performance in weaned kids under relatively high-hygienic farming conditions.

A copro-antigen ELISA for the detection of ascarid infections in chickens.

A reliable method of diagnosing the most prevalent helminth infections in chickens is vital for developing effective control strategies. Ascaridia galli and Heterakisgallinarum are phylogenetically close nematode species that can elicit the development of cross-reactive antibodies in chickens. Therefore, an enzyme-linked immunoassay (ELISA) based on Ascaridia galli antigens in faeces of chickens to detect and quantify infections with both A. galli and H. gallinarum was developed. The ELISA utilised polyclonal antibodies that were obtained from rabbits immunised with soluble antigens isolated from A. galli. In two separate experiments, chickens were kept as uninfected controls or were orally infected with either 100 or 1000 of embryonated eggs of A. galli or H.gallinarum. Faecal samples were collected after 28-30 weeks post-infection. The ELISA was then used to quantify the concentration of soluble worm antigens in faecal samples, i.e., the amount of antigen per gram faeces, APG. The APG from infected chickens was significantly higher than non-infected groups in both experiments (P 0.001). Both 100 and 1000 infection dose groups were not significantly different (P = 0.999) in the experiment with H. gallinarum, whereas in the experiment with A. galli, APG was significantly higher in the 1000 infection group (P 0.001). A receiver operation characteristics (ROC) analysis that evaluates the qualitative performance of diagnostics tests was used to calculate the assay parameters within each mono-infection experiment. The result showed that the assay had a high diagnostics accuracy with an area-under-curve (AUC) of 0.99 in detecting infection in A. galli infected chickens and a moderate-high accuracy (AUC = 0.89) in birds infected with H. gallinarum. The diagnostic sensitivity and specificity of the assay at the optimal cut-off point equivalent to Youden index were 93% and 100% for detecting infections in A. galli experiment and 85% and 92% in H. gallinarum experiment, respectively. The correlation between faecal antigen concentration and all worm burden parameters was positive but generally low (r < 0.33), which provided less information about infection intensities. Nonetheless, these results indicate that a reliable and accurate qualitative diagnosis of the two most prevalent intestinal nematodes in chickens can be achieved using a non-invasive copro-antigen ELISA assay.

Growth efficiency, intestinal biology, and nutrient utilization and requirements of black soldier fly (Hermetia illucens) larvae compared to monogastric livestock species: a review.

In recent years, interest in the larvae of black soldier fly (BSF) (Hermetia illucens) as a sustainable protein resource for livestock feed has increased considerably. However, knowledge on the nutritional and physiological aspects of this insect, especially compared to other conventional farmed animals is scarce. This review presents a critical comparison of data on the growth potential and efficiency of the BSF larvae (BSFL) compared to conventional monogastric livestock species. Advantages of BSFL over other monogastric livestock species includes their high growth rate and their ability to convert low-grade organic waste into high-quality protein and fat-rich biomass suitable for use in animal feed. Calculations using literature data suggest that BSFL are more efficient than broilers, pigs and fish in terms of conversion of substrate protein into body mass, but less efficient than broilers and fish in utilization of substrate gross energy to gain body mass. BSFL growth efficiency varies greatly depending on the nutrient quality of their dietary substrates. This might be associated with the function of their gastrointestinal tract, including the activity of digestive enzymes, the substrate particle characteristics, and their intestinal microbial community. The conceived advantage of BSFL having an environmental footprint better than conventional livestock is only true if BSFL is produced on low-grade organic waste and its protein would directly be used for human consumption. Therefore, their potential role as a new species to better close nutrient cycles in agro-ecological systems needs to be reconsidered, and we conclude that BSFL is a complementary livestock species efficiently utilizing organic waste that cannot be utilized by other livestock. In addition, we provide comparative insight into morpho-functional aspects of the gut, characterization of digestive enzymes, gut microbiota and fiber digestion. Finally, current knowledge on the nutritional utilization and requirements of BSFL in terms of macro- and micro-nutrients is reviewed and found to be rather limited. In addition, the research methods to determine nutritional requirements of conventional livestock are not applicable for BSFL. Thus, there is a great need for research on the nutrient requirements of BSFL.

Pathobiology of Heterakis gallinarum mono-infection and co-infection with Histomonas meleagridis in layer chickens.

Little is known about the induction and modulation of gut-associated immune reactions after nematode infection in the chicken. The objective of the present study was to compare the pathogenesis, induction of immune reactions and electrophysiological changes of the gut after mono-infection with Heterakis gallinarum and after dual infection with H. gallinarum and Histomonas meleagridis in layer chickens. In two experiments 3-week-old chickens were inoculated with embryonated H. gallinarum eggs, which were positive for H. meleagridis. While birds of the first experiment were left untreated, those of the second experiment were treated with dimetridazol to prevent H. meleagridis co-infection. Mild to moderate histological lesions and local immune reactions with a significant increase in CD4(+), CD8α(+), TCRαß(+) and TCRδγ(+) cells in the lamina propria and induction of the T-helper type 2 (Th2) cytokine interleukin-13 dominated the H. gallinarum immune response at 2 weeks post infection. Co-infection with H. gallinarum and H. meleagridis induced an increase in mRNA expression of the T-helper type 1 (Th1) cytokine interferon-γ, a decrease in splenic CD4(+) cells and severe destruction of the caecal mucosa in association with strong T-cell infiltration in the caecal lamina propria. There was no obvious effect on the chloride secretion of the caecal epithelium, which was investigated once the mucosa had almost recovered from the infection, in either experiment. These results suggest that the local T-cell reactions to nematode infections in chickens may be comparable with mammals and may be shifted from a Th2-dominated to a Th1-dominated response when accompanied by a protozoan infection.

Impact of Nematode Infections on Non-specific and Vaccine-Induced Humoral Immunity in Dual-Purpose or Layer-Type Chicken Genotypes.

Nematode infections may induce immune-modulatory effects and influence host-immune responses to other pathogens. The aim of the study was to investigate whether a mixed nematode-infection influences non-specific and vaccine-induced humoral immunity against Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), and Avian Metapneumovirus (AMPV) in already vaccinated hens of a dual-purpose (Lohmann Dual, LD) or a layer genotype (Lohmann Brown Plus; LB). Until 17 weeks-of-age, LD (n = 70) and LB (n = 109) hens were vaccinated against major bacterial and viral diseases and coccidiosis. At 24 weeks-of-age, the hens received either a placebo or an oral inoculation of 1,000 infectious eggs of A. galli and H. gallinarum. Plasma total immunoglobulin (Ig) isotypes (IgY, IgM, IgA) levels and vaccine-induced antibody titers against NDV, IBV, and AMPV were determined from 2 to 18 weeks post-infection (wpi). Infections had no suppressing effect on total Ig isotypes IgY, IgM, and IgA as well as on vaccine-induced antibody titers against NDV, IBV, and AMPV (P > 0.05). Overall, LB hens had higher levels of IgY, IgM, and IgA than those of LD hens (P < 0.05). There were no differences between IBV titers of the two genotypes (P > 0.05). Independent of infection status of the hens, NDV titers were higher in LB hens than in LD hens at wpi 2 (P < 0.05), but not in following weeks (P > 0.05). Uninfected LD hens had lower AMPV titers than their infected counterparts at 6 and 14 wpi (P < 0.05). Regardless of nematode infection, LD hens revealed a higher risk of responding weak to vaccination against NDV (odds ratio = 5.45; P = 0.021) and AMPV (odds ratio = 13.99, P < 0.001) than did LB hens (P > 0.05). We conclude that nematode infections have no adverse effects on non-specific and vaccine-induced humoral immunity in either genotype. LB hens have higher levels of total immunoglobulin isotypes than LD hens. Except for IBV, vaccine-induced humoral immune responses show a dependency on genotype. Dual-purpose hens show lower responsiveness to vaccinations against NDV and AMPV, possibly due to factors associated with increased body fat reserves in this genotype.

Excretion of Histomonas meleagridis following experimental co-infection of distinct chicken lines with Heterakis gallinarum and Ascaridia galli.

BACKGROUND: Histomonosis is a severe re-emerging disease of poultry caused by Histomonas meleagridis, a protozoan parasite which survives in the environment via the cecal worm Heterakis gallinarum. Following infection, the parasites reside in the ceca and are excreted via host feces. In the present work, male birds of conventional broiler (Ross 308, R), layer (Lohmann Brown Plus, LB) and a dual-purpose (Lohmann Dual, LD) chicken line were infected with 250 embryonated eggs of Ascaridia galli and Heterakis gallinarum, respectively, with the latter nematode harboring Histomonas meleagridis, to investigate a co-infection of nematodes with the protozoan parasite in different host lines. METHODS: In weekly intervals, from 2 to 9 weeks post infection (wpi), individual fecal samples (n = 234) from the chickens were collected to quantify the excretion of H. meleagridis by real-time PCR and to determine the number of nematode eggs per gram (EPG) in order to elucidate excretion dynamics of the flagellate and the nematodes. This was further investigated by indirect detection using plasma samples of the birds to detect antibodies specific for H. meleagridis and worms by ELISA. The infection with H. meleagridis was confirmed by histopathology and immunohistochemistry to detect the flagellate in the cecum of representing birds. RESULTS: The excretion of H. meleagridis could already be observed from the 2nd wpi in some birds and increased to 100% in the last week of the experiment in all groups independent of the genetic line. This increase could be confirmed by ELISA, even though the number of excreted H. meleagridis per bird was generally low. Overall, histomonads were detected in 60% to 78% of birds with temporary differences between the different genetic lines, which also showed variations in the EPG and worm burden of both nematodes. CONCLUSIONS: The infection with H. gallinarum eggs contaminated with H. meleagridis led to a permanent excretion of the flagellate in host feces. Differences in the excretion of H. meleagridis in the feces of genetically different host lines occurred intermittently. The excretion of the protozoan or its vector H. gallinarum was mostly exclusive, showing a negative interaction between the two parasites in the same host.

Differentially Expressed Gene Patterns in Ascarid-Infected Chickens of Higher- or Lower-Performing Genotypes.

Here, we describe the first transcriptomic investigation of the peripheral blood of chickens exposed to Ascaridia galli and Heterakis gallinarum infections. We investigated differentially expressed gene (DEG) patterns in two chicken genotypes with either a higher (Lohmann Brown Plus, LB) or lower (Lohmann Dual, LD) laying performance level. The hens were experimentally coinfected with A. galli and H. gallinarum, and their worm burdens and infection parameters were determined six weeks post infection. Based on most representative infection parameters, the hens were clustered into lower- and higher-infection intensity classes. We identified a total of 78 DEGs contributing to infection-related phenotypic variation in the two genotypes. Our data showed significant upregulation of Guanylate Binding Protein 7 (GBP7) in LD hens, making it a promising candidate for tolerance to ascarid infections in chickens. Gene ontology analysis revealed higher transcriptome activity related to biological processes such as "response to external stimulus" in LB hens, implying a higher stress response in this genotype. In contrast, LD hens showed higher transcriptomic expression of genes related to ontology classes that are possibly associated with a higher tolerance to infections. These findings may help explain why lower-performing genotypes (i.e., LD) are less sensitive to infections in terms of maintaining their performance.

The Effect of Dietary Protein Imbalance during Pregnancy on the Growth, Metabolism and Circulatory Metabolome of Neonatal and Weaned Juvenile Porcine Offspring.

Protein imbalance during pregnancy affects women in underdeveloped and developing countries and is associated with compromised offspring growth and an increased risk of metabolic diseases in later life. We studied in a porcine model the glucose and urea metabolism, and circulatory hormone and metabolite profile of offspring exposed during gestation, to maternal isoenergetic low-high (LP-HC), high-low (HP-LC) or adequate (AP) protein-carbohydrate ratio diets. At birth, LP-HC were lighter and the plasma acetylcarnitine to free carnitine ratios at 1 day of life was lower compared to AP offspring. Plasma urea concentrations were lower in 1 day old LP-HC offspring than HP-LC. In the juvenile period, increased insulin concentrations were observed in LP-HC and HP-LC offspring compared to AP, as was body weight from HP-LC compared to LP-HC. Plasma triglyceride concentrations were lower in 80 than 1 day old HP-LC offspring, and glucagon concentrations lower in 80 than 1 day old AP and HP-LC offspring. Plasma urea and the ratio of glucagon to insulin were lower in all 80 than 1 day old offspring. Aminoacyl-tRNA, arginine and phenylalanine, tyrosine and tryptophan metabolism, histidine and beta-alanine metabolism differed between 1 and 80 day old AP and HP-LC offspring. Maternal protein imbalance throughout pregnancy did not result in significant consequences in offspring metabolism compared to AP, indicating enormous plasticity by the placenta and developing offspring.

Accuracy and precision of McMaster and Mini-FLOTAC egg counting techniques using egg-spiked faeces of chickens and two different flotation fluids.

Faecal egg counting techniques (ECTs) are useful tools for assessing anthelmintic efficacy and selecting hosts resistant to parasite infection. McMaster (MM) is one of the most commonly used ECTs, but it suffers from low sensitivity and precision. Mini-FLOTAC (MF) has been proposed to replace MM, but so far has not been evaluated for gastro-intestinal nematode infections in chickens. This study compared sensitivity, precision, and accuracy of MM and MF with two trials using egg-spiked faecal samples ranging from 50-1250 eggs per gram of faeces (EPG). In addition, effects of two flotation fluids with different specific gravities (SG), namely salt (SG = 1.20) and sucrose solutions (SG = 1.32), on accuracy and time-spent for both ECTs were evaluated. Overall sensitivity based on the composite reads across all EPG-levels was 97.1 % for MM and 100 % for MF. MF was, however, more sensitive (P = 0.003) or tended to (P = 0.087) be more sensitive than MM at only the lowest EPG-level (i.e. 50 EPG) using one of the duplicate reads, whereas there was no significant difference at any EPG-level using composite reads. Overall average precision of MF (79.5 %) was higher (P < 0.001) than that of MM (63.4 %) across all EPG-levels. Precision of MM increased from 22 to 87 % with increasing EPG-levels from 50-1250 EPG. Corresponding precision estimates for MF ranged from 76 to 91 %. Overall recovery rate of MM (74.6 %) was significantly higher (P < 0.001) than that of MF (60.1 %). There was no significant difference in recovery rate of spiked-eggs among different EPG-levels (P = 0.833). Recovery rate of MM ranged from 64 % to 79 % across different EPG-levels, while it ranged from 54 % to 64 % with MF without an interaction between ECT and EPG-level (P = 0.701). It took more time (P < 0.001) to process (prepare and read) samples with MF than with MM using the same flotation fluid. The sugar solution tended to (P = 0.100) increase egg-recovery with both ECTs, while increasing (P < 0.001) time-spent for processing the samples. Our data collectively suggest that MM is less sensitive than MF only at around minimum detection level of MM when using unrepeated reads. We conclude that McMaster is faster, relatively more accurate but less precise than Mini-FLOTAC. The sugar solution with higher SG increases accuracy of both techniques at the expense of increased labour time.