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.

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.

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.

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.

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.

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.

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.

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.

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.

Transcript profile of skeletal muscle lipid metabolism genes affected by diet in a piglet model of low birth weight.

Dysregulated skeletal muscle metabolism (DSMM) is associated with increased inter- and intramuscular fat deposition in low birth weight (L) individuals. The mechanisms behind DSMM in L individuals are not completely understood but decreased muscle mass and shifts in lipid and carbohydrate utilisation may contribute. Previously, we observed lower fat oxidation in a porcine model of low birth weight. To elucidate the biological activities underpinning this difference microfluidic arrays were used to assess mRNA associated with lipid metabolism in longissimus dorsi (LD) and semitendinosus (ST) skeletal muscle samples from thirty-six female L and normal birth weight (N) pigs. Plasma samples were collected from a sub-population to measure metabolite concentrations. Following overnight fasting, skeletal muscle and plasma samples were collected and the association with birth weight, diet and age was assessed. Reduced dietary fat was associated with decreased LD intermuscular fat deposition and beta-oxidation associated mRNA, in both birth weight groups. Lipid uptake and intramuscular fat deposition associated mRNA was reduced in only L pigs. Abundance of ST mRNA associated with lipolysis, lipid synthesis and transport increased in both birth weight groups. Lipid uptake associated mRNA reduced in only L pigs. These changes were associated with decreased plasma L glucose and N triacylglycerol. Post-dietary fat reduction, LD mRNA associated with lipid synthesis and inter- and intramuscular fat deposition increased in L, whilst beta-oxidation associated mRNA remains elevated for longer in N. In the ST, mRNA associated with lipolysis and intramuscular fat deposition increased in both birth weight groups, however this increase was more significant in L pigs and associated with reduced beta-oxidation. Analysis of muscle lipid metabolism associated mRNA revealed that profile shifts are a consequence of birth weight. Whilst, many of the adaptions to diet and age appear to be similar in birth weight groups, the magnitude of response and individual changes underpin the previously observed lower fat oxidation in L pigs.

Resistance and tolerance to mixed nematode infections in relation to performance level in laying hens.

Modern chickens have been genetically developed to perform high under optimal conditions. We hypothesized that high-performance is associated with a higher sensitivity to environmental challenges in laying hens. By using nematode infections as an environmental stressor, we assessed performance-level associated host responses in a high (i.e. Lohmann Brown Plus, LB) and in a lower performing, a so-called dual-purpose chicken genotype (i.e. Lohmann Dual, LD). The hens were infected with 1000 eggs of Ascaridia galli and Heterakis gallinarum at 24 weeks of age. Hen performance parameters, humoral immune responses in plasma and egg yolks and worm burdens were assessed at several occasions over a period of 18 weeks post infection (wpi). While infections had no significant effect on feed intake (P = 0.130) and body weight in both genotypes (P = 0.392), feed conversion efficiency was negatively affected by infections (P = 0.017). Infections reduced both laying rate and egg weight and thereby per capita egg mass in both genotypes (P < 0.05). While laying rate in infected LB hens decreased significantly (P < 0.05) in the early infection period (i.e. by 3 wpi), the decrease in LD hens appeared much later (i.e. by 14 wpi). Worm burdens resulting from the experimental infection were not different between the genotypes for both worm species (P > 0.05), whereas LB hens were more susceptible (P < 0.05) to re-infections than LD hens. Changes in humoral immune responses (i.e. ascarid-specific IgY antibodies in plasma and egg yolks) of the two genotypes over time reflected closely the corresponding changes in larval counts of the hens, descending from both experimental and subsequent natural infections in both genotypes. Infections caused a shift in egg size classes, leading to smaller frequency of larger eggs in both genotypes. Infections reduced egg weight (P = 0.018) and led to a reduced fat content in the egg yolks (P = 0.045). The proportion of poly-unsaturated fatty acids (PUFA), especially n-6-PUFA, was also lower in egg yolks of the infected hens (P = 0.032). We conclude that tolerance to nematode infections in laying hens is dependent on host-performance level. The impairment in host tolerance was both genotype and time dependent, likely due to differences in genetic programming for production peak and persistency of the two genotypes. The two genotypes exhibited similar levels of resistance after a fully controlled experimental infection, but the high performing hens were more susceptible to subsequent natural infections. Infections negatively affected economically important egg-quality traits, including egg weight, fat content and fatty acid profiles in egg yolks.

Resistance and tolerance to mixed nematode infections in chicken genotypes with extremely different growth rates.

Fast growing broilers are less able to cope with fitness related challenges. As the allocation of metabolic resources may be traded off between performance and defence functions in parasitized hosts, we hypothesized that fast growing broilers are more sensitive to mixed nematode infections compared with slower growing genotypes under the same environmental conditions. Therefore, we compared male birds of genotypes selected for either meat production (Ross-308, R) or egg production (Lohmann Brown Plus, LB) or for both purposes (Lohmann Dual, LD), to assess their resistance and tolerance to mixed nematode infections with Ascaridia galli and Heterakis gallinarum. While infections reduced feed intake in all three genotypes, feed conversion efficiency was not affected. Infections impaired growth performance only in R birds, indicating lower tolerance in the fast growing genotype compared with slower growing LB and LD genotypes. Impaired tolerance in R birds was associated with a relative nutrient scarcity due to an infection-induced lower feed intake. Resistance to experimentally induced infections depended on host genotype as well as on the worm species involved. Overall, the A. galli burden was higher in R than LB, whereas the burden of LD was not different from that of R and LB. In contrast, the H. gallinarum burden of first generation worms was similar in the three genotypes. Susceptibility to re-infection with H. gallinarum was higher in LB than in LD, whereas very low levels of re-infection were observed in R birds. Our data collectively suggest that resistance and tolerance to mixed nematode infections are sensitive to growth rate in chickens. These differences amongst genotypes may partly be associated with a mismatch between the actual nutrient supply and genotype-specific nutrient requirements.

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.

Co-expulsion of Ascaridia galli and Heterakis gallinarum by chickens.

Worm expulsion is known to occur in mammalian hosts exposed to mono-species helminth infections, whilst this phenomenon is poorly described in avian hosts. Mono-species infections, however, are rather rare under natural circumstances. Therefore, we quantified the extent and duration of worm expulsion by chickens experimentally infected with both Ascaridia galli and Heterakis gallinarum, and investigated the accompanying humoral and cell-mediated host immune responses in association with population dynamics of the worms. Results demonstrated the strong co-expulsion of the two ascarid species in three phases. The expulsion patterns were characterized by non-linear alterations separated by species-specific time thresholds. Ascaridia galli burden decreased at a daily expulsion rate (e) of 4.3 worms up to a threshold of 30.5 days p.i., followed by a much lower second expulsion rate (e = 0.46), which resulted in almost, but not entirely, complete expulsion. Heterakis gallinarum was able to induce reinfection within the experimental period (9 weeks). First generation H. gallinarum worms were expelled at a daily rate of e = 0.8 worms until 36.4 days p.i., and thereafter almost no expulsion occurred. Data on both humoral and tissue-specific cellular immune responses collectively indicated that antibody production in chickens with multispecies ascarid infections is triggered by Th2 polarisation. Local Th2 immune responses and mucin-regulating genes are associated with the regulation of worm expulsion. In conclusion, the chicken host is able to eliminate the vast majority of both A. galli and H. gallinarum in three distinct phases. Worm expulsion was strongly associated with the developmental stages of the worms, where the elimination of juvenile stages was specifically targeted. A very small percentage of worms was nevertheless able to survive, reach maturity and induce reinfection if given sufficient time to complete their life cycle. Both humoral and local immune responses were associated with worm expulsion.

Time- and dose-dependent development of humoral immune responses to Ascaridia galli in experimentally and naturally infected chickens.

Factors affecting the development of Ascaridia galli-specific humoral responses and their protective roles are largely unknown. We investigated the effects of time and infection dose on A. galli-specific IgY antibody levels following experimental infection. The acquisition and development of new infections and reinfections were also monitored by using tracer birds. Relationships between the retrospective IgY and the final worm burden of the birds were investigated to determine whether humoral immune responses generated during infection provide protection to the host animal. Young chickens were infected (+) with either 100 or 1000 embryonated eggs of A. galli (100+: n = 45; 1000+: n = 45) or kept as uninfected controls (CON: n = 10). Uninfected birds were also added to each infection group as tracer (T) birds (T100+; n = 5 and T1000+; n = 5). Faecal egg counts and IgY antibody concentrations in plasma and egg yolk were determined at selected intervals. Final worm burdens were quantified at 28 weeks post infection (wpi). The plasma antibody (PAB) and egg yolk antibody (EAB) levels of experimentally infected birds were compared to those of control and tracer birds throughout the study period, and PAB levels were found to depend initially on the infection dose but thereafter mainly on reinfections. Starting at wpi 2, 1000+ had consistently higher PAB levels than CON did (P < 0.05). With exceptions at wpi 0, 2 and 12, PAB levels were also higher (P < 0.05) or tended to be higher (P < 0.10) in 100+ than in CON. An earlier and higher increase was observed in the PAB levels of T1000+ than in those of T100+, implying that (re-)infection occurrence depended on the infection dose. Although 1000+ showed higher (P < 0.05) EAB levels than CON did at both wpi 14 and 18, EAB levels were higher in 100+ than in CON only at wpi 28 (P < 0.05). The total worm burdens in the initial experimentally infected birds were similar (P = 0.257); they were also highly comparable between experimentally and naturally infected birds, indicating that final worm burden was mainly determined by the naturally occurring infections resulting from continuous exposure. When all available information on the retrospective plasma and egg yolk IgY levels was collectively evaluated to estimate the larval or total worm burdens of the experimentally infected birds, both PAB and EAB levels at particular wpi were significantly associated with worm burden, especially with larval count. In conclusion, our data support the hypothesis that the number of larvae, rather than the number of mature worms, affects the antibody levels in both plasma and egg yolk. Despite the increased levels of A. galli-specific antibodies in plasma and egg yolk throughout the study period, only a weak indication was found that antibodies might be directly associated with protection.

A comprehensive evaluation of an ELISA for the diagnosis of the two most common ascarids in chickens using plasma or egg yolks.

BACKGROUND: Classical faecal egg counts (FEC) provide less reliable diagnostic information for nematode infections in chickens. We developed an ELISA based on Ascaridia galli antigens and tested two hypotheses, as follows: (i) IgY antibodies developed against A. galli will also be useful to identify Heterakis gallinarum infections, and (ii) circulating antibodies stored in egg yolks are as good as plasma samples, so a non-invasive diagnosis is possible. The aim of this study, therefore, was to compare the diagnostic accuracy of the ELISA system with FEC, using both plasma and egg yolks from experimentally infected hens. In addition, naturally infected animals were evaluated to validate the assay. RESULTS: The assay quantified large differences (P < 0.001) in plasma or in egg-yolk IgY concentrations between infected and uninfected animals in two experiments, each performed with either of the nematode species. The assay performed with high accuracy as quantified with the area under the ROC curve (AUC) values of > 0.90 for both nematodes using either plasma or egg yolks. Sensitivity of the assay was 94 and 93% with plasma and egg yolk samples, respectively, whereas FEC yielded in a sensitivity of 84% in A. galli experiment. Total test accuracy of the assay with plasma samples (AUC = 0.99) tended to be higher (P = 0.0630) than FEC (AUC = 0.92) for A. galli, while the assay with either sample matrix performed similar to FEC (AUC ≥ 0.91) for H. gallinarum. Among the three tests, the FECs correlated better with A. galli burden than the ELISA. Although 90% of naturally infected hens were correctly identified by the ELISA, 45% of the infected hens tested negative with FEC, indicating the validity of the higher test accuracy of the ELISA. CONCLUSIONS: Antigens of A. galli can be used successfully to identify H. gallinarum-infected animals, indicating that chickens develop cross-reactive antibodies against the two closely related species. Egg yolks are as informative as plasma samples, so that animal welfare-friendly sampling is possible. Although the assay with plasma samples reveals qualitative information of higher quality than FECs on the infection status of naturally infected birds, the latter is still a better tool to assess the intensity of A. galli but not of H. gallinarum infections.

Maternal protection against Ascaridia galli?

Maternally derived antibodies can provide partial protection against certain bacterial and viral infections. We investigated whether chicks descending from nematode-infected hens are more resistant against Ascaridia galli, a prevalent gastrointestinal nematode, than chicks from nematode-free mothers. One-day-old chicks (N=153) from infected (mab+; maternal antibody+) or uninfected control dams (mab-) were experimentally infected with A. galli at two different levels (100 or 1000 eggs/chick). The worm burdens of the chicks were determined at 6 weeks post infection. There was a high correlation (r=0.89) between A. galli-specific antibody concentrations in dam plasma and egg yolk. There was no difference between worm burdens of chicks descending from infected or uninfected dams (P=0.892), indicating no maternally derived protection against A. galli. Chicks receiving the higher infection dose had higher worm burdens (P<0.05). Although there was no difference (P>0.05) between worm counts of female and male chicks infected with 100 eggs, females chicks infected with 100 eggs harboured longer and heavier female worms. We conclude that there is no protective maternal immunity against A. galli infection.