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

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

Maternal protection against Ascaridia galli?

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

Embryonation ability of Ascaridia galli eggs isolated from worm uteri or host faeces.

Experimental infection models for Ascaridia galli rely on the use of eggs isolated either directly from worm uteri or from host faeces. We investigated whether A. galli eggs isolated from the two sources differ in their embryonation ability. A. galli eggs originating from 12 worm infrapopulations were isolated both from faeces of the living host (faecal eggs) and directly from worm uteri after host necropsy (uterine eggs). The isolated eggs from each infrapopulation and source were incubated in Petri dishes (n=24) containing a potassium-dichromate (0.1%) medium for 28 days (d) at room temperature. Starting from the day of egg isolation (d0), in ovo larval development was evaluated every second day by examining morphological characteristics of 200 eggs/petri dish. A total of 72,000 eggs were classified into undeveloped, early development, vermiform or fully embryonated stages. Isolation procedures caused similar damage to uterine and faecal eggs (2.2% and 0.5%, respectively; P=0.180). The first sign of in ovo embryonic development in faecal eggs (7%) was observed during the 24-h period when faeces were collected. On d28, a higher percentage of uterine eggs remained undeveloped when compared with faecal eggs (58.6% vs 11.0%; P<0.001). Although a higher (P<0.001) percentage of faecal eggs entered both the early developmental and vermiform stages, which took place primarily within the first two weeks of incubation, there was no time-shift between the development of faecal and uterine eggs. Starting from day 10, higher (P<0.05) percentages of faecal eggs completed embryonation compared with uterine equivalents. Eggs from both sources reached a plateau of embryonation by the end of 2nd week of incubation, with faecal eggs having a greater than two-fold higher embryonation ability. Cumulative mortality was higher in uterine eggs (14.3%) than in faecal eggs (0.2%). We conclude that faecal eggs have a higher embryonation ability than uterine eggs possibly due to maturation differences.