Assay for the simultaneous detection of Raillietina spp. (R. echinobothrida, R. tetragona, and R. cesticillus) and Ascaridia galli infection in chickens using duplex loop-mediated isothermal amplification integrated with a lateral flow dipstick assay.

Raillietina species and Ascaridia galli are two of the significant intestinal parasites that affect chickens in a free-range system production. They destroy the intestinal mucosa layer, leading to several clinical symptoms such as weight loss, a slowed growth rate, and economic value loss. Thus, the objective of this study was to develop an assay for simultaneously detecting Raillietina spp. (R. echinobothrida, R. tetragona, and R. cesticillus) and A. galli in a single reaction using duplex loop-mediated isothermal amplification (dLAMP) coupled with a lateral flow dipstick (LFD) assay. The analytical specificity of the dLAMP-LFD assay showed a high specific amplification of Raillietina spp. and A. galli without non-target amplification. Regarding the analytical sensitivity, this approach was capable of simultaneously detecting concentrations as low as 5 pg/µL of mixed-targets. To evaluate the efficiency of the dLAMP assay, 30 faecal samples of chickens were verified and compared through microscopic examination. The dLAMP-LFD assay and microscopic examination results showed kappa values of Raillietina spp. and A. galli with moderate (K= 0.615) to high (K= 1) agreements, respectively, while the McNemar's test indicated that the efficiency between assays was not significantly different. Therefore, the developed dLAMP-LFD assay can be used as an alternative screening method to the existing classical method for epidemiological investigation, epidemic control, and farm management, as well as for addressing poultry health problems.

Comparative Effect of Ascaridia Galli Infection on the Two Laying Hen Lines on the Liver Function and Immune Response.

Chicken production is quickly rising due to the low associated costs and the capability of poultry to convert nutrients into biological protein along with chicken meat accounting for 30% of all animal protein eaten by humans. Despite advances in poultry production, parasitic illnesses in laying hens remain a problem. Farm birds reared in semi-intensive and free-range systems are more prone to parasite infections due to the absorption of polluted water and food from scavenging behaviors and waste droppings. In this study, the effects of Ascaridia galli infection on the immune response and liver function of two laying hen lines are compared, and their infection resistance is determined. In total, 50 laying hens at eight weeks of age were used (25 Lohmann brown-classic and 25 Lohmann lsl-lite), and each line was divided into two groups: an infected group (n=15), which was orally infected with a single dose of 500 A. galli embryonated eggs, and a control group (n=10), which was given normal saline. After four and eight weeks, blood was collected from the wing vein to assess the serum's AST, ALT, total protein, and IgY levels. The results demonstrated that the infected Lohmann brown-classic and Lohmann lsl-lite chickens presented significantly increased (P≤0.05) AST, ALT, and IgY, compared to the respective control group. Moreover, Lohmann brown-classic hens presented a significantly increased (P≤0.05) IgY concentration four weeks after infection, compared to Lohmann lsl-lite hens. From our results, it can be concluded that genetic variation plays a crucial role in the immune response against A. galli, where the Lohmann brown-classic line was found to be more resistant, compared to the Lohmann lsl-lite line.

Ascaridia galli infection in chicken: Pathobiology and immunological orchestra.

BACKGROUND: Ascaridia galli is the largest gut-dwelling helminth of chickens, which confers adverse effects on meat and egg production; thus, on the animal protein supply and the economy. Both adult and immature parasites affect gut health, but larval stages play a major role in pathology. AIMS: Here, we present immunology and pathology of A. galli in chickens. MATERIALS AND METHODS: Literatures were surveyed through online platforms such as PubMed, Google Scholar and Researchgate. RESULTS: The larvae cause excessive mucus production, damage to the intestinal gland, hemorrhage, anemia, diarrhea, and malnutrition. The adult worms can cause death by intestinal obstruction and intussusception. Although both cellular and humoral immunity are involved in fighting against ascariasis, the role of naturally acquired immunity is poorly defined. In cellular immunity, Th-2 cytokines (IL-4, IL-5, IL-9, and IL-13), goblet cells (mucin), gut-associated lymphoid tissues, CD8α+ intraepithelial cells, TCRγδ + T cells, and TGF-ß4 form a protective band. Type 2 immunity provides protection by forming a network of endogenous damage-associated molecular patterns, chitin, and parasitic antigens. Among antibodies, IgY is the most prominent in chickens and provides temporary humoral protection. During parasitic infection, infiltration of various immune cells is evident, especially in the intestinal epithelium, lamina propria, and crypts of the duodenum and jejunum. In chickens older than 12 weeks, gradual reduction of worm burden is more successful than the younger birds. Female chickens exert a short-lived but higher level of protection by passing IgY to chicks in the form of egg yolk antibodies. In laying conditions, immunity differs between breeds. This review provides an overview of the silent but inevitable pathological changes induced by A. galli and the interaction of host immunity with the parasite.

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.

Pumpkin seeds, lemongrass essential oil and ripleaf leaves as feed additives for Ascaridia galli infected laying hens.

The present study was performed to evaluate the in vivo efficiency of Curcurbita pepo (pumpkin) seeds, Cymbopogon citratus (lemongrass) essential oil and Plantago lanceolata (ripleaf) leaves against helminth infections in laying hens. In the first experiment, 75 Lohmann LSL Classic hens naturally infected with Ascaridia galli were assigned to groups of five; groups were randomly assigned to one of three treatments with five replicates each (untreated control; lemongrass oil: 1 g/bird/day; pumpkin seeds: 10 g/bird/day). Feed consumption and egg production were continuously recorded, individual faecal egg counts were determined weekly, and E. coli and Lactobacillus spp. three times during the experimental period of 29 days. After slaughter, intestinal worms were counted and sexed. Pumpkin improved feed conversion as compared to the control (p = 0.008) and to lemongrass (p = 0.021); no treatment effect on any other parameter was found. In the second experiment, 75 LSL pullets were artificially infected with 3 × 200 A. galli eggs, randomly divided into groups of five and assigned to one of three treatments (untreated control, lemongrass oil: 1 g/bird/day; ripleaf: 5% of ration). After 109 days of sampling as described above, hens were slaughtered and worm burdens determined. Performance of the animals did not change regardless of the treatment and none of the treatments resulted in changes of the microbiological and parasitological parameters. In conclusion, with the exception of improved feed conversion in the pumpkin group, no positive nor negative effects of the additives on performance, parasitological and microbiological parameters of naturally and artificially A. galli infected laying hens were observed.

Pharmacokinetics and therapeutic efficacy of levamisole in Ascaridia galli experimentally infected ducks.

This study investigated the pharmacokinetics and therapeutic efficacy of levamisole (LVS) after intravenous (i.v.) and oral administrations to healthy and Ascaridia galli-infected ducks by developing an infection model. Twenty-four two-week old ducklings were experimentally infected with A. galli. The ducks were monitored for the development of infection and after 8 weeks they were administered with LVS at a single dose of 30 mg/kg by oral or i.v. administration. Sixteen healthy ducks were subjected to the same treatment and served as control. Serial blood samples were taken for LVS determination with HPLC-UV and pharmacokinetic analysis was carried out based on the non-compartmental approach. The LVS therapeutic efficacy was determined 1 week post drug administration by intestinal worm count at necropsy. In vivo data on development of ascariasis in ducks showed that 8 weeks post inoculation the number of eggs per gram of feces reached at least 100 in each bird. After a single dose of LVS, no parasites were recovered upon necropsy. Results of the pharmacokinetic study showed no statistical differences between infected and non-infected birds for both routes of administration. The mean oral bioavailability was slightly below 50% in both experimental groups. In conclusion, the pharmacokinetics of LVS in ducks was not affected by experimentally-induced ascariasis. A single dose of LVS was found to be efficient against experimental ascariasis in ducks induced by in field isolates of A. galli.

Ascaridia galli eggs obtained from fresh excreta, worm uteri or worms cultured in artificial media differ in embryonation capacity and infectivity.

Ascaridia galli infection models use eggs isolated from chicken excreta, worm uteri and worms cultured in artificial media. The aim of this study was to compare the infectivity of A. galli eggs isolated from these sources under two infection regimens. A 3 × 2 factorial arrangement was employed to test the infectivity of A. galli eggs from the three sources and two modes of infection (single or trickle infection). One hundred and fifty-six Isa-Brown one day-old cockerels randomly assigned to the six treatment groups (n = 26) were orally infected with embryonated A. galli eggs obtained from the three A. galli egg sources (worm uteri, excreta or eggs shed in vitro) administered either as single dose of 300 eggs at one day-old or trickle infected with 3 doses of 100 eggs over the first week of life. Twenty-two negative control birds remained uninfected. Eggs obtained from cultured worms or excreta exhibited a higher embryonation capacity (P = 0.003) than eggs obtained from worm uteri. There were higher worm establishment (infectivity) rates from embryonated eggs originating from cultured worms and worm uteri compared with eggs obtained from fresh excreta (P < 0.0001). Trickle infection resulted in a significantly higher total worm burden (P = 0.002), establishment rate (P = 0.002) and excreta egg counts (EEC, P = 0.025) than single infection. Worm length was greater in birds infected with embryonated eggs from excreta than from uteri or cultured worms (P < 0.0001). However, mode of infection did not affect worm length (P = 0.719) and weight (P = 0.945). A strong significant positive linear correlation was observed between EECs and female worm counts at 12 weeks of post infection sampling (r = 0.75; P < 0.0001). Body weight of birds was negatively correlated with both worm burden (r = - 0.21; P < 0.01) and EEC (r = - 0.20; P < 0.05) at 12 weeks post infection. In conclusion, our results show that eggs shed by cultured worms or isolated from worm uteri had greater infective capacity than eggs harvested from excreta and that trickle rather than bolus infection resulted in higher worm establishment. These factors should be taken into account when considering artificial infection protocols for A. galli.

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.

Method optimisation for prolonged laboratory storage of Ascaridia galli eggs.

Eggs in the infective stage of the chicken nematode Ascaridia galli are often required for in vivo and in vitro studies on this parasite. The reliability of any artificial A. galli infection depends on the viability and embryonation capacity of A. galli eggs. The aim of this study was to determine ideal storage conditions for maximising the viability of A. galli eggs and maintaining viability for the longest period. A 2 × 2 × 3 × 5 factorial experimental design was employed to investigate the effects of storage temperature (4°C or 26°C), storage condition (aerobic or anaerobic), storage medium (water, 0.1 N H2SO4 or 2% formalin) and storage period (4, 8, 12, 16 and 20 weeks). The viability of eggs was assessed after eggs in all treatment groups were held aerobically at 26°C for 2 weeks after the storage period to test embryonation capacity. Based on morphological characteristics, they were categorised as undeveloped, developing, vermiform, embryonated or dead. The maintenance of viability during storage at 4°C was optimal under anaerobic conditions while at 26°C it was optimal under aerobic conditions. Anaerobic conditions at 26°C led to a rapid loss of viability while aerobic conditions at 4°C had a less severe negative effect on maintenance of viability. Egg storage in 0.1 N H2SO4 resulted in a significantly higher viability overall (54.7%) than storage in 2% formalin (49.2%) or water (37.3%) (P < 0.0001). Untreated water was the least favourable storage medium when eggs were stored at 26°C while it was a medium of intermediate quality at 4°C. The viability of A. galli eggs decreased significantly with storage time (P < 0.0001) depending on the other factors. The lowest rate of decline was seen with storage of eggs under anaerobic conditions at 4°C or aerobic conditions at 26°C in 0.1 N H2SO4. Eggs in these treatments retained up to 72% of overall viability at 20 weeks with a decline rate of approximately 2% per week with no significant difference between the two. Therefore, this study has clearly revealed opposing aerobic conditions required for prolonged storage of A. galli eggs in the pre-embryonated state at 4°C. It has also identified that 0.1 N H2SO4 provides the best preservation against degradation during storage, particularly at 26°C under aerobic conditions. Achieving strictly anaerobic conditions can be difficult to achieve so storage aerobically at 26°C may be preferred for simplicity.

Infection with Ascaridia galli Does Not Significantly Alter Intestinal Microbiota and Is Cleared After Changes in the Expression of Cytokines.

Because of the trend of cage-free egg production, infections with the nematode Ascaridia galli are receiving increased attention. The aim of this study was to establish a timeline for the influence of A. galli on the expression of key cytokines related to a parasitic immune response, and on the composition of the jejunal microbiota. Twenty-eight male layer-type birds were challenged at 24, 25, and 26 days of age. An additional 28 birds were kept as uninfected controls. Starting on Day 31, three birds of each group were euthanized every week until 8 wk postinfection (PI). The number of larvae isolated from the intestinal wall decreased over time, until no larvae were seen at 7 and 8 wk PI. At 5 wk PI, there was a numerical upregulation of all cytokines (TGF-ß, IFN-γ, IL-4, IL-8, IL-10, IL-13) in the infected group, but this change was only statistically significant for IL-13. At this time point, larvae were expected to have developed into adults that would have shed eggs in the feces. However, no adult worms were seen and there was no egg shedding. For the microbiota analysis, there were significant differences in the alpha diversity (Faith's phylogenetic diversity) between challenge and control groups, and the beta diversity analysis showed slight differences between samples, suggesting that the age of the birds was the main reason for the separation of groups. These findings suggest that the upregulation of all cytokines evaluated in Week 5 might be the reason for resolution of the infection. Possible explanations are that a high infection dose and the fact that birds were fed with a more nutritionally dense feed might have contributed to the birds' immune system clearing the infection before the worms were able to reach maturity.

La infección por Ascaridia galli no altera significativamente la microbiota intestinal y se elimina tras cambios en la expresión de citocinas. Debido a la tendencia de la producción de huevos libres de jaulas, las infecciones con el nematodo Ascaridia galli están recibiendo una mayor atención. El objetivo de este estudio fue establecer una línea de tiempo para la influencia de A. galli en la expresión de citoquinas clave relacionadas con una respuesta inmune parasitaria y en la composición de la microbiota yeyunal. Veintiocho aves macho de tipo postura fueron desafiadas a los 24, 25 y 26 días de edad. Se mantuvieron 28 aves adicionales como controles no infectados. A partir del día 31, se practicó la eutanasia a tres aves de cada grupo cada semana hasta las 8 semanas posteriores a la infección (PI). El número de larvas aisladas de la pared intestinal disminuyó con el tiempo, hasta que no se observaron larvas a las 7 y 8 semanas después de la infección. A las cinco semanas post-infección, hubo una regulación ascendente numérica de todas las citoquinas (TGF-ß, IFN-γ, IL-4, IL-8, IL-10, IL-13) en el grupo infectado, pero este cambio solo fue estadísticamente significativo para IL-13. En ese momento, se esperaba que las larvas se hubieran convertido en adultos que eliminarían huevos en las heces. Sin embargo, no se observaron nemátodos adultos y no hubo eliminación de huevos. Para el análisis de microbiota, hubo diferencias significativas en la diversidad alfa (diversidad filogenética de Faith) entre los grupos de desafío y control y el análisis de diversidad beta mostró ligeras diferencias entre las muestras, lo que sugiere que la edad de las aves fue la razón principal de la separación de los grupos. Estos hallazgos sugieren que la regulación al alza de todas las citocinas evaluadas en la semana 5 podría ser el motivo de la resolución de la infección. Las posibles explicaciones son que una dosis alta de infección y el hecho de que las aves fueran alimentadas con un alimento más denso desde el punto de vista nutricional podrían haber contribuido a que el sistema inmunitario de las aves eliminara la infección antes de que los nemátodos pudieran alcanzar la madurez.

Morphological and molecular characterization of Ascaridia columbae in the domestic pigeon (Columba livia domestica) and the assessment of its immunological responses.

Parasitism is a divesting problem that is frequently overlooked and may result in severe prominent clinical manifestation. This study aimed to investigate the seasonal and sexual prevalence of the gastrointestinal nematode Ascaridia columbae (A. columbae) infection among domestic pigeons in Giza governorate, Egypt, during the period from 2020 to 2021. One hundred and sixty suspected pigeons were clinically investigated. Blood & tissue samples were collected from infected birds to estimate serum zinc concentration, malondialdehyde (MDA), and nitric oxide levels. As well as tumor necrosis factor-alpha (TNF-α), interleukin 1ß (IL1ß) activity, and histopathological examination were estimated; also, worms were collected for morphological identification using electron microscope (SEM) and molecularly identified using polymerase chain reaction (PCR), further sequenced, and submitted in GenBank with accession number MZ343369. The average ascarid (length × breadth) were 72.4 ± 3.3 µm (70.5 - 79.9 µm) × 39.9 ± 2.5 µm (37.6 - 42.3 µm). The distinguishing morphological characteristics that have been noticed in ascarid worms were creamy white, cylindrical worm with triradiate lips with wide cephalic alae extending on both the lateral sides and filariform esophagus. In males, spicules were almost equal with the presence of precloacal chitinous-rimmed sucker. The prevalence of A. columbae infection was (63.1%) with a higher incidence in females (79.2%) than males (46.1%). The highest seasonal prevalence was observed in winter (92.5%), followed by summer and spring (87.5% and 55%), respectively while, the lowest prevalence was observed in autumn (17.5%). The intensity of worms in the infected intestine varied from 5 to 120 adult worms. The histopathological examination revealed the presence of chronic diffuse moderate catarrhal enteritis with roundworms in the lumen. Infected birds showed a significant increase in nitric oxide and MDA levels while serum zinc levels were lowered in infected pigeons. Infected pigeons revealed a marked increase in IL1-ß and TNFα than apparently healthy ones.

Ascaridia galli challenge model for worm propagation in young chickens with or without immunosuppression.

With the continued growth of free-range egg production, the importance of the chicken roundworm Ascaridia galli is increasing. Investigations into this parasite would be facilitated by the availability of characterised strains and clear guidelines on optimal methods of multiplication and maintenance. Currently, there is lack of well-defined in vivo models for maintaining A. galli and the potential of using host immunosuppression to boost parasite development and worm egg output has not been investigated. To determine the most efficient way of propagating A. galli in young chickens an experiment with a 2 × 3 × 4 × 2 factorial design involving age of chicken at infection (day-old or 14 days old), immunosuppression (dexamethasone (DEX), cyclophosphamide (CY) or sham), infective egg dose (0, 100, 300 or 900 embryonated eggs/bird) and time of worm recovery after infection (8 or 10 weeks post-infection) was conducted. The experiment used a total of 384 layer cockerel chicks. Infection was delivered orally in 3 split doses over one week and immunosuppressants were administered by intramuscular injection concurrently with the infections. Body weight, excreta egg counts, intestinal worm count and worm establishment rate were assessed. The only sign of ascaridiosis noted was mild diarrhoea at the time of slaughter in some birds with a significant- positive association with worm count. Infection caused a significant dose dependent reduction in body weight in non-immunosuppressed birds but this effect was ameliorated by immunosuppression. Age at infection had no significant effect on the studied variables although both worm and egg counts were numerically higher in the day-old infected groups. Egg dose significantly influenced the prevalence of infection, worm establishment rate, worm egg production and mean worm count. The 300 and 900 egg doses resulted in significantly higher worm count and egg production than the 100 egg dose. A significant negative correlation was observed between egg dose and worm establishment rate indicating an inverse relationship. Immunosuppression with DEX, but not CY resulted in significantly higher mean worm burden than in control chickens with excreta egg counts also considerably higher in DEX treated birds. Our results suggest that trickle infection at day-old with infective doses of 300 eggs coupled with immunosuppression with DEX would provide the most efficient way to propagate A. galli worms in vivo, as using older birds or a higher egg dose did not provide any advantage.

Anthelmintic efficacy evaluation against different developmental stages of Ascaridia galli following individual or group administration in artificially trickle-infected chickens.

The efficacy of commercially available anthelmintics against mature and immature stages (including ovicidal effects) of two Australian field isolates of Ascaridia galli was evaluated in two separate experiments. The anthelmintics tested were levamisole (LEV), piperazine (PIP) and flubendazole (FBZ) plus LEV-PIP. A total of 192 artificially trickle-infected young cockerels (96 birds per isolate) were randomized into sixteen experimental groups of 12 cockerels each (7 treatments and 1 untreated control per isolate). Chickens received label-recommended doses of LEV (28 mg/kg), PIP (100 mg/kg) or LEV-PIP co-administered at their full individual doses as a single oral dose or in group drinking water at recommended concentrations of 0.8 mg/ml or 2.5 mg/ml over eight hours for 1 and 2 days respectively and FLBZ (30 ppm) in the feed over 7 days. Anthelmintic efficacies were assessed by worm count reduction (WCR%) and excreta egg count reduction (EECR%) estimated by two methods. Ten days post treatment, all untreated control birds harboured mixed worm population of 10.1 and 12.3/bird for each isolate respectively which was significantly higher (P < 0.0001) than counts in all treatment groups. Luminal or histotrophic larvae comprised 50-57 % of the total worm count. For LEV, PIP and LEV-PIP, individual oral administration provided a somewhat higher efficacy than group medication in drinking water. EECR% values were inconsistent with WCR% and found to be only an indicator of efficacy against adult worms. All developmental stages of the two A. galli isolates were highly susceptible to FLBZ (100 %) followed by LEV-PIP (92.4-100 %) and LEV (87.7-100 %). PIP exhibited good efficacy against adult worms (92-97 %) but reduced efficacy against luminal (79-84 %) and histotrophic (61-72 %) larvae. Embryonation capacity of eggs recovered from worms expelled after treatment with LEV (47-54 %), PIP (44-54 %) or LEV-PIP (45-48 %) did not differ from those from untreated birds (50-51 %) whereas eggs from FLBZ treated worms had a significantly lower (P < 0.05) capacity to embryonate (≤ 2 %). Put together, our results demonstrate no evidence of resistance of the test A. galli isolates to the tested anthelmintics but a significant advantage of FLBZ, followed by LEV-PIP and LEV over PIP in the control of A. galli, specifically with regard to immature stages. A. galli worms expelled after treatment with LEV, PIP or their combination, but not FLBZ contain viable eggs. This has epidemiological implications and may also provide an option for isolating eggs from mature worms for A. galli propagation experiments without having to sacrifice birds.

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.

Molecular characterization of Ascaridia galli from Bangladesh and development of a PCR method for distinguishing A. galli from Heterakis spp.

We analyzed the nuclear ribosomal internal transcribed spacer (ITS) 1 and ITS2 sequences for Bangladesh isolates of Ascaridia galli, and we determined that the sequences were unreliable as molecular markers for distinguishing A. galli from other Ascaridia species, because the sequences showed high identity with that of A. columbae. However, the ITS1 sequences were available for designing PCR primers distinguishable between Ascaridia galli and Heterakis spp. Bangladesh isolates of A. galli constituted a monophyletic clade along with other geographical isolates in the cytochrome c oxidase subunit I (COI) phylogenetic tree, however, we could not clarify the phylogenetic relationships between A. galli and other Ascaridia spp., because their available sequences in GenBank were very few. The developed PCR method using DNA from A. galli and Heterakis spp. eggs would enable differential diagnosis of the individual infections in the future.

Viability and development of Ascaridia galli eggs recovered in artificial media followed by storage under different conditions.

Eggs oviposited by Ascaridia galli females in artificial media are commonly used as a source of infective material. We investigated the rate of egg production by cultured mature females (n = 223), and changes in egg viability under different storage and incubation conditions. Eggs recovered after 1, 2 or 3 days of culture were subjected to either (1) storage in water at 4°C (1, 4 or 8 weeks) followed by incubation in 0.1 N H2SO4 at 26°C (2, 4 or 6 weeks); or (2) prolonged storage at 4°C (up to 14 weeks). Egg development and viability was assessed by morphology coupled with a viability dye exclusion test of hatched larvae. Of the 6,044 eggs recovered per mature female 49.2, 38.5 and 12.3% were recovered on days 1, 2 and 3 of worm incubation respectively with similar initial viability (≥99%) between days. Eggs recovered on different days had only minor differences in viability after storage. The prolonged storage period at 4°C significantly affected both viability and embryonation ability resulting in decline in viability of 5.7-6.2% per week. A smaller but significant decline in egg (2.0%) and hatched larval (1.4%) viability per week of incubation at 26°C was also observed. We conclude that storage and incubation conditions, not the day of egg recovery, are the main factors affecting A. galli egg viability. Our findings indicate that under aerobic conditions storage at 26°C may be preferable to 4°C whereas other studies indicate that under anaerobic conditions storage at 4°C is preferable.

Effect of a targeted treatment strategy against Ascaridia galli on egg production, egg quality and bird health in a laying hen farm.

Worm control is an important aspect of the successful management of the egg production industry. Of particular concern is Ascaridia galli, which at high parasite loads affect health and production in layers. Application of a targeted treatment strategy (TT) to control A. galli has shown promise. The purpose of this study was to explore the effect of such a strategy on welfare indicators and production performance of layers. Six flocks (F1-6) on a commercial farm were allocated to three treatment groups. Flocks F1 and F4 were treated (TT) with fenbendazole at 22, 27 and 36 weeks post-placement (WPP). Flocks F2 and F5 were treated at 27 WPP (conventional treatment, CT) and hens in flocks F3 and F6 served as untreated (UT) control groups. At 19, 35 and 45 WPP twenty-five hens plus thirty eggs per flock were randomly selected. Hens were weighed and their plumage conditions (PC) were assessed. The eggs were subjected to various external and interior quality analyses. Production data such as number of eggs/hen/week, egg mass and feed conversion ratio (FCR) were calculated from raw data obtained from all flocks on a weekly basis. The number of eggs/hen/week, egg mass and FCR were higher (P < 0.05) in the TT flocks and hens had better PC both at 35 and 45 WPP compared with other flocks. No differences in body weight and physical egg quality were observed between groups except for egg shell strength which was higher (P < 0.05) in the CT flocks. These data suggest that better production performance and plumage, which suggests improved health, can be achieved through the application of a TT strategy. The insights gained from this research should help to justify the extra cost and labor associated with the TT strategy.

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.

Gastroenteric parasite of wild Galliformes in the Italian Alps: implication for conservation management.

This study provides insights about the diversity, prevalence and distribution of alpine wild galliformes gastrointestinal parasite community, trying to fill a gap in the scientific information currently available in scientific literature. The analysis included three host species: 77 rock partridge (Alectoris graeca saxatilis), 83 black grouse (Tetrao tetrix tetrix) and 26 rock ptarmigan (Lagopus muta helveticus) shot during the hunting seasons 2008-2015. Parasites isolated were Ascaridia compar, Capillaria caudinflata and cestodes. The rock ptarmigan was free from gastrointestinal parasites, whereas the most prevalent helminth (37%) was A. compar in both black grouse and rock partridge. C. caudinflata occurrence was significantly higher in black grouse (prevalence = 10%, mean abundance = 0.6 parasites/sampled animal) than in rock partridge (prevalence = 1.20%, mean abundance = 0.01 parasites/sampled animal). Significant differences were detected among hunting districts. A. compar was found with a significant higher degree of infestation in the hunting districts in the northern part of the study area whereas cestodes abundance was higher in Lanzo Valley. Quantitative analysis of risk factors was carried out using a generalized linear model (GLM) only on the most common parasite (A. compar). Latitude was the only factors associated with infestation risk (OR = 52.4). This study provides information on the composition and variability of the parasite community in the alpine Galliformes species.