Gallinarum Eggs Research Articles

Evaluation of the detection method by a flotation method using a wire loop for gastrointestinal parasites.

Infections by gastrointestinal parasites are found in a variety of animals worldwide. For the diagnosis of such infections, the flotation method is commonly used to detect parasitic microorganisms, such as oocysts or eggs, in feces. Instead of adding a flotation solution after the final centrifugation step and using a cover slip to collect the parasites, the method using a wire loop for the recovery of the organisms has been reported as one of alternative methods. However, the recovery rates of microorganisms from the flotation method have not been analysed. In the present study, the utility of a flotation method with the use of a wire loop of 8mm in diameter (the loop method) was evaluated using different numbers of E. tenella oocysts and Heterakis gallinarum eggs, and chicken fecal samples collected at the farms. Consequently, we found that the oocysts and eggs in tubes could be collected at a ratio of 2.00 to 3.08. Thus, our results indicate that the loop method is a simple and time saving method, implicating the application for the estimated OPG/ EPG (Oocysts/Eggs per gram) of the samples.

Influence of Tempetature on Viability and Deveplopment of Heterakis gallinarum (Nematoda, Heterakidae) Eggs

Pinworms of the genus Heterakis Dujardin, 1845 parasitize a wide range of hosts, including a number of species of domestic and wild birds. These nematodes are ecologically adapted to the climatic conditions of many geographical zones, which contributes to their widespread distribution. The aim of the work was to investigate the influence of different temperature regimes at laboratory conditions on the growth, development and viability of eggs isolated from the gonads of female nematodes Heterakis gallinarum Schrank, 1788. The conducted studies established that, depending on the cultivation temperature, nematode eggs developed from 6 to 56 days, and their survival rate was from 67.7 to 83.7 %. The temperature of 25 °С was the most favourable for the development of H. gallinarum eggs, the development lasted 12 days and the highest number of eggs with motile infective larvae was formed (83.7 %). It was found that the period of egg development in cultures gradually decreased with increasing temperature: it was 56 days at 15 °C, 18 days at 20 °C, 12 days at 25 °C and 6 days at 30 °C. The survival rate of H. gallinarum eggs gradually increased with increasing temperature in regimes of 15 °С, 20 °С and 25 °С to 67.7 %, 80.7 % and 83.7 %, respectively. At a temperature of 30 °C, egg survival decreased slightly to 78.7 %. It was found that the development of H. gallinarum eggs from the zygote stage to the formation of motile infective larvae, depending on the temperature regime of cultivation, is accompanied by a decrease in egg length by 4.9–5.4 %, an increase in egg width by 4.1–7.6 % and a thinning of the shell by 9.5–28.6 %. Under different temperature regimes, the morphometric parameters of eggs during their development differ in terms of their length, width and shell thickness.

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

BackgroundA 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).MethodsFaecal, 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.ResultsTime-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.ConclusionAscarid 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.Graphical

Fenbendazole resistance in Heterakis gallinarum, the vector of Histomonas meleagridis, on a broiler breeder farm in South Carolina.

Parasites are highly prevalent in poultry; thus, the management of parasites is a key component in the profitable production of poultry. The most common nematode parasite of poultry, Heterakis gallinarum, typically causes no direct pathology but is the vector of Histomonas meleagridis, a highly pathogenic protozoan parasite that causes blackhead disease. There are no approved treatments for H. meleagridis, making control reliant on controlling the helminth vector. In the United States, the benzimidazole anthelmintic fenbendazole (FBZ) is the only approved treatment for H. gallinarum. We were contacted by an industry veterinarian regarding clinical problems with histomoniasis despite frequent anthelmintic treatments. Given that we had recently diagnosed FBZ resistance in the closely related parasite Ascaridia dissimilis, we were interested to determine if H. gallinarum had also evolved resistance. An initial on-farm pilot study using 20 birds suggested that FBZ was poorly effective, therefore a larger controlled study was initiated. Heterakis gallinarum eggs were isolated from litter at the farm and used to infect 118 chicks. Treatment groups included a non-treated control, a label-, and a 2×-label dose of FBZ, with 36 birds per group divided into two replicates of 18 birds. Three weeks post-hatch, birds were infected with 150 embryonated eggs. Two weeks post-infection treated birds were administered either a label- or 2× label-dose of FBZ in water for five days (SafeGuard® Aquasol, 1mg/kg BW). To increase the likelihood that all birds consumed the full intended dose, the dosage was calculated using 1.25 times the average body weight. One-week post-treatment, birds were euthanized, and parasites enumerated. There were no significant differences in worm numbers recovered from any of the three groups (p-value=0.3426), indicating that both dosages of FBZ failed to provide the expected levels of efficacy. These data provide strong evidence that H. gallinarum has developed resistance to FBZ on this farm. Consequently, on this farm, or any farm with FBZ-resistant H. gallinarum, H. meleagridis will continue to cycle in an unrestricted manner despite administration of anthelmintic treatments. Given recent evidence of increasing problems with histomoniasis, and the fact that resistance was documented on the first farm we investigated, further investigations are needed to determine the prevalence of resistance in H. gallinarum on poultry farms. These data, when viewed together with our recent findings of FBZ resistance in A. dissimilis on multiple farms, suggest that drug resistance in ascarid nematodes may be an emerging problem in the US poultry industry.

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

BackgroundHistomonosis 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.MethodsIn 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.ResultsThe 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.ConclusionsThe 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.Graphic abstract

A novel duplex ddPCR assay for detection and differential diagnosis of Ascaridia galli and Heterakis gallinarum eggs from chickens feces

Since the EU ban on battery cages, many studies have listed Ascaridia galli and Heterakis gallinarum as the most common roundworms in the European laying hen population. A complicating factor is that the eggs of these parasites are almost identical. Thus, lack of molecular diagnostic approaches has driven epidemiological studies to take on necropsy for species discrimination, which is labor and cost intensive. Here, we describe a novel diagnostic tool based on droplet digital PCR for simultaneous identification and absolute quantification of the eggs of both of these ascarids in chickens’ droppings using two different genus-specific primer-probe sets targeting the second internal transcribed spacer region (ITS-2) in the nuclear ribosomal (rRNA) gene array. No cross-reaction was observed when different combinations of DNA and species-specific primers and probes were tested. The lowest obtained frequency threshold for the detection of H. gallinarum in the presence of a constant A. galli DNA concentration was determined to be 0.8 %. After validation, we used the assay to analyze field samples collected from several Swedish laying hen farms. Out of 134 samples, 86 (64 %) were positive for A. galli while 11 (8.3 %) samples were positive for H. gallinarum. These samples were initially analyzed with flotation technique for detection of ascarid eggs. The results of the Cohen’s kappa indicated substantial agreement (85.8 %) between the two tests. In conclusion, we have validated a novel molecular-based diagnostic tool for quantification and differentiation between intestinal parasites of major importance in chickens with high precision. Although this study focuses on identification of parasites of laying hens, the findings may well have a bearing on all types of chicken production systems. The present study lays the groundwork for future research into epidemiology of these two important chicken parasite species.

Germination capacity of the Pochonia chlamydosporia fungus after its passage through the gastrointestinal tract of domestic chickens (Gallus gallus domesticus)

This study evaluated the germination capacity of Pochonia chlamydosporia (VC4) fungus after its passage through the gastrointestinal tract of domestic chickens and its interaction with Ascaridia galli and Heterakis gallinarum eggs. Twenty-two domestic chickens were divided in two groups: control group (G1) received shredded corn substrate without VC4; and treatment group (G2) received a single dose of 29 g corn substrate containing 3.3 × 106 conidia/chlamydospores (VC4). Subsequently, chicken fecal samples were collected at intervals of 0, 6, 8, 10, 12, 18 and 24 h. Petri dishes from fecal samples of the treated group (G2) were subdivided (G2a and G2b), and then replicated in 2% agar-water medium for the microbiological test. After VC4 growth, approximately 200 eggs of A. galli (G2a) and H. gallinarum (G2b) were added to each subgroup to evaluation of ovicidal activity. There was fungal viability after passage through chicken gastrointestinal tract and egg predation of 59.9% and 43.2% for A. galli and H. gallinarum, respectively. The present work demonstrates the ability of the fungus P. chlamydosporia to survive after passing through the gastrointestinal tract of domestic chickens, an extreme environment (low pH, enzymes, microbiota and mechanical action), and still germinate after being excreted with feces.

Occurrence of Ascaridia galli and Heterakis gallinarum in Guinea Fowl (Numida meleagris) in the State of Rondônia, Brazil

Background: Parasitic diseases are among the problems that most affect birds. The Ascaridia spp. is one of the genera which is responsible for the main infections, and the Ascaridia. galli is the most frequent species found in birds. The Ascaridia spp. egg is oval and present a smooth shell which could be mistaken with the Heterakis gallinarum eggs, once they are the most frequent parasites in birds. Therefore, this case report describes the occurrence of Ascaridia galli and Heterakis gallinarum in a guinea fowl (Numida meleagris) feces and gut.Case: In January 2017, rain season in northern Brazil, a visit was performed to a free range avian breeding property in the municipality of São Miguel do Guaporé in the state of Rondônia, Brazilian Amazonian Region. The property breeds two chicken species: Gallus gallus domesticus and Numida meleagris as well as one quail species. The problem was related to the guinea fowl breeding which was composed by 50 adults and 45 young birds about two or three weeks old. All 45 young guinea fowl were presenting various clinical signs, which included: yellowish catarrhal diarrhea, isolation, loss of appetite, body weight, ruffled feathers, drooping wings and sternal prominence. All animals that presented the clinical signs as ruffled feathers, drooping wings and sternal prominence died on an average of 24 h and the mortality was of 65.9% (29/45). Subsequently, a three-week old bird corpse was necropsied, and it was observed that its intestines presented a large amount of endoparasites besides presenting thickening. As there were a lot of helminths, they were collected along with the intestinal content for examination. The parasitological examinations of the feces were performed by Willys Molay's technique. As a result of the fluctuation technique, were observed and identified large quantities of Heterakis gallinarum and Ascaridia galli eggs. The adult worms of H. gallinarum and A. galli were identified using dichotomous key.Discussion: Ascaridia galli and Heterakis gallinarum infection were reported in guinea fowl in a free range chicken breeding property in the municipality of São Miguel do Guaporé in the state of Rondônia, Brazilian Amazonian Region. Ascaridia galli and Heterakis gallinarum eggs have already been identified in free-range chickens (Gallus gallus domesticus) by flotation techniques, as Willys Mollay and Faust and they are the main species of helminths housed by chickens. However, these parasites are not commonly reported in guinea fowl worldwide, especially in the Numida meleagris species. Regarding necropsy, intestinal wall thickening is generally a common finding which has been reported in chickens, however it is not commonly reported in guinea fowl. Additionally, the presence of clinical signs of nematode parasitosis frequently occurs in birds with high parasitic load. In this report, we believe that the guinea fowls births on the rain season was one of the factors to the high parasites incidence found, since both nematodes species are frequently identified in the rain seasons. Additionally, the management adopted by the breeder, using concomitantly the same nursery for guinea fowl and chicken, added up to inadequate sanitary management, seems to have directly influenced the contamination and recontamination of the different brood litter of guinea fowls. It is concluded that guinea fowl (Numida meleagris) located in the state of Rondônia were affected by high amounts of Ascaridia galli and Heterakis gallinarum and caused high mortality in the young specimens and consequently causing economic losses to the proprietary.

PCR detection of Heterakis gallinarum in environmental samples

Heterakis gallinarum is a widely distributed cecal nematode that parasitizes gallinaceous birds including chickens and turkeys. H. gallinarum infection poses a problem for the poultry industry as the nematode egg serves as a vector for the protozoan parasite, Histomonas meleagridis, the causative agent of histomonosis. The only means of detecting H. gallinarum in the environment is microscopic identification of the eggs in soil or feces; however, H. gallinarum eggs are often mistaken for those of Ascaridia galli. Three primer sets were designed from sequences cloned from the H. gallinarum genome to develop a diagnostic PCR. Each of these primer sets amplified a single product from H. gallinarum, but were unable to amplify DNA from H. meleagridis, Ascaridia galli, or Cestode sp. H. gallinarum DNA was amplified from Lumbricus sp. (earthworms) and Alphitobius diaperinus (darkling beetles), confirming that the earthworm acts as a paratenic host for H. gallinarum and suggesting that the darkling beetle may be a carrier for this nematode.

An In Vitro Assay of Disinfectants on the Viability of Heterakis gallinarum Eggs.

Nematodes are widespread and common in poultry. Disinfectants are used to reduce infection rates in poultry houses, but there is little documentation of their effectiveness. An in vitro assay was developed to test the efficacy of products to damage Heterakis gallinarum eggs, and nine disinfectants and chemicals commonly used in the poultry industry were tested. Embryonated eggs of H. gallinarum were pipetted into wells of plastic cell culture plates (250-300 eggs/well in water). Measured amounts of test articles were added to the suspensions for 2, 4, 6, or 24 hr. After exposure, eggs were washed with water and treated with trypan blue (1 ml of 0.4% solution, added to each well) at room temperature for 2 min. Eggshell integrity was determined microscopically by counting the number of eggs that were clear (intact) or that contained blue dye (compromised). As a test of embryo viability, five eggs per well from treatments containing compromised eggs were transferred to a Petri dish and hatched manually, using forceps to open the eggshell. Released larvae were then observed for signs of controlled movement. In a test of Clorox bleach (NaOCl), Green Klean, Decon7, Kem San, PLT, Virkon S, NaCl, dry limestone (CaCO3), and diesel fuel, only NaOCl (bleach) and Green Klean damaged the eggshell, and only 20,625 ppm of NaOCl rendered the larvae nonviable.

A survey of the incidence of worm parasites in laying chickens in Abak Local Government Area, Akwa Ibom State, Nigeria

This research is based on parasitic helminthes of laying birds conducted in July 2018 in the Department of Animal Science, Akwa Ibom State University, Akwa Ibom State, Nigeria. This work aimed at identifying the types of worm parasites that may be found in laying birds (poultry) farms and to determine their prevalent rates in layers together with the percentage infestation of these worm parasites in Abak Local Government Area. A total of 270 sterile samples were collected randomly from 10 farms in 3 weeks at Abak Local government Area. These sterile samples were transported immediately for analysis in the laboratory using floatation method with saturated salt solution. With the aid of x10 microscope, it was discovered that out of the 270 (or 100%) samples examined for helminthes only two types of nematodes were identified. A sum total of 196 (or 73%) nematode eggs were found and a total of 113 (or 42%) Ascardia gallinarum eggs were tested positive and 83 (or 31%) Heterakis gallinarum were tested positive while a total of 74 (or 27%) fecal sample tested negative. The high prevalence of gastrointestinal helminthes as observed in this area has a strong relationship with their mode of feeding and living conditions which lead to reduced body weight, decreased egg production and deaths thus affecting the meat quality and nutrient content of the chicken. Laying birds should be dewormed every three months and high level of hygiene should be maintained in the farm. It was concluded that there is a high prevalence of helminthes in Abak Local government Area in laying birds, which may lead to high losses. Thus, improved farm management and disease control to enhance their potentials is necessary.

Effect of the nematophagous fungus Pochonia chlamydosporia on soil content of ascarid eggs and infection levels in exposed hens

BackgroundThe nematophagous fungus Pochonia chlamydosporia can degrade ascarid (e.g. Ascaridia galli) eggs in agar and soil in vitro. However, it has not been investigated how this translates to reduced infection levels in naturally exposed chickens. We thus tested the infectivity of soil artificially contaminated with A. galli (and a few Heterakis gallinarum) eggs and treated with P. chlamydosporia. Sterilised and non-sterilised soils were used to examine any influence of natural soil biota.MethodsUnembryonated eggs were mixed with sterilised (S)/non-sterilised (N) soil, either treated with the fungus (F) or left as untreated controls (C) and incubated (22 °C, 35 days) to allow eggs to embryonate and fungus to grow. Egg number in soil was estimated on days 0 and 35 post-incubation. Hens were exposed to the soil (SC/SF/NC/NF) four times over 12 days by mixing soil into the feed. On day 42 post-first-exposure (p.f.e.), the hens were euthanized and parasites were recovered. Serum A. galli IgY level and ascarid eggs per gram of faeces (EPG) were examined on days -1 and 36 (IgY) or 40 p.f.e. (EPG).ResultsEgg recovery in SF soil was substantially lower than in SC soil, but recovery was not significantly different between NF and NC soils. SF hens had a mean worm count of 76 whereas the other groups had means of 355–453. Early mature/mature A. galli were recovered from SF hens whereas hens in the other groups harboured mainly immature A. galli. Heterakis gallinarum counts were low overall, especially in SF. The SF post-exposure IgY response was significantly lower while EPG was significantly higher compared to the other groups.ConclusionsPochonia chlamydosporia was very effective in reducing ascarid egg numbers in sterilised soil and thus worm burdens in the exposed hens. However, reduced exposure of hens shifted A. galli populations toward a higher proportion of mature worms and resulted in a higher faecal egg excretion within the study period. This highlights a fundamental problem in ascarid control: if not all eggs in the farm environment are inactivated, the resulting low level infections may result in higher contamination levels with associated negative long-term consequences.

Density related effects on lifetime fecundity of Heterakis gallinarum in chickens.

BackgroundDensity related effects, both inverse density- and density-dependent, contribute to regulating population dynamics of parasites. We investigated whether density related effects are directly controlling lifetime fecundity of Heterakis gallinarum.MethodsDaily total numbers of H. gallinarum eggs in faeces samples (N = 1365) from chickens (N = 39) were quantified starting from 3 weeks (wk) post-infection (p.i.). The birds were necropsied 8 wk p.i., and intensity and demographic characteristics of infrapopulations were determined. Density related effects on cumulative egg excretion (CEE), lifetime fecundity and worm length were investigated with a segmented regression analysis.ResultsFor CEE, lifetime fecundity and female worm length, we determined highly similar parasite intensity thresholds (52–54 worms), which separated infrapopulations for influences of inverse density- and density dependence. CEE increased as parasite intensity increased up to an intensity of 52 worms. After this threshold, the relationship followed more of a horizontal line indicating impaired worm fecundity at higher parasite intensities. Lifetime fecundity was enhanced linearly in infrapopulations with up to 54 worms, but thereafter decreased gradually with increasing infrapopulation size. Female worm length increased linearly with elevating parasite intensity up to a threshold of 54 worms and thereafter declined with a rate of -0.014 mm for each additional worm. Lifetime fecundity and female worm length did not significantly differ between infrapopulations below and above the thresholds (P > 0.05). Lifetime fecundity was positively associated with the percentage of male worms (r = 0.44; P < 0.001), but negatively with absolute deviation from the theoretically expected sex-ratio in the infrapopulations (r = -0.56; P = 0.005). These relationships were stronger in infrapopulations below the threshold (r = 0.51 and -0.61, respectively), and were not significantly different from zero in the infrapopulations above the threshold (P > 0.05).ConclusionsEgg production of H. gallinarum is regulated by the effects of both inverse density- and density-dependent mechanisms, which result in similar average lifetime fecundity below or above intensity thresholds. In infrapopulations below the intensity thresholds, inverse density dependence effects on lifetime fecundity appear to result partly from sex-ratio fluctuations and impaired mating success of the nematode.

Precision, repeatability and representative ability of faecal egg counts in Heterakis gallinarum infected chickens

This study investigated whether a precise and repeatable quantification of Heterakis gallinarum egg excretion, which considerably reflects the actual worm burdens, can be achieved based on collection of the daily total amount of faeces from chickens.Three-week-old birds (N=64) were infected with 200 embryonated eggs of H. gallinarum, and placed into individual cages 3wk after infection for 5wk to collect daily faeces (N=2240). The total daily faeces was mixed and a randomly taken sample per bird was analyzed to estimate the numbers of eggs per gram of faeces (EPG) and total number of eggs excreted within 24h (EPD). A total of 235 daily faecal collections were randomly selected and further examined to determine between and within sample variations of EPG counts as a measure of precision. For this, two random faecal samples were taken from the daily produced faeces by a bird, and the EPG was determined for each of the samples (EPG1 and EPG2). The second faecal sample was analyzed once more to determine a parallel EPG2 count (EPG2a) of the suspended sample. Precision of an EPG count was defined as its relative closeness to the average of two EPG counts using a relative asymmetry index (IndexEPG). At an age of 11wk, i.e. 8wk p.i. the birds were slaughtered and their worm burdens were determined.There were no significant differences between EPG1 and EPG2 (P=0.764) nor between EPG2 and EPG2a (P=0.700), suggesting that the differences between or within the samples were not different from zero. Correlations between EPG counts, as between and within sample coherences, were r=0.85 and r=0.86, respectively. Precision of EPG counts, as measured by IndexEPG, was not influenced by consistency (P=0.870) and total amount of faeces (P=0.088). However, concentration of eggs in faeces (mean EPG) had a significant effect on the precision of the EPG counts (P<0.001). Similar results were also observed for the within sample precision (IndexEPG2). A segmented regression analysis indicated an abrupt change in the precision of EPG counts as the response to changing egg concentration in the examined faecal samples. The precision of analyses remarkably heightened up to a breakpoint with an EPG count of ≤617. A similar breakpoint was also determined for within sample precision (EPG2≤621). Moderate repeatabilities (R=0.49) for EPG and EPD were estimated in the first week of egg excretion, whereas the estimates were higher (R=0.67–0.84) in the following weeks. Correlations between number of female worms with daily measured EPG and EPD increased to an almost constant level (r≥0.70; P<0.05) in a few days after the nematode excreted eggs and predominantly remained so for the rest of the sampling period.It is concluded that mixing daily total faeces provides samples with random homogenous distribution of H. gallinarum eggs. Precision of the EPG counts increases as the egg concentration in faecal sample increases. Egg excretion of H. gallinarum, quantified either as EPG or EPD, is highly repeatable and closely correlated with the actual worm burden of birds starting as early as in 5thwk of infection.

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+, TCRαβ+ 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.

Poultry litter as a source of gastrointestinal helminth infections

The aim of this study carried out in 6 commercial layer houses was to examine the effect of litter management on water content, helminth egg count and litter infectiousness with the intestinal nematodes Ascaridia galli, Heterakis gallinarum, and Capillaria spp. Three types of litter management were established in each layer house in parallel: in compartment A, litter was left undisturbed, in compartment B, wet litter was replaced and in compartment C, new litter material was added weekly. Dry matter (DM) contents of the litter and parasitological parameters (helminth egg concentration in litter samples, faecal egg counts (FECs) in the permanent layer flocks, helminth prevalence and burdens in two series of tracer animals) were determined every 4 weeks during the first 32 weeks of one laying period. DM contents of the litter varied in a broad range (48–95%); 8 weeks after onset of the study, there were significant differences between sites ( P < 0.001) but not between management regimes. A. galli/ H. gallinarum eggs were isolated from 91% of the litter samples, whereas eggs of Capillaria spp. were only extracted from 13% of the samples. Egg concentrations in litter remained at a similar level during the observation period. Neither management regime reduced helminth egg concentrations in the litter compared to the unmanaged regime. Laying hens started excreting helminth eggs 8 weeks after introduction to the layer house. In treatment C (litter added) FECs were lower than in the unmanaged treatment A in weeks 8 ( P < 0.0001), 20, and 28 (both P < 0.1). There was no correlation between the concentration of helminth eggs in the litter and the FECs of the layer flocks. The prevalence of A. galli in tracer animals was lower (<10%) than the prevalences of H. gallinarum (68–80%) and Capillaria spp. (30–58%). Prevalences and H. gallinarum burdens did not differ significantly between management regimes. Although high helminth egg concentrations were found in litter, the prevalence and worm burdens in tracer animals were low compared to a similar study with tracers kept in poultry runs. The reasons for this may be that poultry litter negatively affects viability and infectiousness of helminth eggs. However, underlying mechanisms need to be clarified.

Genetic resistance to Heterakis gallinarum in two chicken layer lines following a single dose infection

Groups of White Leghorn and New Hampshire male and female chicks were reared under helminth-free conditions and divided into two groups at an age of 8 weeks. One group was artificially infected with 100 embryonated Heterakis gallinarum eggs, while the other group was kept as uninfected control. After 8 weeks all animals were harvested and worm burden was established. Maximum of 60 worms per bird randomly selected were measured for length. Body weights were recorded at 8 and 16 weeks of age. Total worm burden was significantly ( P < 0.0001) higher in infected males than in infected females. Furthermore, White Leghorns showed a significantly ( P = 0.011) higher number of worms when compared with New Hampshire animals. Average worm lengths were not significantly different between the breeds ( P = 0.155) and sexes ( P = 0.062). Correlations between worm burden and worm length was 0.08 ( P = 0.140). The daily weight gains between 8 and 16 weeks of age of the infected animals were significantly ( P = 0.038) lower when compared with the control group. There was no significant correlation between daily weight gain and worm burden. The estimated heritabilities for logarithm (ln) worm burden were 0.41 (S.E. ± 0.086) in White Leghorn and 0.31 (S.E. ± 0.126) in New Hampshire, respectively. Based on the estimated heritabilities it is possible to select for H. gallinarum resistance in both genetically distinct chicken breeds. This approach may be of importance for chickens kept in floor husbandry systems.

Observations on the occurrence of Heterakis gallinarum in laying hens kept on soil

The occurrence of Heterakis gallinarum infection in a flock of Rhode Island Red laying hens is described. These hens were entirely kept in houses on a farm for commercial egg production, where a deep litter production system was adopted. Faecal samples from 120 hens selected at random were examined by common flotation technique and modified McMaster’s technique. H. gallinarum eggs were detected in 50% of the examined samples with very low faecal egg counts (<50 eggs per gram of faeces). There was no evidence of clinical signs, gross pathological lesions, and consequences on production level linkable to heterakiasis. H. gallinarum is transmitted by direct ingestion of infective eggs from the soil and is one of the most important intestinal helminths of poultry due to the role it plays as vector of histomoniasis. In accordance with European legislation on the welfare of laying hens, a progressively increasing number of farmers can adopt breeding programs on soil. Periodic coprological examinations of chickens reared on commercial farms from areas throughout Italy are thus advisable to determine the exact distribution of H. gallinarum and the extent to which heterakiasis may influence health status and production of chickens in this country.

THE RELATIONSHIP BETWEEN EIMERIA TENELLA AND HETERAKIS GALLINARUM INFECTION IN BROILER CHICKENS

The present investigation dealt with the effect of concurrent infections of E. tenella and Heterakis gallinarum in broiler chickens. Six groups of 10 chicks (15 day old) coccidia free Hubbard chicken were used in this work. Group I was infected with 50.000 sporulated oocysts of E. tenella per chick and considered as Eimeria positive control. Group 2 was infested with 150 embryonated Heterakis gallinarum and consid-ered as Heterakis gallinarum positive control. The third group of chicken received 150 embryonated Heterakis gallinarum eggs at 6th day before inoculation with 50.000 sporulated oocysts of E. tenella. The4thgroup received embryonated eggs atthe 6thday after inoculation with sporulated oocysts. Group 5 received both parasites at the same time Heterakis gallinarum followed by E.tenella one hour later. Group 6 was kept without treatment and considered as a negative control. The results indicated that weight gain of infected chicken with Heter-akis gallinarum and Eimeria tenella was significantly decreased than that of non infested negative control and the chicken inoculated with embryonated Heterakis gallinarum eggs at 6 days before; on the same day and at 6 days after receiving Eimeria sporulated oocysts produced significantlylowerEimeriaoocyst count than positive coccidialcontrol group. Otherwise,chickens receiving oocysts and embryonated eggs on the same day did not have significant variation in the count of Heterakis gallinarum worms. Also, hypoprteinamia, hypoalbuminamia and hypoglobulinamia was noticed in the infected groups comparing with control ones.

Stochasticity accelerates nematode egg development.

Day-degrees models of nematode development assume that temperature stochasticity has no effect on the development rate of infective stages as long as the mean temperature is held constant. This assumption was tested in this study. Unembryonated Heterakis gallinarum eggs were subjected to nocturnal and diurnal daily temperature cycles at 12 and 17 C. respectively, and embryonation was compared with eggs subjected to similar stochastic daily cycles, in which random normal variations in the temperature were added to the 2 temperatures. The prediction that there is no effect of stochasticity was refuted. Embryonation of eggs subjected to variable daily cycles occurred significantly earlier than that of eggs subjected to deterministic daily cycles, suggesting that stochastic variation in temperature accelerated embryonation even though mean temperatures were the same. These findings show that the development time of H. gallnarum eggs is decreased by stochastic variation in temperature, which may have important implications for the effects of climate change on parasite availability.