An automated faecal egg count system for detection of Ascaridia galli ova in chickens.

Chicken production has increased over the past decade, resulting in a concomitant rise in the demand for more humane options for poultry products including cage-free, free-range, and organic meat and eggs. These husbandry changes, however, have come hand-in-hand with increased prevalence of Ascaridia galli infection, which can cause clinical disease in chickens as well as the occasional appearance of worms in eggs. Additionally, development of anthelmintic resistance in closely related helminths of turkeys highlights the need for closely monitored anthelmintic treatment programs. Manual faecal egg counts (FECs) can be time-consuming and require specialist training. As such, this study sought to validate an automated FEC system for use in detection and quantification of A. galli eggs in chicken faeces. Automated counts using the Parasight System (PS) were compared to traditional manual McMaster counting for both precision and correlation between methods. Overall, ten repeated counts were performed on twenty individual samples for a total of 200 counts performed for each method. A strong, statistically significant correlation was found between methods (R2 = 0.7879, P < 0.0001), and PS counted more eggs and performed with statistically significant higher precision (P = 0.0391) than manual McMaster counting. This study suggests that PS is a good alternative method for performing A. galli FECs and provides a new tool for use in helminth treatment and control programs in chicken operations.

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.

A molecular and morphological study of Ascaris suum in a human-pig contact scenario in northeastern Brazil / Estudo molecular e morfológico de Ascaris suum em um cenário de contato entre suínos e humanos no nordeste brasileiro

The aim of the present study was to assess morphologic and genetic data on ascariasis in swine (Sus scrofa domesticus) and humans in low-resource rural and periurban communities in the state of Piauí, Brazil. Our cross-sectional survey included 100 fecal samples obtained from swine and 682 samples from humans. Fifteen pigs were necropsied. Human and porcine fecal samples were examined to identify Ascaris eggs. Parasites obtained in the swine necropsies were studied using scanning electron microscopy (SEM), and the mitochondrial gene encoding the cytochrome oxidase 1 (cox1) enzyme was partially amplified and sequenced for molecular taxonomy and phylogenetic analyses. The overall prevalence of Ascaris eggs in the swine fecal samples was 16/100 (16%). No Ascaris eggs were identified in the human fecal samples. SEM of six worms recovered from pigs demonstrated morphological characteristics of A. suum. Cox1 sequences were compatible with A. suum reference sequences. Original and reference (GenBank) nucleotide sequences were organized into clusters that did not segregate the parasites by host species or and region. The largest haplogroups were dominated by haplotypes H01, H02 and H31. In the communities studied, there was no epidemiological evidence of the zoonotic transmission of ascariasis at the human-swine interface.(AU)

O presente estudo teve como objetivo acessar dados morfológicos e genéticos sobre a ascaridíase em suínos (Sus scrofa domesticus) e humanos, em comunidades rurais e periurbanas no estado do Piauí. O estudo transversal incluiu 100 amostras fecais de suínos e 682 amostras obtidas de humanos. Quinze suínos foram necropsiados. Amostras fecais suínas e humanas foram examinadas para detecção de ovos de Ascaris. Os parasitas adultos, obtidos nas necropsias, foram estudados através de microscopia eletrônica de varredura (MEV), e o gene mitocondrial codificante da enzima citocromo oxidase 1 (cox1) foi parcialmente amplificado e sequenciado para análises filogenéticas e de taxonomia molecular. A prevalência de Ascaris em amostras fecais de suínos foi 16/100 (16%), não sendo identificado nenhum caso de infecção por este parasita em humanos. A análise por MEV de parasitas recuperados de suínos demonstrou características morfológicas de Ascaris suum. As sequências nucleotídicas de cox1 foram compatíveis com A. suum. As sequências originais e de referência (obtidas no GeneBank) foram organizadas em clusters que não segregaram os parasitas por hospedeiro ou região geográfica. Os maiores haplogrupos foram dominados pelos haplótipos H01, H02 e H31. Nas comunidades estudadas, não foi evidenciada transmissão zoonótica de A. suum na interface suíno-humana.(AU)

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.

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.

Ascaridiasis hepatobiliar en una adulta joven / Hepatobiliary ascaridiasis in a young adult woman

Se presenta el caso clínico de una paciente de 27 años de edad, con antecedentes de colecistectomía convencional desde hacía 10 meses por aparente colecistitis, quien acudió al Hospital Luis Vernaza de Guayaquil, Ecuador, por presentar ictericia y dolor abdominal. Teniendo en cuenta los hallazgos clínicos, de laboratorio e imagenológicos se le diagnosticó sepsis de foco abdominal, colangitis y coledocolitiasis. Durante la intervención quirúrgica se observó la presencia de 2 Ascaris lumbricoides y cálculo de colesterol en la vía biliar, por lo que se le realizó una derivación bilioentérica. Después de algunas complicaciones como insuficiencia respiratoria y descompensación hemodinámica, la paciente egresó de la institución a los 25 días de operada, con seguimiento por consulta externa durante 2 meses.

The case report of a 27 years patient is presented, with history of conventional cholecystectomy for 10 months due to apparent cholecystitis who went to Luis Vernaza Hospital in Guayaquil, Ecuador, presenting jaundice and abdominal pain. Taking into account the clinical, laboratory and imaging findings a sepsis of abdominal focus, cholangitis and choledocolithiasis was diagnosed. During the surgical intervention the presence of 2 Ascaris lumbricoides and cholesterol calculi in the bile duct was observed, reason why a bilioenteric bypass was carried out. After some complications such as breathing failure and hemodynamic upset, the patient was discharged from the institution 25 days after the surgery, with follow up in outpatient clinics during 2 months.

Ascaridia galli Case as a Pseudohelminth in a Human Arm Skin

A 25-year-old male patient, who had terminal stage skin cancer (malignant melanocytic neoplasia) on his skin of arm was infected with white, round, worm parasites in the lesion area of the skin. Parasites were collected with a thin forceps in a petri dish, cleaned in warm physiological saline, fixed by 70% hot ethyl alcohol and clarified in lactophenol, respectively. All 14 female and 7 male nematodes, some of which had partial autolysis, were identified as Ascaridia galli, a chicken nematode. Due to the fact that it is a very rare case in the medical literature, we wish to report it.

Reliability of three common fecal egg counting techniques for detecting strongylid and ascarid infections in horses.

The detection and quantification of nematode eggs using fecal egg count techniques have an irreplaceable role in equine parasitic control. The reliability, particularly precision and accuracy, of individual techniques have been described only for strongylid infections. The aim of this study was to compare three fecal egg count techniques used for the detection of the two most common equine nematode infections: strongylid and ascarid. The Simple McMaster, Concentration McMaster and Mini-FLOTAC techniques were tested on spiked fecal samples with various levels of egg concentration (50, 100, 200, 500, 1000 and 3000 eggs per gram) and naturally infected mixed strongylid-ascarid samples with 30 replicates. The Simple McMaster, Concentration McMaster and Mini-FLOTAC techniques had precision coefficients of variation of 44.33, 35.64 and 18.25% for the strongylid infection and 62.95, 35.71 and 18.95% for the ascarid infection, and percent accuracies (mean count/number of eggs spiked) of 97.53, 88.39 and 74.18% for the strongylid infection and 65.53, 83.18 and 90.28% for the ascarid infection, respectively. Accuracy depended greatly on the type of nematode, but precision did not. The Mini-FLOTAC technique was more precise than the Simple and Concentration McMaster techniques regardless of nematode type. Simple McMaster was the most accurate technique for detecting strongylid eggs, and Mini-FLOTAC was the most accurate technique for detecting ascarid eggs. Our results indicated that none of the current techniques were universally and sufficiently reliable for the simultaneous quantification of both of these common equine nematodes.

Analysis of antibody levels in egg yolk for detection of exposure to Ascaridia galli parasites in commercial laying hens.

Ascaridia galli is one of the most abundant nematode parasites in poultry. A. galli infections can significantly impact the profitability of egg farms and have negative implications for bird health and welfare. The main objectives of this study were to determine whether A. galli specific antibodies in egg yolks can be used to detect prior or current exposure to A. galli in laying hens, and to distinguish between eggs obtained from caged and free-range hens. Twenty-two laying hen flocks from different production systems (10 free-range, 2 barn-housed, and 9 caged flocks) were enrolled in the study. An in-house enzyme-linked immunosorbent assay was used to analyze levels of A. galli specific antibodies in yolk. The numbers of A. galli eggs in hen excreta were also determined in a subset of farms. Free-range flocks had higher and also more variable levels of anti-A. galli antibodies in the egg yolk compared to those of the cage flocks (0.50 ± 0.39 vs. 0.16 ± 0.13 OD units) (P < 0.001). Results also confirmed that excreta from free-range and barn-housed flocks contained higher numbers of A. galli eggs than did excreta from caged flocks in which no A. galli eggs were detected. In conclusion, analysis of anti-A. galli antibodies in the egg yolk can be used to detect worm exposure in commercial layer flocks. However, the method used in this study cannot be used in isolation to distinguish between eggs from cage and free-range production systems as anti-A galli antibodies were detected in egg yolk samples from all production systems, and the range of antibody levels overlapped between production systems.

Detection of Ascaridia galli infection in free-range laying hens.

Reliable methods for detection of A. galli infection using excreta egg count (EEC) and ELISA assays to determine A. galli specific IgY levels in serum and yolk samples were compared from hens infected naturally and artificially. Artificially infected hens were used to generate samples for analysis of preferred detection methods and to generate contaminated ranges for use in the naturally acquired infection study in which Lohmann Brown hens (n = 200) at 16 weeks of age were randomly assigned to four treatments with five replicate pens. Hens of negative control (NC) ranged on a decontaminated area, hens of low infection, medium infection and positive control (PC) ranged on the areas previously contaminated by hens artificially infected with 250, 1000 and 2500 A. galli eggs/hen, respectively. Additionally, hens of PC were orally infected with 1000 A. galli eggs/hen. Anti A. galli antibody levels in hen serum (SIgY) and yolk (YIgY) were measured before range access, and 2, 7 and 12 weeks after access to the contaminated ranges. In a natural infection study, eggs were detected in the excreta of all hens 4 weeks after range access, with the exception of NC in which no eggs were detected. EEC increased to reach maximum value (2204 ±â€¯307 eggs/g) after 11 weeks of range access and then declined at 12 weeks (905 ±â€¯307eggs/g) (p < 0.01). While SIgY OD values were not different in hens between any groups before range access, after 2 weeks, both SIgY and YIgY gradually increased in hens of PC (1.17 ±â€¯0.03 and 0.88 ±â€¯0.04) and medium infection (1.07 ±â€¯0.03 and 0.96 ±â€¯0.04) compared to low infection (0.38 ±â€¯0.03 and 0.29 ±â€¯0.04) (p < 0.01) and NC. After 12 weeks, SIgY were similar in hens of PC, medium and low groups whereas YIgY was higher in hens of low infection group (p < 0.01). Sensitivity of the serum and egg yolk antibody levels assay to detect A. galli infection was 100% and 96%, respectively, whereas the pooled EEC method yielded a sensitivity of 93%. The results of this study suggest that hens naturally infected with A. galli produce both SIgY and YIgY at different levels depending on the infection intensity and duration of exposure which allows the diagnosis of prior infection or early diagnosis of current infection. Use of the practical and non-invasive method of yolk sample analysis for detecting IgY can be just as informative as using serum samples to detect A. galli infection.

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.

Aspectos clínicos e radiológicos da ascaridíase no trato biliar / Clinical and radiological aspects of biliary tract ascariasis

O áscaris é o verme cilíndrico mais comum do intestino humano, com aproximadamente 1 bilhão de pessoas infectadas no mundo. O verme pode se infiltrar nas vias biliares causando sintomas de cólicas biliares e icterícia. A ultrassonografia de abdômen tem se mostrado ser um exame não invasivo com alta acurácia no seu diagnóstico.

Ascaris is the most common and the largest roundworm parasite of the human intestine. Approximately 1 billion people worldwide are infected. The worm can infiltrate the biliary tract causing symptoms of biliary colic and jaundice. Ultrasound of abdomen proved to be a noninvasive test with high diagnostic accuracy.

Ascaridia galli in chickens: intestinal localization and comparison of methods to isolate the larvae within the first week of infection.

This study was conducted to observe the localization and to compare methods for isolation of minute Ascaridia galli larvae in chicken intestine. Firstly, six 7-week-old layer pullets were orally infected with 2,000 embryonated A. galli eggs and necropsied either at 3, 5 or 7 days post infection (dpi). More than 95 % of the recovered larvae were obtained from the anterior half of the jejunoileum, suggesting this part as the initial predilection site for A. galli larvae. Secondly, the intestinal wall of one layer pullet infected with 20,000 A. galli eggs 3 days earlier was digested in pepsin-HCl for 90 min. The initial 10 min of digestion released 51 % of the totally recovered larvae and the last 30 min of continuous digestion yielded only 5 %. This indicates that the majority of larvae were located superficially in the intestinal mucosa. Thirdly, 48 7-week-old layer pullets were infected with 500 A. galli eggs and necropsied at 3 dpi to compare three different larval isolation methods from the intestinal wall, viz., EDTA incubation, agar-gel incubation and pepsin-HCl digestion, resulting in mean percentages of the recovered larvae: 14.4, 18.2 and 20.0 %, respectively (P = 0.15). As conclusion, we recommended Pepsin-HCl digestion as the method of choice for larval recovery from the intestinal wall in future population dynamics study due to high efficiency and quick and simple detection. The agar-gel method was considered to be a prerequisite for molecular and immunological investigations as the larvae were more active and fully intact.

Partial purification and characterization of Ascaridia galli diagnostic worm antigen.

Partial purification of Ascaridia galli whole worm extract was conducted by Cyanogen bromide Sepharose 4B immunoaffinity column chromatography. The resulted fraction was characterized by sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The fraction was found to be consisted of six bands of 207 KDa, 157 KDa. 110 KDa, 103 KDa, 76 KDa and 41 KDa. This profile was compared with that of whole worm and excretory-secretory antigens. Both antigens were resolved into multiple bands in both high and low molecular weight ranges. The isoelectric focusing of the fraction displayed 8 bands of isoelectric points 7.5, 7.0, 6.8, 6.5, 6.2, 5.8. 5.3 and 4.6. The potency of this fraction in the diagnosis of natural ascaridiosis in chickens was assessed by ELISA compared with that of whole worm and ES antigens. The affinity purified fraction showed higher potentials in the diagnosis of A. galli infection in chickens than whole worm antigen at any sera dilution and than ES antigen at high sera dilutions. While ES antigen of the worms revealed higher diagnostic capabilities than whole worm extract. The current research recommends utilization of the affinity isolated fraction in the diagnosis of natural ascaridiosis in chickens.